method for recovering butanol, methods for producing butanol and composition

专利摘要:
  METHOD TO RECOVER BUTANOL, METHODS TO PRODUCE BUTANOL AND COMPOSITIONThe present invention relates to a method for producing butanol by microbial fermentation, in which the butanol product is removed duringextraction fermentation in a water-immiscible organic extractor in thepresence of at least one electrolyte in a concentration that is at leastenough to increase the partition coefficient of butanol in relation to thecoefficient in the presence of the salt concentration of the basal medium for fermentation. The electrolyte may comprise a salt that dissociates in the fermentation medium, or in the aqueous phase of a biphasic fermentation medium, to form free ions. Also provided is a method and composition for recovering butanol from a fermentation medium.
公开号:BR112012012219A2
申请号:R112012012219-3
申请日:2010-11-23
公开日:2021-02-02
发明作者:Michael Charles Grady；Ranjan Patnaik
申请人:Butamax (Tm) Advanced Biofuels Llc.；
IPC主号:

专利说明:

| 1 “METHOD TO RECOVER BUTANOL, METHODS TO PRODUCE | 'BUTANOL AND COMPOSITION ”| : CROSS REFERENCE FOR RELATED ORDERS This order claims the priority benefit for the Order | 5 - Provisional Patent US 61 / 263,519, filed on November 23, | 2.009, the disclosure of which is incorporated herein by reference in its | wholeness. | FIELD OF THE INVENTION | The present invention is related to the field of | 10 biofuels. More specifically, the invention relates to a method for producing butanol by means of microbial fermentation, in which at least one electrolyte is present in the fermentation medium in a concentration at | less enough to increase the partition coefficient of butanol in | compared to that in the presence of the salt concentration of the basal medium for fermentation, and the butanol product is removed by extraction in a water-immiscible organic extractor.
BACKGROUND OF THE INVENTION Butanol is an important industrial chemical product that has several applications, for example, its use as a fuel additive, as a mixing component for diesel fuel, as a raw material for chemicals in the plastics industry and as a food grade extractor in the food and seasoning industry. Each year, about 10 to 12 million tons of butanol are produced by petrochemical means. As the need for butanol is growing, the interest in producing this chemical from renewable resources such as corn, sugar cane, or cellulosic sources through fermentation is expanding. In a fermentation process to produce butanol, removal of the product in situ advantageously reduces the inhibition of micro-
| j | butanol and improves fermentation rates by controlling 'butanol concentrations in the fermentation broth.
Technologies for | «Removal of the product in situ include extraction, adsorption, pervaporation, | membrane solvent extraction and liquid-liquid extraction.
In the extraction | liquid-liquid, an extracting agent is contacted with the fermentation broth | for the partition of the butanol between the fermentation broth and the extractor phase.
O | butanol and the extractor are recovered by a separation process, for example, by distillation.
JJ Malinowski and AJ Daugulis, A / IChE Journal (1994), 40 (9): 1459-1465, describe experimental studies to evaluate the effect of adding salt on the extraction of 1-butanol, ethanol and acetone from aqueous solutions diluted using cyclopentanol, n-valeraldehyde, tert-amyl alcohol, and Adol 85SNF (composed mostly of oleyl alcohol) as extractors.
The authors note in their conclusions that, despite the advantages that the addition of salt offers in the extraction of ethanol, 1-butanol and acetone from dilute aqueous solutions typically found in fermentation processes, the practical application of such a configuration process is currently limited.
How to | an in situ recovery strategy (extractive fermentation) the relatively high concentrations of salts that may be needed could have severely deleterious effects on cells resulting from osmotic shock.
Published Patent Application US 2009/0171129 A1 discloses methods for the recovery of C3-C; k alcohols from dilute aqueous solutions, such as fermentation broths.
The method includes increasing the activity of C3-CÊ alcohol, in a portion of the aqueous solution so that there is - at least C3 alcohol saturation; -Cg in the portion.
According to an exemplary embodiment of the invention, increasing the activity of C3-C alcohol; may comprise the addition of a hydrophilic solute to the aqueous solution.
Sufficient hydrophilic solute is added to allow the formation of a second liquid phase, either solely by adding the hydrophilic solute or by combining it with other process steps.
The additional hydrophilic solute can: be a salt, an amino acid, a water-soluble solvent, a sugar or combinations thereof.
US Patent Application 12/478389 filed on June 4, 2009, describes methods for the production and recovery of butanol from a fermentation broth, the methods comprise the step of contacting the fermentation broth with an organic extractor immiscible in water selected from the group consisting of C12 to C22 fatty alcohols, C12 to Cx fatty acids, C12 to C22 fatty acid esters, C12 to C22 fatty aldehydes, and mixtures thereof, to form a two phase mixture comprising an aqueous phase and an organic phase containing butanol.
Provisional Patent Applications US 61/168640, US 61/168642 and US 61/168645; deposited simultaneously on April 13, 2009; and US 61/231697, US 61/231698 and US 61/231699; deposited simultaneously on August 6, 2009, they disclose methods for the production and recovery of butane! from a fermentation medium, the methods comprise the step of contacting the fermentation medium with a water-immiscible organic extractor comprising a first solvent and a second solvent, the first solvent being selected from the group consisting of fatty alcohols C12 to Cr2, fatty acids C12 to C27, esters of fatty acids C12 to C22, fatty aldehydes C12 to C22, and mixtures thereof, and the second solvent being selected from the group consisting of C alcohols; Ci11, OC carboxylic acids; to C411, esters of C carboxylic acids; to C11, C aldehydes; to Cu, and mixtures thereof, to form a two-phase mixture comprising an aqueous phase and an organic phase containing butanol.
Improved methods for the production and recovery of butanol from a fermentation medium are constantly being used.
| | wanted. A process for removing the butanol product in situ is desired | : in which the addition of electrolyte to a fermentation medium provides * improved butanol extraction efficiency and acceptable biocompatibility with the micro-organism.
BRIEF DESCRIPTION OF THE INVENTION The present invention provides a method for recovering the | butanol from a fermentation medium comprising water, butanol, at least one electrolyte, and a genetically modified microorganism that produces butanol from at least one fermentable carbon source. The electrolyte is present in the fermentation medium in a concentration at least sufficient to increase the partition coefficient of butanol in relation to that in the presence of the salt concentration of the basal medium for fermentation. The present invention also provides methods for the production of butanol using such a microorganism and an added electrolyte. The - methods include contacting the fermentation medium with i) a first water-immiscible organic extractor and, optionally, ii) a second water-immiscible organic extractor, separating the organic phase containing butanol from the organic phase, and recovering the butane! from the organic phase containing butanol. In an example of an embodiment of the invention, a method is provided for recovering butanol from a fermentation medium, comprising said method: a) providing a fermentation medium comprising butanol, water, at least one electrolyte in at least a sufficient concentration to increase the partition coefficient of butanol in relation to that in the - presence of the salt concentration of the basal medium for fermentation, and a micro- | genetically modified organism that produces butanol from at least one fermentable carbon source; b) put the fermentation medium in contact with i) a first
| water-immiscible organic extractor selected from the group consisting of fatty alcohols C12 to C22, fatty acids C12 to C> 2, fatty acid esters C12 to "C> 22, fatty aldehydes C12 to C22, fatty amides C12 to C22 and mixtures thereof; and optionally, ii) a second water-immiscible organic extractor selected from —part of the group consisting of C fatty alcohols; to C22, C fatty acids; to C22, C fatty acid esters; to C22, C fatty aldehydes ; C22, C fatty amides; Cx and mixtures thereof to form a two-phase mixture comprising an aqueous phase and an organic phase containing butanol; Cc) optionally separating the organic phase containing butanol from the aqueous phase; and d) recovering the butane from the organic phase containing butanol to produce recovered butanol.
In some examples of embodiments, a portion of the butanol is removed simultaneously from the fermentation medium by a process comprising the steps of: a) stripping the butanol from the | fermentation medium with a gas to form a gas phase containing butanol; and b) recovering the butanol from the gas phase containing butanol.
According to the methods of the invention, the electrolyte can be added to the fermentation medium, to the first extractor, to the optional second extractor, or in a combination thereof.
In some embodiments, the electrolyte comprises a salt having a cation selected from the group consisting of lithium, sodium, potassium, rubidium, cesium, magnesium, calcium, strontium, barium, ammonium, phosphonium, and combinations thereof.
In some embodiments, the electrolyte comprises a salt having an anion - selected from the group consisting of sulfate, carbonate, acetate, citrate, lactate, phosphate, fluoride, chloride, bromide, iodide, and combinations thereof.
In some embodiments, the electrolyte is selected from the group consisting of sodium sulfate, sodium chloride, and combinations thereof.
According to the methods of the invention, in some examples of | achievements the genetically modified micro-organism is selected from; of the group consisting of bacteria, cyanobacteria, filamentous fungi and yeasts. In some examples, the bacteria are selected from the group consisting of Zymomonas, Escherichia, Salmonella, Rhodococcus, - Pseudomonas, - Bacillus, - Lactobacillus, - Enterococcus, Pediococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium and Brory . In some examples of realization, the | yeast is selected from the group consisting of Pichia, Candida, | 10 Hansenula, Kluyveromyces, Issatchenkia, and Saccharomyces. According to the methods of the invention, the first extractor can be selected from the group consisting of oleyl alcohol, alcohol! | behenyl, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, methyl oleate, lauric aldehyde, 1-dodecanol and mixtures thereof. In some embodiments, the first extractor comprises oleyl alcohol. In some embodiments, the second extractor can be selected from the group consisting of 1-nonanol, 1-decanol, 1-undecanol, 2-undecanol, 1-nonanal, and a combination of these.
In some embodiments, the butanol is 1-butanol. In some embodiments, butanol is 2-butanol. In some embodiments, butanol is isobutanol. In some exemplary embodiments, the fermentation medium further comprises ethanol, and the butanol-containing organic phase contains ethanol.
In an example of an embodiment of the invention, a method for producing butanol is provided, comprising said method: a) providing a genetically modified microorganism that produces butanol from at least one fermentable carbon source;
u RR 7 | b) to grow the microorganism in a fermentation medium: biphasic comprising an aqueous phase ei) a first organic extractor + water immiscible selected from the group consisting of fatty alcohols C12 to C22, fatty acids C12 to C22, esters of fatty acids Ci2 to Co2, —C2 fatty aldehydes, C12 to C22 fatty amides, and mixtures thereof, and | optionally, ii) a second water-immiscible organic extractor selected from the group consisting of C alcohols; to C> 22, C carboxylic acids; to C> 22, esters of C carboxylic acids; to C22, C aldehydes; to C22, C fatty amides; à Co, and mixtures thereof, in which the biphasic fermentation medium | 10 additionally comprises at least one electrolyte, in a concentration at least sufficient to increase the partition coefficient of butane! in relation to that in the presence of the salt concentration of the basal medium for fermentation, for a time sufficient to allow the extraction of the butanol in the organic extractor to form an organic phase containing butanol; | C) optionally separating the butanol-containing organic phase from the aqueous phase; and d) recovering the butanol from the organic phase containing butanol to produce recovered butanol.
In an example of an embodiment of the invention, a "method for producing butanol, comprising said method, is provided: a) providing a genetically modified microorganism that produces butanol from at least one fermentable carbon source; b) growing the micro-organism in a fermentation medium in which the micro-organism produces butane! in the fermentation medium to produce a medium fermentation containing butanol; c) add at least one electrolyte to the fermentation medium to supply the electrolyte in a concentration at least sufficient to increase the partition coefficient of the butane! in relation to the one in the presence ta. | l | the concentration of salt in the basal medium for fermentation; : d) put at least a portion of the butanol-containing fermentation medium in contact with i) a first water-immiscible organic extractor selected from the group consisting of Ci2 to —Cz2 fatty alcohols, Ci7 to Cpo fatty acids, esters of fatty acids C12 to C22, fatty aldehydes Ci7 a Cor, fatty amides Ci, a C », and mixtures thereof, and optionally, ii) a second water-immiscible organic extractor selected from the group consisting of C alcohols; to C22, C carboxylic acids; to C22, esters of C carboxylic acids; to C22, C aldehydes; to C22, fatty amides C; aCrx and mixtures thereof, to form a mixture of two phases comprising an aqueous phase and an organic phase containing butanol; e) optionally, separating the butanol-containing organic phase from the aqueous phase; f) recovering butanol from the organic phase containing butanol; and g) optionally, returning at least a part of the aqueous phase to the fermentation medium. In some examples of realization, the microorganism | genetically modified comprises a modification that inactivates a pathway that competes for the flow of carbon. In some embodiments, the genetically modified microorganism does not produce acetone.
BRIEF DESCRIPTION OF THE FIGURES AND SEQUENCE DESCRIPTIONS Figure 1 schematically illustrates an example of realization of the methods of the present invention, in which the first extractor and the second extractor are combined in a container before coming into contact with —the fermentation method in the fermentation vessel . Figure 2 schematically illustrates an example of carrying out the methods of the present invention, in which the first extractor and the second extractor are added separately to a fermentation vessel in which the fermentation medium is contacted with the extractors. : Figure 3 schematically illustrates an embodiment of the. methods of the invention, wherein the first and second extractor are added separately in different fermentation vessels. Figure 4 schematically illustrates an example of realization | of the methods of the present invention, in which the extraction of the product takes place downstream of the fermenter and the first extractor and the second extractor are combined in a container before contact of the fermentation medium with | extractors in a different container.
Figure 5: schematically illustrates an example of realization of the methods of the present invention, in which the extraction of the product takes place downstream of the fermenter and the first extractor and the second extractor are added separately to a container in which the fermentation medium is brought into contact with extractors.
Figure 6: schematically illustrates an example of realization of the methods of the present invention, in which the extraction of the product occurs downstream of the fermenter and the first extractor and the second extractor are added separately in a different container for contact with the fermentation medium .
Figure 7 illustrates schematically an example of realization of | methods of the present invention, in which extraction of the product occurs in at least | a batch fermenter (batch - or batch) through the co-current flow of | a water-immiscible organic extractor at or near the bottom of a fermentation must to fill the fermenter with the extractor that flows out of the fermenter at a point at or near the top of the fermenter.
The following strings comply with Title 37 of the | CFR $ 1,821-1,825 ("Requirements for Patent Applications Containing Nucleotide Sequences and / or Amino Acid Sequence Disclosures - the
Sequence Rules ") and are consistent with the World Intellectual Property Organization (OMPINVIPO), Standard ST.25 (1998) and the EPO and PCT sequence listing requirements (Rules 5.2 and 49.5 (a-bis), and in section 208 and in Annex C of the administrative instructions.) TABLE 1A
SEQ ID NUMBERS OF CODING AND PROTEINS. SEQ ID NO: SEQ ID NO: Description nucleic acid amino acid Klebsiella pneumoniae budB (acetolactate synthase) E. coli ilvC (acetohydroxy acid reductoisomerase) pg ni cn E. coli ilvD (acetohydroxy acid dehydratase) Pg Lactococcus lactis kivD (a-keto acid decase) (codon-branched chain) optimized) Achromobacter xylosoxidans sadB (butanol dehydrogenase) Bacillus subtilis alsS (acetolactate synthase) S. cerevisiae IL V5 (acetohydroxy acid "reductoisomerase;" KARI ") Mutant KARI (encoded by Pf5.ilvC) | as as Streptococcus mutans ilvD (acetohydroxy acid ”dehydratase) Bacillus subtilis kivD (19 keto acid decarboxylase (codon 20 | branched chain) optimized) Horse liver dehydrogenase alcohol (HADH) 56 (codon 57 optimized) E. coli pflB (pyruvate lyase format) E. coli frdB (73 7 fumarate reductase enzyme complex subunit) and E. coli IdhA (lactate dehydrogenase) E. coli adhE (alcohol dehydrogenase) | | E. coli frdA (subunit of the fumarate reductase enzyme complex) E. coli frdC (subunit of the ss fumarate reductase enzyme complex) E. coli frdD (subunit of the fumarate reductase enzyme complex) |
| | TABLE 1B: SEQ ID NUMBERS USED IN CONSTRUCTION, PRIMERS AND VECTORS o 2 | remm | 'e | |
ERR [FERE Poe Ag [FS TRA ma] [SRT mg [eme Ro ao Rg Pere
Ms 12 Bei seat | GPD1 promoter fragment [| - and AÊ BF | FE A | the FETUS RR
RR E PRS423 FBA ilvD (Strep) | 8 Fo a [FERE
It's the FCEE
DETAILED DESCRIPTION OF THE INVENTION | The present invention provides methods for recovering butanol from a microbial fermentation medium comprising at least one electrolyte by extraction in a water-immiscible organic extractor to form a two-phase mixture comprising an aqueous phase and an organic phase containing butanol. The electrolyte is present in the fermentation medium in a concentration at least sufficient to increase the partition coefficient of butanol in relation to that in the presence of the salt concentration of the basal medium for fermentation. The organic phase containing butanol is separated from the aqueous phase and the butanol can be recovered. Methods for the production of butanol are also provided.
DEFINITIONS 'The following definitions are used in this disclosure. . The term "electrolyte" refers to a solute that ionizes or dissociates | in an aqueous solution and can function as an ionic conductor. The term "butanol" refers to 1-butanol, 2-butanol and / or isobutane!, Individually or as mixtures thereof. | The term “water immiscible” refers to a chemical component, | such as an extractor or solvent, which is unable to mix with a | aqueous solution, such as a fermentation broth, in order to form a phasic liquid. | The term "extractor" as used herein refers to one or more organic solvents that are used to extract the butane! from a fermentation broth. The term "biphasic fermentation medium" refers to a two-stage growth medium comprising a fermentation medium (i.e., an aqueous phase) and a suitable amount of a water-immiscible organic extractor.
The term "organic phase" as used in the present, refers to the non-aqueous phase of a biphasic mixture obtained by contacting a fermentation broth with a water-immiscible organic extractor.
The term "aqueous phase" as used herein, refers to the phase of a biphasic mixture, obtained by contacting an aqueous fermentation medium with a water-immiscible organic extractor, which comprises water.
The term "removal of the product in situ", as used herein, means the selective removal of a specific fermentation product from a biological process, such as fermentation to control the concentration of the product in the biological process.
The term “fermentation broth” as used in this f | | means the mixture of water, sugars, dissolved solids, solids in: suspension, microorganisms that produce butanol, butane product! and all other constituents of the material in the fermentation vessel in which product butanol is being produced by the reaction of sugars in —butanol, water and carbon dioxide (CO> z) by the microorganisms present. The fermentation broth may comprise one or more sources of fermentable carbon, such as the sugars described herein. The fermentation broth is the aqueous phase in the biphasic fermentative extraction. From time to time, as used in the present, the term "fermentation medium" can be used as a synonym for "fermentation broth".
The term "fermentation vessel" or just "vessel" as used herein, means the vessel in which the fermentation reaction, through which the butanol product is produced from sugars, is carried out. The term "fermenter" can be used as a synonym for the term "fermentation vessel".
The term "fermentable carbon source" refers to a carbon source capable of being metabolized by the microorganisms disclosed herein. Suitable fermentable carbon sources include, but are not limited to, monosaccharides, such as glucose or fructose; disaccharides , such as lactose or sucrose; oligosaccharides; polysaccharides, such as starch, cellulose; substrates of a carbon; and a combination of these, which can be found in the fermentation medium. Fermentable carbon sources include renewable carbon, which is carbon obtained by other non-petroleum sources, including carbon from agricultural raw materials, algae, cellulose, —hemicellulose, lignocellulose, or any combination thereof.
The term "fatty acid" as used in the present invention refers to a carboxylic acid having a long, aliphatic chain of C; to C> 22 carbon atoms that are saturated or unsaturated.
O o E E Ea
15 'The term "fatty alcohol" as used in the present invention º refers to an alcohol having a long C aliphatic chain; a Cx atoms: carbon that is saturated or unsaturated.
The term "fatty aldehyde" as used in the present invention refers to an aldehyde having a long C aliphatic chain; to C >> | carbon atoms that are saturated or unsaturated. | The term "grease amide" as used in the present invention refers to an amide having a long aliphatic chain of C12 to C >> carbon atoms that is saturated or unsaturated.
The term "partition coefficient", abbreviated as K ,, means the relationship between the concentration of a compound in two phases of a mixture of two immiscible solvents in equilibrium.
A partition coefficient is a measure of the differential solubility of a compound between two immiscible solvents.
As used herein, the "partition coefficient for butanol" refers to the ratio of butanol concentrations between the organic phase comprising the extractor and the aqueous phase comprising the fermentation medium.
The partition coefficient, as used at present, is synonymous with the term distribution coefficient.
The term “separation” as used herein is synonymous | 20 “recovery” and refers to the removal of a chemical compound from an initial mixture to obtain the compound in greater or greater purity | concentration than the purity or concentration of the compound in the initial mixture.
The term "butanol biosynthetic pathway" as used herein refers to an enzymatic pathway for the production of 1-butanol, 2-butanol or isobutanol.
The term “1-butanol biosynthetic pathway” as used in | present, refers to an enzymatic pathway for the production of 1-butanol from | acetyl-coenzyme A (acetyl-CoA).
The term "2-butanol biosynthetic pathway" as used herein refers to an enzymatic pathway for the production of 2-butanol from. pyruvate. The term "isobutanol biosynthetic pathway" as used in the - present, refers to an enzymatic pathway for the production of isobutane! from pyruvate. The term "effective title" as used in the present invention, refers to the total amount of butanol produced by fermentation per liter of | fermentation medium. The total amount of butanol includes: (i) the amount of | 10 butanol in the fermentation medium; (ii) the amount of butanol recovered at | from the organic extractor; and (iii) the amount of butanol recovered from the gas phase, if gas desorption is used. The term "effective rate" as used in the present invention, refers to the total amount of butanol produced by fermentation per liter of | 15 —my fermentation per hour of fermentation.
The term "effective yield" as used herein, refers to the amount of butanol produced per unit of fermentable carbon substrate consumed by the biocatalyst.
The term "aerobic conditions", as used in the present invention, means growth conditions in the presence of oxygen.
The term "microaerobic conditions", as used in the present invention, means growth conditions with low levels of oxygen (i.e., below normal atmospheric oxygen levels).
The term "anaerobic conditions", as used in the present invention, means growth conditions in the absence of oxygen.
The term "minimal medium", as used herein, refers to growth media that have the least possible nutrients for growth, usually without the presence of amino acids. A minimal medium usually contains a source of fermentable carbon and various salts, which may vary between microorganisms and growing conditions. salts generally provide essential elements, such as magnesium, nitrogen, phosphorus and sulfur, to allow the microorganism to synthesize proteins and nucleic acids.
The term “defined medium” as used herein, refers to growth media that have known amounts of all the ingredients present, for example, a defined carbon source and nitrogen source, and trace elements and vitamins needed for micro- body.
The term "biocompatibility" as used herein refers to the measure of a microorganism's ability to use glucose in the presence of an extractor. A biocompatible extractor allows the microorganism to use glucose. A non-biocompatible extractor (ie , biotoxic) does not allow the microorganism to use glucose, for example, at a rate greater than about 25% of the rate when the extractor is not present. The term "ºC" means degrees Celsius. The term "OD ”Means optical density. | The term“ ODçov ”refers to the optical density in the length of - around 600 nm.
The term "ATCC", as used herein, refers to the American Collection of Type Cultures, "American Type Culture Collection", Manassas, VA.
The term "sec" means second (s).
The term "min" means minute (s).
The term "h" means hour (s).
The term "mL" means milliliter (s).
The term "L" means liter (s).
The term "g" means grams. 'The term “mmol” means millimol (es). . The term "M" means molar. The term "ul" means microliter. The term "ug" means microgram. | The term "ug / mL" means microgram per liter milliliter. | The term "mL / min" means milliliters per minute. The term "g / L" means gram per liter. The term "g / L / h" means gram per liter per hour. | 10 The term "mmoi / min / mg" means millimoles per minute per milligram. The term "temp" means temperature. The term "rpm" means rotations per minute. | The term “HPLC” means high pressure liquid gas chromatography.
The term "GC" means gas chromatography.
All publications, patents, patent applications, and others | references mentioned in the present invention are expressly and | fully incorporated into the present by the reference for all purposes. | In addition, when a quantity, concentration or other value or parameter | is given as a range, preferred range, or a list of preferred upper and lower preferred values, these should be understood as being specifically disclosed in all ranges formed from any pair of any upper limit range or values - preferred and or preferential values, regardless of whether the ranges are disclosed separately or not. If a range of numerical values is recited at present, unless otherwise stated, the range is intended to include the end points of the range, and all integers and fractions within the range.
The scope of the invention is not intended to be limited to the specific values recited when one. interval is defined.
GENETICALLY MODIFIED MICRO-ORGANISMS Microbial hosts for the production of butanol can be | selected from bacteria, cyanobacteria, filamentous fungi and | yeasts.
The microbial host used must be tolerant to the produced butanol product, so that the yield is not limited by the toxicity of the product to the host.
The selection of a microbial host for the production of butanol is described in detail below. | Microbes that are metabolically active at high levels of butanol titer are not well known in the art.
Although the | butanol-tolerant mutants have been isolated from solventogenic Clostridia, little information is available on the tolerance to —butanol from other potentially useful bacterial strains.
Most studies on comparing alcohol tolerance in bacteria suggest that butanol is more toxic than ethanol (de Cavalho et al.
Microsc.
Res.
Tech. 64: 215-22 (2004) and Kabelitz et al.
FEMS Microbiol.
Lett. 220: 223-227 (2003)). Tomas et al. (J.
Bacteriol. 186: 2006-2018 (2004)) described that the | 20 yield of 1-butanol during fermentation of Clostridium acetobutylicum | may be limited by the toxicity of butanol.
The main effect of 1-butanol on Clostridium acetobutylicum is the disruption of membrane functions (Hermann et al.
Appl.
Environ.
Microbiol. 50: 1238-1243 (1985)). The microbial hosts selected for the production of —butanol must be tolerant to butanol and must be able to convert carbohydrates to butanol using an introduced biosynthetic pathway, such as the path described below.
The criteria for selecting the appropriate microbial hosts include the following: Intrinsic tolerance to butanol, high rate of
| THE . | | use of carbohydrates, availability of genetic tools for 'gene manipulation, and ability to generate chromosomal changes. stable.
Suitable host strains with a tolerance to butanol - can be identified by screening based on the intrinsic tolerance of the strain.
The intrinsic tolerance of microbes to butanol can be measured by determining the concentration of butanol which is responsible for 50% inhibition of growth rate (ICso) when grown in minimal medium.
ICso values can be determined using methods known in the art.
For example, microbes of interest can be grown in the presence of varying amounts of butane! and the monitored growth rate | by measuring the optical density at 600 nanometers.
The doubling time can be calculated from the logarithmic part of the growth curve and used as a measure of the growth rate.
The concentration of butanol, which produces 50% growth inhibition, can be | determined from a percentage of growth inhibition graph | depending on the butanol concentration.
Preferably, the host strain | must have an IC55 value for butane! greater than about 0.5%. Most suitable is a host strain with an ICso for butanol that is greater than about 1.5%. Especially suitable is a host strain with an ICsº5 for butanol that is greater than about 2.5%. | Microbial hosts for butanol production too | should use glucose and / or other carbohydrates at a high rate.
À | most microbes are able to use carbohydrates.
However, certain | environmental microbes cannot use carbohydrates efficiently and, | therefore, they would not be suitable hosts. | The ability to genetically modify the host is essential for the production of any recombinant microorganism.
Modes
| 21 of gene transfer technology that can be used include 'electroporation, conjugation, transduction or natural transformation. A wide range of host conjugative plasmids and drug resistance markers is currently available. The cloning vectors used with an organism are adapted to the host organism depending on the nature of antibiotic resistance markers that can work on the same host. The host microorganism can also be manipulated in order to inactivate pathways that compete for the carbon flow through the inactivation of several genes. This requires the availability of transposons or chromosomal integration vectors for direct inactivation. In addition, production hosts that are susceptible to chemical mutagenesis may experience improvements in intrinsic tolerance to butanol through chemical mutagenesis and selection of mutants.
As an example of inactivating competing carbon flow pathways, pyruvate decarboxylase can be reduced or eliminated (see, for example, Published Patent Application US 20090305363). In realizations, butane! L is the main product of the microorganism. In embodiments, the microorganism does not produce acetone.
Based on the criteria described above, suitable microbial hosts for butanol production include, but are not limited to, members of the genera Zymomonas, Escherichia, Salmonella, Rhodococcus, | Pseudomonas, - Bacillus, - Lactobacillusó, - Enterococcus, - Pediococcus, | Alcaligenes, - Klebsiella, —Paenibacillus, —Arthrobacter, - Corynebacterium, | 25 - Brevibacterium, Píchia, Candida, Hansenula, Kluyveromyces, Issatchenkia and | Saccharomyces. Preferred hosts include: Escherichia coli, Alcaligenes | eutrophus, Bacillus licheniformis, Paenibacilus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus j "e | | 22 | | | | | | | faecium, Enterococcus gallinarium, Enterococcus faecalis, Pediococisi The microorganisms "mentioned above can be genetically modified to convert fermentable carbon sources to butanol, specifically 1-butanol, 2-butanol or isobutanol, using methods known in the art. Suitable microorganisms include Escherichia, Lactobacillus, and Saccharomyces. Suitable microorganisms include E. coli, L. plantarum and S. cerevisiae. In addition, the microorganism may be a butanol-tolerant strain of one of the microorganisms listed above, which is isolated using the method described by Bramucci et a /. (US Patent Application 11/761497 and WO 2007/146377). An example of such a strain is Lactobacillus plantaru m PNO512 strain (ATCC: PTA- 7727, biological deposit made on July 12, 2006 for patent application US11 / 761497). | The biosynthetic pathways suitable for the production of butanol are | known in the art, and some suitable routes are described herein. In examples of embodiments, the butanol biosynthetic pathway comprises at least one gene that is heterologous to the host cell.
In some examples of embodiments, the butanol biosynthetic pathway comprises | more than a gene that is heterologous to the host cell. In some | realization examples, the butanol biosynthetic pathway comprises genes | heterologues encoding polypeptides corresponding to each stage of a biosynthetic pathway.
Likewise, certain suitable proteins having the ability to catalyze the substrate indicated for product conversions are | described herein and other suitable proteins are provided in the prior art. For example, Published Patent Applications US 20080261230,
US 20090163376, and US 20100197519 describe acetohydroxy acid | ] isomeroreductases as well as application US 12/893077, filed on September 29, 2010; Published Patent Application US 20100081154 describes dihydroxy-acid dehydratases; alcohol dehydrogenases are described in Published Patent Application US20090269823 and Provisional Patent Application US 61/290636. | Microorganisms can be genetically modified to contain a 1-butanol biosynthetic pathway to produce 1-butanol.
Appropriate modifications include those described by Donaldson et al. in WO 2007/041269. For example, the microorganism can be genetically modified to express a 1-butanol biosynthetic pathway comprising the following enzyme-catalyzed substrates for product conversions: a) acetyl-CoA to acetoacetyl-CoA; b) acetoacetyl-CoA in 3-hydroxybutyryl-CoA; | c) 3-hydroxybutyryl-CoA in crotonyl-CoA; d) crotonyl-CoA in butyryl-CoA; | e) butiri-CoA in butyraldehyde; and | f) butyraldehyde in α-butanol. | 20 Microorganisms can also be genetically | modified to express a 2-butanol biosynthetic pathway to produce 2-butanol.
Appropriate modifications include those described by Donaldson et al. in US Patent Application Publications 2007/0259410 and US 2007/0292927, and in PCT Application WOZ2007 / 130518 and WO2007 / 130521. For example, in one embodiment, the microorganism can be genetically modified to express a 2-butane biosynthetic pathway! | comprising the following enzyme-catalyzed substrates for product conversions:
a) pyruvate in alpha-acetolactate; ] b) alpha-acetolactate in acetoin; 'C) acetoin in 2,3-butanediol; d) 2,3-butanediol in 2-butanone; and | 5 e) 2-butanone in 2-butanol.
Microorganisms can also be genetically modified to express an isobutanol biosynthetic pathway to produce isobutanol. Appropriate modifications include those described by Donaldson et al. in US Patent Application 2007/0092957 and WO 2007/050671. For example, the microorganism can be genetically modified to contain an isobutanol biosynthetic pathway comprising the following enzyme-catalyzed substrates for product conversions: | a) pyruvate in acetolactate; b) acetolactate in 2,3-dihydroxyisovalerate; | C) 2,3-dihydroxyisovalerate in α-ketoisovalerate; d) a-ketoisovalerate in isobutyraldehyde; and e) isobutyraldehyde in isobutane! l.
The Escherichia coli strain can comprise: (a) an isobutane biosynthetic pathway! encoded by the following genes: budbs (SEQ ID NO: 1) from Klebsiella pneumoniae encoding acetolactate synthase (given as SEQ ID NO: 2), ilvC (given as SEQ ID NO: 3) from E. coli encoding acetohydroxy acid reductoisomerase (given as SEQ ID NO: 4), ifvD (given as SEQ ID NO: 5) from E. coli encoding acetohydroxy acid dehydratase (given as SEQ ID NO: 6), kivD (given as SEQ ID NO: 7 ) of Lactococeus lactis encoding branched-chain keto acid decarboxylase (given as SEQ ID NO: 8), and sadB (given as SEQ ID NO: 9) from Achromobacter xylosoxidans which encodes butanol dehydrogenase (given as SEQ ID NO: 10) . Enzymes encoded by genes in the isobutanol biosynthetic pathway
catalyze the substrate for product conversions for converting pyruvate in isobutanol, as described above. Specifically, acetolactate 'synthase catalyzes the conversion of pyruvate to acetolactate, to acetohydroxy acid reductoisomerase * “catalyzes the conversion of acetolactate to 2,3- dihydroxyisovalerate, to acetohydroxy acid dehydratase catalyzes the conversion of 2,3-dihydroxyisovalerate to a-c , branched-chain keto acid decarboxylase catalyzes the conversion of a-ketoisovalerate to isobutyraldehyde and butanol dehydrogenase catalyzes the conversion of isobutyraldehyde to isobutanol. This recombinant strain of Escherichia coli can be constructed using methods known in the art (see Copendant Patent Application Publications US 12 / 478,389 and 12 / 477,946) and / as described below. It is contemplated that suitable strains can be constructed comprising a sequence that has about at least 70-75% identity, about at least 75-80% identity, and about at least 80-85% identity, or at least 85-90% identity to the protein sequences described herein.
The Escherichia coli strain may comprise deletions of the following genes to eliminate competing pathways that limit production of isobutanol, pflB given as SEQ ID NO: 71 (which encodes the pyruvate | 20 lyase format), / dhA, given as SEQ ID NO: 73, (encoding lactate dehydrogenase), | adhE, given as SEQ ID NO: 77 (encoding alcohol dehydrogenase), and at least one gene comprising the frdABCD operon (encoding fumarate reductase), specifically, frdA, given as SEQ ID NO: 90, frdB, given as SEQ ID NO: 75, frdC given as SEQ ID NO: 92, and frdD given as SEQ ID NO: 94 The Saccharomyces cerevisiae strain can comprise: an isobutanol biosynthetic pathway encoded by the following genes: Bacillus a / sS coding region subtilis (SEQ ID NO: 11) encoding acetolactate
| 26 Of the synthase (SEQ ID NO: 12), S. cerevisiae ILV5 (SEQ ID NO: 13) encoding 'acetohydroxy acid reductoisomerase (KARI; SEQ ID NO: 14) and / or a KARI. mutant such as that encoded by Pf5.//lvC-Z4B8 (SEQ ID NO: 15; protein SEQ ID NO: 16), Streptococcus mutans ilvD (SEQ ID NO: 17) encoding acetohydroxy acid dehydratase (SEQ ID NO: 18 ), Bacillus subtilis kivD (codon optimized given as SEQ ID NO: 19) encoding branched-chain keto acid decarboxylase (SEQ ID NO: 20), and sadB of Achromobacter xylosoxidans (SEQ ID NO: 9) encoding a butanol dehydrogenase (SEQ ID NO: 10). The enzymes encoded by the genes of the isobutane! 1 biosynthetic pathway catalyze the substrate for product conversions for converting pyruvate to isobutanol, as described herein. It is contemplated that suitable strains can be constructed comprising a sequence that has about at least 70-75% identity, about at least 75-80% identity, and about at least 80-85% identity, or at least 85-90% identity with the protein sequences described herein.
A yeast strain that expresses an isobutanol pathway with acetolactate synthase (ALS) activity in the cytosol and with deletions of the endogenous pyruvate decarboxylase (PDC) genes is described in US Patent Application 12/477942. This combination of cytosolic ALS and reduced expression of PDC has been shown to significantly increase the flow of pyruvate to acetolactate, which then flows into the isobutanol production pathway. Such a recombinant strain of Saccharomyces cerevisiae can be constructed using methods known in the art and / or described herein. Other suitable yeast strains are known in the art. Additional examples are provided in Provisional Orders US 61/379546, US 61/380563, and Order US 12/893089.
Additional modifications suitable for microorganisms
| . used in conjunction with the processes provided include modifications that reduce glycerol-3-phosphate dehydrogenase, as described in Publication | . of US Patent Application 20090305363, modifications to a host cell that provides carbon flow through an increase in the pathway — Entner-Doudoroff reduced the balance of equivalents as described in US Patent Application Publication 20100120105. Active yeast strains increased number of heterologous proteins that require the binding of an Fe-S center for their activity are described in the Request for Publication | US patent 20100081179. Other modifications include modifications to an endogenous polynucleotide that encodes a polypeptide having paper | double in hexokinase activity, described in Provisional Application US 61/290639, and the integration of at least one polynucleotide encoding a polypeptide | that catalyzes a step in a biosynthetic pathway that uses the described pyruvate | in US Provisional Order 61/380563. | In addition, host cells comprising at least one deletion, mutation and / or substitution of an endogenous gene encoding a polypeptide that affects the biosynthesis of Fe-S centers are described in Provisional US Patent Application 1/305333, and host cells comprising a heterologous polynucleotide encoding a polypeptide with phosphocetolase activity and host cells comprising a heterologous polynucleotide encoding a polypeptide with phosphotransacetylase activity are described in Provisional Patent Application US 61/356379. | CONSTRUCTION OF AN APPROPRIATE YEAST CRAFT | 25 NGI-049 is an example of a suitable strain of Saccharomyces cerevisiae. NGI-049 is a strain with endogenous insertion-inactivation of PDC1, PDC5 and PDC6 genes, containing the expression vectors pLH475-Z4B8 and pLH468. The PDC1, PDC5 and PDC6 genes encode the three
DEE ENA ALLA SNC main isoenzymes of pyruvate decarboxylase.
The strain expresses genes that | The. encode enzymes for an isobutanol biosynthetic pathway that are integrated: or are in plasmids.
The construction of the NGI-049 strain is provided at present.
Endogenous pyruvate decarboxylase activity in yeast | converts pyruvate to acetaldehyde, which is then converted to ethanol or acetyl-CoA via acetate.
Therefore, endogenous pyruvate decarboxylase activity is a target for reducing or eliminating by-product formation.
Examples of other yeast strains with reduced pyruvate decarboxylase activity due to disruption of genes encoding pyruvate decarboxylase have been described for Saccharomyces in Flikweert et al. (Yeast (1996) 12: 247-257), for Kluyveromyces in Bianchi et al (Mol.
Microbiol. (1996) 19 (1): 27-36), and the interruption of the regulatory gene in Hohmann, (Mol Gen Genet. (1993) 241: 657-666). Strains of Saccharomyces that do not have pyruvate decarboxylase activity are available from the ATCC (Accession No. f200027 and 200028). CONSTRUCTION OF THE PDC6 INTEGRATION CASSETTE :: GPMP1-SADB AND PDC6 DELETION | A pdc6 :: GPM1p-sadB-ADH1t-URA3r integration cassette was made by joining the GPM-sadB-ADHt (SEQ ID NO: 21) segment of PpRS425 :: GPM-sadB (SEQ ID NO: 63) with the URA3r gene puC19-URA3r.
O | PUC19-URA3r (SEQ ID NO: 22) contains the URS3Z marker from pRS426 (ATCC | t 77107) flanked by 75 bp of homologous repeat sequences to allow for homologous recombination in vivo and removal of the URA3 marker. The two DNA segments were joined by SOE-PCR (as described by Horton et al (1989) Gene 77: 61-68.) Using the plasmid DNAs of pRS425 :: GPM-sadB and pUC19-URA3r as a template, with the polymerase "Phusion DNA polymerase" "(New England Inc.
Biolabs, Beverly, MA; catalog F-
5408) and primers 114117-11A through 114117-11D (SEQ ID NOs: 23, 24, 25 and 26), and "> 114117-13A and 114117-13B (SEQ ID NOs: 27 and 28).
z External primers for SOE PCR (114117-13A and 114117-13B) contained 5 'and 3' homologous regions of - 50 bp upstream and downstream of the PDCG6 promoter and terminator, respectively. The fragment from the complete PCR cassette was transformed into BY4700 (ATCC * 200866) and the | Transformants were maintained in a complete synthetic medium without uracil and supplemented with 2% glucose at 30 ºC by standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202). The transformants were screened by PCR using primers 112590-34G and 112590-34H (SEQ ID NOs: 30 and 31), and 112590-34F and 112590-49E (SEQ ID NOs: 29 and 32) to verify integration at the PDC6 locus with the deletion of the PDC6 coding region. The URA3r marker was recycled by plating in a complete synthetic medium supplemented with 2% glucose and 5-FOA, at 30 ºC following the standard protocols.
The removal of the markers was confirmed by plating colonies from 5-FOA plates in SD-URA medium to verify the absence of growth. The resulting strain identified had the genotype: BY4700 pdc6: PePmr-sSadB-ADH1t. CONSTRUCTION OF THE INTEGRATION CASSETTE PDC1 :: PDC1-ILVD AND DELETION OF PDC1 An integration cassette pdc1 :: PDC1p-ilvD-FBA1t-URA3r was made by joining the segment iflvD-FBA1t (SEQ ID NO: 33) from pLH468 as URA3r gene from puUC19-URA3r by means of SOE PCR (as described by Horton et a / (1989) Gene 77: 61-68), using the plasmid DNAs of plH468 and pUC19-URA3r as a template, with the DNA polymerase “Phusion DNA polymerase ”(New England Biolabs Inc., Beverly, MA; .. catalog
F-5408) and primers 114117-27A through 114117-27D (SEQ ID NOs: 34, 35, 36 and 37). o External primers for SOE PCR (114117-27A and 114117- - 27D) contained 5 'and 3' homologous regions of - 50 bp downstream of the PDCT1 promoter and downstream of the PDC71 coding region. The fragment of the complete PCR cassette was transformed into BY4700 pdc6 :: Perm1-SsadB-ADH1t and the transformants were maintained in a complete synthetic medium without uracil and supplemented with 2% glucose at 30 ºC by standard genetic techniques
(Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold | | Spring Harbor, NY, pp. 201-202). The transformants were analyzed by | 10 PCR using primers 114117-36D and 135 (SEQ ID NOs: 38 and 39), and primers 112590-49E and 112590-30F (SEQ ID NOs: 32 and 40) to verify integration into the PDC1 locus with the deletion of the coding region of PDC1. The URA3r marker was recycled by plating in a complete synthetic medium | supplemented with 2% glucose and 5-FOA, at 30 ºC following the standard protocols.
The removal of the markers was confirmed by plating colonies from 5-FOA plates in SD-URA medium to verify the absence of growth.
The resulting strain identified “NYLA67” had the genotype:
BY 4700 pdc6 :: GPM1p-sadB-ADH1t pde1 :: PDC1p-ilvD-FBA1t.
HIS3 DELETION
| 20 To delete the endogenous HIS3 coding region, a his3 :: URA3r2 cassette was amplified by PCR from the URA3r2 template DNA (SEQ ID NO: 41). The URA3r2 contains the URS3 marker from pRS426 (ATCC t 77107) flanked by 500 bp of homologous repeat sequences to allow for homologous recombination in vivo and removal of the URAS marker.
The PCR was | carried out using the DNA polymerase "Phusion DNA polymerase" and the primers' 114117-45A and 114117-45B (SEQ ID NOs: 42 and 43) to generate a product from
| PCR of - 2.3 kb.
The HIS3 portion of each primer was derived from the 5 'to | upstream of the HIS3 promoter and region 3 'downstream of the coding region, so
:: | MN 31 forms that the integration of the URA3r2 marker results in the replacement of the> HIS3 coding region. The PCR product was transformed into NYLA67 using 'standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring | Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), and the transformants were selected in a complete synthetic medium. without uracil and | supplemented with 2% glucose at 30 ºC. The transformants were | selected to verify the correct integration by replicating transformants on plates with complete synthetic medium without histidine and supplemented with 2% glucose at 30 ºC. The URA3r marker was recycled by - plating in a complete synthetic medium supplemented with 2% glucose and | 5-FOA, at 30 ºC following the standard protocols. The removal of the markers was | confirmed by plating colonies from 5-FOA plates in medium | SD-URA to check for lack of growth. The resulting strain identified “NYLA73” had the genotype: BY4700 pdc6 :: GPM1p-sadB-ADH1t pdet: PDC1p-ilvD-FBA1t Ahis3.
| CONSTRUCTION OF THE PDC5 INTEGRATION CASSETTE :: KANMX AND PDC5 DELETION | The pdc5 :: kanMX4 cassette was amplified by PCR from the chromosomal DNA of the strain YLR1I34W (ATCC * 4034091), using the DNA polymerase "Phusion DNA polymerase" "and the primers PDC5 :: KanMXF and PDC5: KanMXR (SEQ ID NOs: 44 and 45), which generated a PCR product from -
2.2 kb. The PDCS5 portion of each primer was derived from the 5 'region upstream of the PDCS5 promoter and the 3' region downstream from the coding region such that integration of the kanMX4 marker resulted in the replacement of the PDC5 coding region. The PCR product was transformed into NYLA73 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), and the transformants were selected in YP medium supplemented with 1 % ethanol! and geneticin (200 ug / mL) at 30 ºC. The transformants were analyzed by PCR to ! :: Í. :
Í 32 | o | verify the correct integration in the PDC locus with the substitution of the PDCS5 coding region using primers PDC5kofor and N175 (SEQ ID NOs: 46, and 47). The correct transformants identified have the genotype: BY4700 pde6 :: GPM1p-sadB-ADH'1t pde1 :: PDC1p-ilvD-FBA1t Ahis3 pdeS5 :: kanMX4. CONSTRUCTION OF PLH475-Z4B8 Plasmid pLH475-Z4B8 (SEQ ID NO: 48) was constructed for the expression of ALS and KARI in yeast. PLH475-Z4B8 is a pHR81 vector (ATCC t 87541), containing the following chimeric genes: 1) the CUP1 promoter (SEQ ID NO: 49), coding region for Bacillus subtilis — acetolactate synthase (AlsS; SEQ ID NO: 11 protein SEQ ID NO: 12) and terminator CYC1 (CYC1-2; SEQ ID NO: 50); 2) promoter / LV5 (SEQ ID NO: 51), coding region for Pf5./lvC-Z4B8 (SEQ ID NO: 15; protein SEQ ID NO: 16) and terminator / LV5 (SEQ ID NO: 52); and 3) FBA1 promoter (SEQ ID NO: 53), from the KARI coding region of S. cerevisiae (ILV5; SEQ ID NO: 13; protein SEQ ID NO: 14) and terminator CYC1 (SEQ ID NO: 54). The coding region for Pf5.I / lvC-Z4B8 is a KARI coding sequence derived from Pseudomonas fluorescens, but containing the mutations, which were described in Patent Application US20090163376. Pf5.1IlvC-Z4B8 encoding KARI (SEQ ID NO: 16;) has the following amino acid changes compared to the natural KARI of Pseudomonas fluorescens: C33L: cysteine at position 33 changed to leucine, i R47Y: arginine at position 47 changed for tyrosine, S50A: serine at position 50 changed to alanine, T52D: threonine at position 52 changed to asparagine, V53A: valine at position 53 changed to alanine, |
| l 33 L61F: leucine at position 61 changed to phenylalanine, and T80I: threonine at position 80 changed to isoleucine,: A156V: alanine at position 156 changed to threonine, and G170A: glycine at position 170 changed to alanine.
The coding region of Pf5./lvC-Z4B8 was synthesized by DNA 2.0 | (Palo Alto, CA; SEQ ID NO: 15) based on the codons that have been optimized | for expression in Saccharomyces cerevisiae. | EXPRESSION VECTOR PLHA468 Plasmid pLH468 (SEQ ID NO: 55) was constructed for the expression of DHAD, KivD and HADH in yeast. The coding regions of B. subtilis ketoisovalerate decarboxylase (KivD) and horse liver alcohol dehydrogenase (HADH) were synthesized by DNA2.0 based on codons that were optimized for expression in Saccharomyces cerevisiaee (SEQ ID NO: 19 and 56, respectively) and provided in plasmids pKivDy-DNA2.0 and pHadhy-DNA2.0. The encoded proteins are SEQ ID NOs: 20 and 57, respectively. Individual expression vectors for KivD and HADH were constructed. To assemble the plLH467 (pRS426 :: Pepp1-kivDy-GPD14), the PNY8 vector (SEQ ID NO: 58; also called pRS426 :: GPD-ald-GPDt, described in Patent Application US20080182308, Example 17), was digested with Ascl and Sfil enzymes, thereby excising the GPD71 promoter (SEQ ID NO: 59) and the a / d coding region. A fragment of the GPD1 promoter (GPD1-2; SEQ ID NO: 60) from pnNY8 was amplified by PCR to add an Ascl site at the 5 'end, and a Spel site at the 3' end using the 5 'primer OT1068 and primer 3 'OT1067 (SEQ ID NOs: 61 and 62). The Asci / Sfil-digested PNY8 vector fragment was ligated with the Ascl and Spel-digested GPDT1 promoter PCR product, and the Spel-Sfil fragment containing the optimized codon of the kivD coding region isolated from the pKivD-DNA2.0 vector. THE
| 34 triple bond generated the vector pLH467 (pRS426 :: Pepp1-kivDy-GPD1t). The pLH467 GG was verified through restriction and sequencing mapping. e pLH435 (pRS425 :: PePpmi-Hadhy-ADH18) was derived from the vector pRS425 :: GPM-sadB (SEQ ID NO: 63), which is described in Patent Application US12 / 477942, Example 3. pRS425 :: GPM -sadB is the pRS425 vector (ATCC * 77106) with a chimeric gene containing the GPM1 promoter (SEQ ID NO: 64), the coding region for a butanol dehydrogenase from Achromobacter xylosoxidans (sadB; SEQ ID NO: 9; protein SEQ ID NO : 10: disclosed in US Patent Application Publication 20090269823), and the ADH1 terminator (SEQ ID NO: 65). PRS425 :: GPMp-sadB contains Bbvl and Pacl sites at the 5 'and 3' ends of the sadB coding region, respectively. An Nhel site was added at the 5 'end of the sadB coding region by oligonucleotide-mediated mutagenesis (site-directed) using primers OT1074 and OT1075 (SEQ ID NO: 66 and 67) to generate the vector pRS425-GPMp- —sadB- Nhel, which has been verified by sequencing. PRS425 :: Pepm1-SadB-Nhel was digested with Nhel and Pacl to remove the coding region of sadB, and ligated with the Nhel-Pacl fragment containing the coding region of the HADH codon optimized from the vector pHadhy-DNAZ2.0 for create the pLH435. To combine the KivD and HADH expression cassettes into a single vector, the yeast vector pRS411 (ATCC * 87474) is digested with Sacl and Notl, and ligated with the Sacl-Sal fragment from pLH467 containing the PerpD1-kivDy-GPD1t cassette along with the Sal / l-Notl fragment from plLH435 that contains the Peopm-Hadhy-ADH1t cassette in a triple bond reaction. This produced the vector pRS411 :: Pepp1-kivDy-Perm-Hadhy (pLH441), which can be verified through restriction mapping.
In order to generate a coexpression vector for all three genes in the isobutane pathway! bottom: ilvD, kivDy and Hadhy, we use the pRS423 FBA ilvD (Strep) (SEQ ID NO: 68), which is described in US Patent Application
12/569636 as the source of the / lvD gene. This carrier vector contains a VC origin of replication F1 (nt 1423-1879) for maintenance in E. coli and a '2 micron origin (nt 8082-9426) for yeast replication. The vector has an FBA promoter (nt 2111-3108; SEQ ID NO: 53) and FBA terminator (nt 4861-5860; SEQID NO: 69). In addition, it carries the His marker (nt 504-1163) for | selection in yeast and ampicillin resistance marker (nt 7092-7949) for selection in E. coli. The i / vD coding region (nt 3116-4828; SEQ ID NO: 17; SEQ ID NO: 18 protein) from Streptococcus mutans UA1I59 (ATCC H 700610) is between the FBA promoter and the FBA terminator forming a chimeric gene for the expression. In addition, there is a “tag lumio” marker merged in the ilvD coding region (nt 4829-4849). The first step was to linearize the pRS423 FBA ilvD (Strep) (also called pRS423-FBA (Spel) -llvD (Streptococcus mutans) -Lumio) with Sacl and Sacll (with the Sacll site ending at blunt ends using T4 DNA polymerase) , to form a vector with a total length of 9,482 bp. | The second step was to isolate the kivDy-hADHy cassette from pLH441 with Sacl and Kpnl (with the Kpnl site forming blunt ends using T4 DNA polymerase), | which formed a 6063 bp fragment. This fragment was linked with | 9482 bp fragment of the vector pRS423-FBA (Spel) -llvD (Streptococcus mutans) -Lumio. This vector generated pl H468 (pRS423 :: PrgarilvD (Strep) Lumio- FBA1t-Peopp1-kivDy-GPD1t-PGPM1-hadhy-ADH1t), - has been confirmed - by | restriction mapping and sequencing.
| The plasmidials plH468 and pLH475-24B8 vectors were simultaneously transformed into BY4700 pdc6 :: GPM1p-sadB-ADH1t pdet :: PDC1p-ilvD-FBA1t Ahis3 pdeS :: kanMX4 strains using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202), and the resulting strain was maintained in complete synthetic medium without histidine and uracil and supplemented with 1% ethanol at 30 ºC.
The resulting strain was called NG1-049. ORGANIC EXTRACTORS
. The extractor is a water-immiscible organic solvent or solvent mixture having characteristics that make it useful for the extraction of | 5 —butanola from a fermentation broth. A suitable organic extractor must meet the criteria of an ideal solvent for a commercial two-stage extractive fermentation for the production or recovery of butanol.
The extractor must specifically (i) be biocompatible with microorganisms, | for example, Escherichia coli, Lactobacillus plantarum, and Saccharomyces | 10 cerevisiae; (ii) be substantially immiscible with the fermentation medium; (iii) having a high partition coefficient (Kp) for the extraction of butanol; (iv) have a low partition coefficient for the extraction of nutrients; (v) have a low | tendency to form emulsions with the fermentation medium; and (vi) be low-cost and not dangerous.
In addition, for process operability | 15 improved and economy, the extractor: (vii) has low viscosity (pu), (viii) has low density (p) in relation to the aqueous fermentation medium; and (ix) have a boiling point suitable for the separation downstream of the extractor and butanol.
In an example of an embodiment, the extractor may be biocompatible with the microorganism, that is, non-toxic to the microorganism or toxic only to the extent that the microorganism is damaged to an acceptable level, so that the micro- organism continues to produce the butanol product in the fermentation medium.
The extent of an extractor's biocompatibility can be determined by the rate of glucose utilization of the microorganism in the presence of the extractor and the butanol product, measured under - defined fermentation conditions.
See, for example, the examples in Provisional Patent Applications US 61/168640; US 61/168642; and US 61 / 168,645. While a biocompatible extractor allows the micro-organism to use glucose, a non-biocompatible extractor does not allow the micro-organism to use glucose at a rate greater than, for example,> about 25% of the rate when the extractor is not gift.
As the presence of the butanol fermentation product can affect the sensitivity of the microorganism to the extractor, the fermentation product must be present during the extractor's biocompatibility tests.
The presence of additional fermentation products, for example, ethanol, can also affect the extractor's biocompatibility.
The use of a biocompatible extractor is | desired for processes in which continuous butanol production is desired after contact of the fermentation broth comprising the micro- | 10 organism with an organic extractor.
In an example of an embodiment, the extractor is selected from the group consisting of C fatty alcohols; to C272, C fatty acids; to C27, esters of C fatty acids; to C22, fatty aldehydes C; to C22, C fatty amides; to C22, and mixtures thereof.
Examples of suitable extractors include an extractor comprising at least one solvent selected from the group consisting of oleyl alcohol, berry alcohol, cetyl alcohol, alcohol! lauryl, myristyl alcohol, stearyl alcohol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, methyl oleate, lauric aldehyde, 1-nonanol, 1-decanol, 1-undecanol, 2-undecanol, 1-nonanal , 2-butyloctanol, 2-butyl- —octanoic acid and mixtures thereof.
In examples of embodiments, the extractor comprises oleyl alcohol.
In examples of embodiments, the extractor comprises a branched chain saturated alcohol, for example, 2-butyloctanol, commercially available as ISOFAL No. 12 (Sasol, Houston, | TX) or Jarcol 1-12 (Jarchem Industries, Inc., Newark, NJ ). In examples of embodiments, the extractor comprises a branched-chain carboxylic acid, for example, 2-butyl-octanoic acid, 2-hexyl-decanoic acid, or 2-decyl-tetradecanoic acid, commercially available as ISOCARBº 12, ISOCARBº 16, and ISOCARBº 24, respectively (Sasol, Houston, TX).
In one example, the first water-immiscible organic extractor is selected from the group consisting of alcohols. fatty acids C12 to C22, fatty acids C12 to C27, esters of fatty acids C12 to C22, fatty aldehydes C12 to C22, fatty amides C12 to C22 and mixtures thereof.
O - First suitable extractor can be selected from the group consisting of 'oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, also referred to as 1-dodecanol, myristyl alcohol, stearyl alcohol, oleic acid, | lauric acid, myristic acid, stearic acid, methyl myristate, oleate | methyl, lauric aldehyde, and mixtures of these.
In an example of realization, the! extractor comprises oleyl alcohol. | In one example, a second organic extractor | optional immiscible water can be selected from the group consisting of | in C fatty alcohols; to C22, C fatty carboxylic acids; to C27, esters of | C fatty carboxylic acids; to C22, fatty aldehydes C; to C22, CC fatty amides; BC »and mixtures thereof.
The second suitable extractor can be additionally selected from the group consisting of 1-nonanol, 1- | decanol, 1-undecanol, 2-undecanol, 1-nonanal, and mixtures thereof.
In | an example of an embodiment, the second extractor comprises 1-decanol.
In an example of an embodiment, the first extractor comprises oleyl alcohol and the second extractor comprises 1-decanol.
When a first and a second extractors are used, the relative amounts of each can vary within a suitable range.
For example, the first extractor can be used in a | amount that is about 30 percent to about 90 percent, or about | 25 —of 40 percent to about 80 percent, or about 45 percent to about 75 percent, or about 50 percent to about 70 percent of the volume | combined first and second extractors.
The ideal range reflects the | maximizing the characteristics of the extractor, for example, balancing a |
| 39. partition coefficient for relatively high butanol with an acceptable level of biocompatibility. For an extractive fermentation in two stages for the. butanol production or recovery, temperature, contact time, butanol concentration in the fermentation medium, relative quantities of fermentation medium and extractor, the first and second specific extractor being used, the relative quantities of the first and second extractors, presence of other organic solutes, the presence and concentration of electrolytes, and the quantity and type of microorganisms are related; thus, these variables can be adjusted as necessary within the appropriate limits to optimize the extraction process as described in the present invention. Suitable organic extractors may be commercially available from a variety of sources, such as Sigma-Aldrich (St. Louis, MO), to varying degrees, many of which may be suitable for use in extractive fermentation to produce or recover butanol. The technical qualities (grades) can contain a mixture of compounds, including the desired component and components with higher and lower molecular weights. For example, a commercially available technical grade oleyl alcohol contains about 65% oleyl alcohol and a mixture of upper and lower fatty alcohols.
ELECTROLYTE According to the present method, the fermentation medium contains at least one electrolyte in a concentration at least sufficient i to increase the partition coefficient of butanol in relation to that in | 25 - presence of the salt concentration of the basal medium for fermentation. The electrolyte may comprise one or more of the salts contained in the basal medium for | fermentation, in which case the electrolyte is present in a concentration above the concentration of total salts contained in the basal medium for fermentation.
The electrolyte may comprise one or more salts that are not present in the GC basal medium for fermentation. The basal fermentation medium can contain, for example, + phosphate, magnesium, and / or ammonium salts and is generally adapted for a specific microorganism. Suggested compositions of basal fermentation media can be found in the manuals of Difco "" & BBL'Y (Becton Dickinson & Company, Sparks, MD 21152, USA). In general, the salts provided by trace elements can be ignored when calculating the total salt concentration of the basal medium for fermentation due to their extremely low concentrations.
The electrolyte may comprise a salt that dissociates in the fermentation medium, or in the aqueous phase of a biphasic fermentation medium, to form free ions. For example, the electrolyte may comprise a salt having a cation selected from the group consisting of lithium, sodium, potassium, rubidium, cesium, magnesium, calcium, strontium, barium, ammonium, phosphonium, and combinations thereof. For example, the electrolyte may comprise a salt having an anion selected from the group consisting of sulfate, carbonate, acetate, citrate, lactate, phosphate, fluoride, chloride, bromide, iodide, and combinations thereof. The electrolyte can be selected from the group consisting of sodium sulfate, sodium chloride, and combinations thereof.
The electrolyte can be commercially available from a variety of sources, such as Sigma-Aldrich (St. Louis, MO), in varying degrees of purity, many of which may be suitable for use in extractive fermentation to produce or recover butanol by methods here | 25 described. The electrolyte can be recovered by methods known in the art from a fermentation medium or from an aqueous phase formed by contacting the fermentation medium with an extractor or by other physical or chemical methods such as precipitation, crystallization, and / or
| 41 | : evaporation.
The recovered electrolyte can be used in a subsequent i Go fermentation. . The amount of electrolyte required to achieve a concentration in the fermentation medium that is at least sufficient to increase the partition coefficient of butane! 1 with respect to that in the presence of the salt concentration of the basal fermentation medium can be determined as described, for example , by the procedures of the EXAMPLES described below.
The range of electrolyte concentrations that have a positive effect on the partition coefficient is determined, for example, by experimentation.
The range of electrolyte concentrations that demonstrates an acceptable biocompatibility with the microorganism of interest is also determined.
The appropriate range of electrolyte concentrations is then selected from the overlap of these two ranges, so that the amount of electrolyte needed to have a positive effect on the partition coefficient of —butanol is balanced with the range of concentrations that provide an acceptable level of biocompatibility with the microorganism.
Economic considerations can also be a factor in selecting the amount of electrolyte to be used.
In one example of an embodiment, the electrolyte may be present in the fermentation medium at a concentration that is biocompatible with the microorganism, that is, a concentration that is non-toxic to the micro-organism or toxic only to a certain extent that the micro -organism is impaired to an acceptable level, so that the microorganism continues to produce the butane product! in the fermentation medium in the presence of the electrolyte.
The extent of an electrolyte's biocompatibility can be determined by the rate of growth of the microorganism in the presence of various concentrations of the electrolyte, as described in Example 2 below.
While a concentration of biocompatible electrolyte allows the microorganism to use glucose (or
| other carbon source) or grow, a non-biocompatible electrolyte concentration does not allow the micro-organism to use glucose (or another carbon source) or to grow at a rate greater than, for example, about 25% of the growth rate when the excess amount of electrolyte is not present. The presence of fermentation products, for example, butanol, can also affect the electrolyte concentration levels that are biocompatible with the microorganism. The use of an electrolyte within | Concentration intervals with biocompatibility are desired for processes in which continuous butanol production is required after contact of the fermentation medium comprising the microorganism with the electrolyte. In processes in which the continuous production of butanol after contact of the fermentation medium comprising the microorganism with the electrolyte is not necessary, an electrolyte can be used in ranges (ranges) of concentration that have little, if any, biocompatibility with the microorganism.
To achieve an electrolyte concentration in the fermentation medium that is at least sufficient to increase the partition coefficient of butanol in relation to that in the presence of the salt concentration of | basal fermentation medium, the electrolyte can be added to the fermentation medium or to the aqueous phase of a biphasic fermentation medium during the growth phase of the microorganism, during the butanol production phase, when the butanol concentration is inhibitory, or in a combination of these phases. The electrolyte can be added to the first extractor, the second extractor, or a combination of these. The electrolyte can be added as a solid, as a slurry, or as an aqueous solution. Optionally, the electrolyte can be added to the fermentation medium and to the extractor (s). The electrolyte can be added in a continuous, semi-continuous, or batch (batch or batch) manner. The electrolyte
| 43 can be added to every flow to which it is introduced, for example, in the middle | that of whole fermentation in a fermenter, or at a partial flow withdrawn from. from one or more containers, for example, a partial flow from a fermenter. In examples of embodiments, the total electrolyte concentration in the fermentation medium is greater than about 0.05M; 0.1M; 0.2M; 0.3M; 0.4M; 0.5M; 0.6M; 0.7M; 0.8M or IM. In some embodiments, the electrolyte concentration in the fermentation is less than about 1M, and in some embodiments, the electrolyte concentration in the fermentation is less than 2M.
FERMENTATION The microorganism can be grown in a suitable fermentation medium in a suitable fermenter to produce butanol. Any suitable fermenter can be used, including a stirred tank fermenter, an airlift-type fermenter, a bubble-bubble fermenter, or any combination of these. Materials and methods for the maintenance and growth of microbial cultures are well known to those skilled in the field of microbiology or fermentation science (see, for example, Bailey et al. Biochemical Engineering Fundamentals, Second Edition, McGraw Hill, New York, 1986 ). As for the appropriate fermentation medium, great attention should be paid to pH, temperature, and requirements for aerobic, microaerobic or anaerobic conditions, depending on the specific needs of the micro-organism, a | fermentation, and the process. The fermentation medium used is not | 25 fundamental, but it must support the growth of the micro-organism used and promote the necessary biosynthetic pathway to produce the butanol product | wanted. A conventional fermentation medium can be used, including, but not limited to, complex media containing sources of organic nitrogen, such as yeast extract and peptone or at least one source of | VV fermentable carbon; minimum half and defined half. Fermentable carbon sources - suitable include, but are not limited to, monosaccharides, such as glucose or fructose; disaccharides, such as lactose or sucrose, oligosaccharides, polysaccharides, such as starch or cellulose, a carbon substrate and mixtures thereof. In addition to a suitable carbon source, the fermentation medium may contain a suitable nitrogen source, such as an ammonium salt, yeast and peptone extract, minerals, salts, cofactors, buffers and other components, known to those skilled in the art (Bailey et al. supra). Suitable conditions for extractive fermentation will depend on the micro-organism used and can be easily determined by a skilled technician in the field using routine experimentation. METHODS FOR BUTANOL RECOVERY USING FERMENTATION
EXTRACTIVE WITH ELECTROLYTE ADDITION Butanol can be recovered from a fermentation medium containing water, butanol, at least one electrolyte in one | at least sufficient concentration to increase the partition coefficient of butanol in relation to that in the presence of the salt concentration of the basal medium for fermentation, optionally in at least one carbon source | fermentable and a microorganism that has been genetically modified (ie | by genetic engineering) to produce butanol via a biosynthetic pathway a | from at least one carbon source. Such microorganisms | genetically modified can be selected from bacteria, | 25 - cyanobacteria, filamentous fungi and yeasts and include, for example, Escherichia coli, Lactobacillus plantarum, and Saccharomyces cerevisiae. À | first step in the process is to put the fermentation medium in contact with | a non-water-miscible organic extractor, and optionally, a second | | |
| 45 | | | water-miscible organic extractor to form a mixture of two phases comprising an aqueous phase and an organic phase containing butanol.
o "Contact" or "put in contact" means that the fermentation medium and the | organic extractor are placed in physical contact at any time during the fermentation process. The electrolyte can be added to the fermentation medium, to the first extractor, to the optional second extractor, or to a | combination thereof. In one embodiment, the fermentation medium further comprises ethanol, and the organic phase containing butanol! may contain ethanol.
When a first and a second extractor are used, contact can be made with the first and second extractor being previously combined. For example, the first and second extractors can be combined in a container, such as a mixing tank, then the combined extractors can be added to a tank containing the fermentation medium. Alternatively, contact can be made with the first and second extractors being combined during the contact. For example, the first and second extractors can be added separately to a container containing the fermentation medium. In one embodiment, the contact of the fermentation medium with the organic extractor further comprises the contact of the fermentation medium with the first extractor before the contact of the fermentation medium and first extractor with the second extractor. In one example, contact with the second extractor takes place in the same container as contact with the first | extractor ran. In one example, contact with the second extractor may occur in a container other than the one in which contact with the first extractor was carried out. For example, the first extractor can be contacted with the fermentation medium in a container, and the contents | transferred to another container in which contact with the second extractor:
| | 46 | occurs.
In these examples, the electrolyte can be added to the fermentation medium, to the first extractor, to the optional second extractor, or to | a combination of them. | The organic extractor can contact the fermentation medium in the | 5 start of fermentation, forming a biphasic fermentation medium. | Alternatively, the organic extractor can contact fermentation medium | after the microorganism has reached a desired amount of growth, | which can be determined by measuring the optical density of the culture.
In an example of realization, the first extractor can contact the means of | 10 fermentation in a container, and the second extractor can contact the medium of | fermentation and the first extractor in the same container.
In another example, the second extractor can contact the fermentation medium and the | first extractor in a different container from the one in which the contact of the first extractor with the fermentation medium occurred.
In these exemplary embodiments, the electrolyte can be added to the fermentation medium, the first extractor, the optional second extractor, or a combination of | themselves. | . In addition, the organic extractor can be placed in contact | with the fermentation medium at a time when the butanol level in the fermentation medium reaches a pre-selected level, for example, before the butanol concentration reaches a toxic or inhibitory level.
The concentration of butanol can be monitored during fermentation using methods known in the art, such as gas chromatography or high performance liquid chromatography.
The electrolyte can be added to the “medium fermentation before or after the concentration of butane! L reaches a toxic or inhibitory level.
In examples of embodiments, the organic extractor comprises fatty acids.
In examples of embodiments, the processes described in the present can be used in conjunction with the E EE DEE Es a a a A processes
| 47! described in Provisional Patent Applications US 61/368429 and US 61/379546 | DV in which the butanol is esterified with an organic acid, such as fatty acid,. using a catalyst, such as a lipase, to form butanol esters.
Fermentation can be carried out under aerobic conditions long enough for the culture to reach a pre-selected level of growth, as determined by measuring the optical density.
The electrolyte can be added to the fermentation broth before or after the pre-selected level of growth is reached.
An inductor can be added to induce the expression of the butane biosynthetic pathway! in the modified microorganism, and the fermentation conditions are changed to microaerobic or anaerobic conditions to stimulate the production of butanol, as described in detail in Example 6 of US Patent Application 12 / 478,389. The extractor can be added after the transition from microaerobiosis or anaerobiosis conditions.
The electrolyte can be added before or after changing the conditions of microaerobiosis or anaerobiosis.
In an example of an embodiment, the first extractor may contact the fermentation medium prior to contact of the fermentation medium and first extractor with the second extractor.
For example, in a batch fermentation process, an adequate period of time may be allowed between the contact time of the fermentation method with the first and second extractors.
In a process of | Continuous fermentation, contact of the fermentation medium with the first extractor can occur in one container, and contact of the contents of the container with the second extractor can occur in a second container.
In these embodiment examples, the electrolyte can be added to the medium | fermentation, to the first extractor, to the optional second extractor, or to a | combination thereof.
After contact of the fermentation medium with the organic extractor in the presence of the electrolyte, the butanol product is partitioned in the organic extractor,
. | decreasing its concentration in the aqueous phase containing the microorganism, | and thus limiting the exposure of the producing microorganism to the product: inhibitory butanol.
The volume of the organic extractor to be used depends on a number of factors, including the volume of the fermentation medium, the size | 5 of the fermenter, the partition coefficient of the extractor for the butanol product, the electrolyte concentration and the chosen fermentation mode, as described below.
The volume of the organic extractor can be about 3% to | about 60% of the working volume of the fermenter.
The ratio between extractor and fermentation medium is from about 1:20 to about 20: 1 on a volume: volume basis, for example, about 1:15 to about 15: 1, or about 1:12 to about 12: 1, or from about 1:10 to about 10: 1, or about 1: 9 to about 9: 1, or from about 1: 8 to about 8: 1 . | The amount of electrolyte to be added depends on several factors, including the effect of the added electrolyte on the growth properties of the butanol-producing microorganism, the effect of the added electrolyte on the Kp of butanol in a two-phase fermentation.
À | Optimal amount of electrolyte to be added may also be dependent on the composition of the basal medium for initial fermentation.
A very high concentration of electrolyte, although possibly increasing the Kp of butanol and | 20 relieve the toxicity effects of butanol to the microorganism, it could be | inhibitor of the microorganism itself.
On the other hand, a very low concentration of electrolyte may not increase the Kp of butanol sufficiently to alleviate the inhibitory effect of butanol on the microorganism.
Therefore, a balance must be found through experimentation to ensure that —the final result of adding excess electrolyte to the fermentation medium results in an overall increase in the rate and titration of butanol production. | In addition, the biocompatibility of salts to the microorganism can be modulated | by the addition of osmoprotectors or osmolites, either exogenously to the environment
| "49 or by genetic modification of the microorganism to produce the osmolyte (s) | | endogenously.
In examples of realizations, the Kp is increased by about. 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, or about 200% compared to Kp without the addition of the electrolyte.
In exemplary embodiments, Kp is increased by at least approximately 2 times, at least approximately 3 times, at least approximately 4 times, at least approximately 5 times, or at least approximately 6 times.
In examples of embodiments, the total electrolyte concentration is selected to increase the Kp by an amount while maintaining the growth rate of the microorganism at a level that is at least about 25%, at least about 50%, at least about 80%, or at least about 90% of the growth rate in the absence of the addition of the electrolyte.
In embodiments, the total concentration of —electrolyte in the fermentation medium is sufficient to increase the effective rate of butanol production by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100% compared to the rate without the addition of the electrolyte.
In embodiments, the total electrolyte concentration in the fermentation medium is sufficient to increase the effective yield of butanol by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, - at least about 80%, at least about 90%, or at least about 100% compared to the yield without the addition of the electrolyte.
In embodiments, the total electrolyte concentration in the fermentation medium is sufficient to increase the effective titration of butanol by at least about i DB 50 | 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about. 70%, at least about 80%, at least about 90%, or at least about 100% compared to the titration without the addition of the electrolyte.
In examples of embodiments, the amount of electrolyte added is sufficient to result in an effective titre of at least about 7 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 25 g / L, at least about 30 g / L, or at least about 40 g / L. In exemplary embodiments, the amount of electrolyte added is sufficient to result in an effective yield of at least about 0.12, at least about 0.15, at least about 0.2, at least about 0.25 , or at least about 0.3. In exemplary embodiments, the amount of electrolyte added is sufficient to result in an effective rate of at least about 0.1 g / L / h, at least about 0.15 g / L / h, at least about 0 , 2 g / L / h, at least about 0.3 g / L / h, at least about 0.4 g / L / h or at least about 0.6 g / L / h, or at least at least about 0.8 g / L / h, or at least about 1 g / L / h, or at least about 1.2 g / L / h. In some examples of realization, the effective rate is about 1.3 | glL / h.
The next step is optionally separating the organic phase containing butanol from the aqueous phase using methods known in the art, including, but not limited to, siphoning, decanting, centrifuging, using a gravity decanter, membrane-assisted phase separation .
| 25 The recovery of butanol from the organic phase containing | butanol can be made using methods known in the art, including, but not limited to, distillation, resorption by resins, separation by molecular sieves and pervaporation. Specifically, distillation can |
| 51 | . | be used to recover the butanol from the organic phase containing butanol. | o The extractor can be recycled for the production and / or recovery process of butanol.
The electrolyte can be recovered from the fermentation medium - or from the aqueous phase of a two-phase mixture by methods known in the art.
For example, the aqueous phase or fermentation medium can be concentrated by distillation, desorption, pervaporation, or other methods to obtain a concentrated aqueous mixture comprising the electrolyte.
Optionally, the electrolyte can be returned to a fermentation medium and thus be recycled in the fermentation process.
Optionally, the electrolyte obtained from a fermentation medium can be added to a fermentation medium to provide a concentration at least sufficient to increase the butane partition coefficient! in relation to that in the presence of the salt concentration of the basal medium for fermentation.
Gas desorption (gas sftripping) 8 can be used | simultaneously with the organic extractor and the addition of the electrolyte to remove | the butanol product from the fermentation medium.
Gas desorption can be done by passing a gas such as air, nitrogen or carbon dioxide through the fermentation medium, thus forming a gas phase containing butanol.
The butanol product can be recovered from the gas phase containing butanol using methods known in the art, such as using a cooled water collector to condense the butanol, or washing the gas phase with a solvent.
Any butanol remaining in the fermentation medium after the completion of fermentation can be recovered by continuous extraction using a new or recycled organic extractor.
Alternatively, the | butanol can be recovered from the fermentation medium using methods
| 52 | | known in the state of the art, such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas desorption, membrane evaporation, pervaporation, and the like.
In the event that the fermentation medium is not recycled to the process, an additional electrolyte can be added to further increase the partition coefficient of butanol and improve the recovery efficiency of butanol.
The two-stage extractive fermentation method can be carried out in a continuous mode in an agitated tank fermenter.
In this mode, the mixture of the fermentation medium and the organic extractor containing butanol are removed from the fermenter.
The two phases are separated by means known in the art, including, but not limited to, siphoning, decanting, centrifuging, using a gravity decanter, membrane-assisted phase separation, and the like as described above.
After separation, the fermentation medium and the electrolyte in it can be recycled to the fermenter or can be replaced with fresh medium at the | which the additional electrolyte is added.
Then, the extractor is treated for | recover the butanol product as described above.
The extractor can be recycled back to the fermenter for additional product extraction. | Alternatively, a new extractor can be continuously added to the fermenter to replace the removed extractor.
This continuous mode of operation offers several advantages.
As the product is continuously removed from the reactor, a smaller volume of organic extractor is required allowing a larger volume of the fermentation medium to be used.
This results in | higher production yields.
The volume of the organic extractor can —be about 3% to about 50% of the working volume of the fermenter, 3% to about 20% of the working volume of the fermenter, or 3% to about 10% volume of the fermentor fermenter work.
It is beneficial to use the smallest possible amount of extractor in the fermenter to maximize the volume of | aqueous phase and thus the quantity of cells in the fermenter. The process can be operated in an entirely continuous mode, in which the. the extractor is continuously recycled between the fermenter and a separator and the fermentation medium is continuously removed from the fermenter and fed with fresh medium. In this entirely continuous mode, the butanol product is not allowed to reach the critical toxic concentration and fresh nutrients are continuously supplied so that fermentation can be carried out for long periods of time. The apparatus that can be used to carry out these two-stage extractive fermentation modes is well known in the art. Examples are described, for example, by Kollerup et a /. in US Patent 4,865,973.
The batch fermentation mode (batch or 'batch') can also be used. Batch fermentation, which is well known in the art, is a closed system, in which the composition of the fermentation medium is defined at the beginning of the fermentation and is not subject to artificial changes during the process. In this mode, the desired amount of complementary electrolyte and a volume of organic extractor are added to the fermenter and the extractor is not removed during the process. The organic extractor can be formed in the fermenter by separately adding the optional first and second extractor, or the first and second extractors can be combined to form the extractor before adding any extractor to the fermenter. The electrolyte can be added to the fermentation medium, the first extractor, the optional second extractor, or a combination thereof. Although this fermentation mode is simpler than the continuous or entirely continuous mode described above, it requires a larger volume of organic extractor to minimize the concentration of the inhibitory butanol product in the fermentation medium. Consequently, the volume of the fermentation medium is smaller and the amount
| 54 of product produced is less than that obtained using the continuous mode. "à The volume of organic extractor in batch mode can be from 20% to about. 60% of the working volume of the fermenter, or from 30% to about 60% of the working volume of the fermenter. It is beneficial to use the smallest volume possible to extract the fermentor, for the reasons described above.
The fed batch method (batch-fed or - "Fed-Batch") can also be used. Fed batch batch is a variation of the standard batch system, in which nutrients, for example , glucose, are increased during fermentation. The amount and rate of nutrient addition can be determined by routine experimentation. For example, the concentration of critical nutrients in the fermentation medium can be monitored during fermentation. easily measurable, such as pH, dissolved oxygen, and partial pressure of residual gases, such as carbon dioxide, can be monitored. From these analyzed parameters, the rate of addition of nutrients can be determined. addition methods in this mode are the same as those used in batch mode, described above. The amount of electrolyte added can be the same as used in other fermentation modes.
The product can be extracted downstream of the fermenter, instead of "in situ". In this external mode, the extraction of the butanol product to the organic extractor is carried out in the fermentation medium removed from the fermenter. The electrolyte can be added to the fermentation medium removed from the fermenter. The amount of extractor used is about 20% to about 60% of the working volume of the fermenter, or 30% to about 60% of the working volume of the fermenter. The fermentation medium can be removed from the fermenter continuously or periodically, and the extraction of the product
| butanol by the organic extractor can be made with or without removing the cells from the fermentation medium. The cells can be removed from the medium. fermentation by means known in the art, including, but not limited to | limiting filtration or centrifugation. The electrolyte can be added to the medium | 5 fermentation, before or after removal of the cells. After the separation of | extractor fermentation medium by the methods described above, the fermentation medium can be recycled in the fermenter, discarded or treated to remove any remaining butanol product. Likewise, isolated cells can also be recycled in the fermenter. After treatment to recover the butanol product, the extractor can be recycled for use in | extraction process. Alternatively, a new extractor can be used. | In this mode, the extractor is not present in the fermenter, so the toxicity of the extractor is much less problematic. If the cells are separated from the fermentation medium before contact with the extractor, the toxicity problem of the extractor can be further reduced. In addition, using this external mode there is less chance of forming an emulsion, and evaporator evaporation is minimized, alleviating environmental concerns. METHODS FOR THE PRODUCTION OF BUTANOL USING EXTRACTIVE FERMENTATION
WITH ADDED ELECTROLYTE An improved method for the production of butane is provided, in which a microorganism that has been genetically modified to produce butanol via a biosynthetic pathway from at least one fermentable carbon source is grown in a two-phase fermentation medium comprising | an aqueous phase and |) a first water-immiscible organic extractor and, optionally, ii) a second water-immiscible organic extractor, and the medium | of biphasic fermentation additionally comprises at least one electrolyte, in a concentration at least sufficient to increase the coefficient of
| | 56 butane partition! in relation to that in the presence of the salt concentration of the basal medium for fermentation. Such genetically modified microorganisms can be selected from bacteria, cyanobacteria, filamentous fungi and yeasts and include, for example, Escherichia coli, —Lactobacillus plantarum, and Saccharomyces cerevisiae. The first water-immiscible organic extractor can be selected from the group consisting of fatty alcohols C127 to C272, fatty acids C12 to Cz7, fatty acid esters C12 to C22, fatty aldehydes C12 to C22, fatty amides C, 7 to C22 and mixtures thereof, and the optional second water-immiscible organic extractor can be selected from the group consisting of C alcohols; Co, carboxylic acids C; to Cao, esters of C carboxylic acids; at C> 2, C aldehydes; to C22, C fatty amides; to C3 ', and mixtures thereof, in which the biphasic fermentation medium comprises from about 10% to about 90% by volume of the organic extractor. Alternatively, the biphasic fermentation medium can comprise from about 3% to about 60% by volume of organic extractor, or from about 15% to about 50%. The micro-organism can be grown in the biphasic fermentation medium for a period sufficient to | extract butanol in the extractor and form an organic phase containing butanol. À | at least sufficient concentration of the electrolyte in the fermentation medium can be achieved by adding electrolyte in the aqueous phase during the | growth of the micro-organism, in the aqueous phase during the production phase | of butanol, in the aqueous phase when the concentration of butanol in the aqueous phase is inhibitory, in the first extractor, in the second extractor, or in a combination thereof.
In one embodiment, the fermentation medium additionally comprises ethanol, and the organic phase containing butanol can contain ethanol. The organic phase containing butanol is then separated from the aqueous phase, as described above. Subsequently, butanol is
| 57 recovered from the organic phase containing butanol, as described. The previously.
| An improved method for the production of butanol is also provided in which a microorganism that has been genetically modified for | 5 producing butanol via a biosynthetic pathway from at least one carbon source is grown in a fermentation medium so that the microorganism produces butanol in the fermentation medium to produce a | fermentation medium containing butanol. Such genetically modified microorganisms can be selected from bacteria, cyanobacteria, filamentous fungi and yeasts and include, for example, Escherichia coli, Lactobacillus plantarum, and Saccharomyces cerevisiae. At least one electrolyte is added to the fermentation medium to provide the electrolyte in a 'concentration at least sufficient to increase the partition coefficient of butanol relative to that in the presence of the salt concentration of the basal medium' for fermentation. In some embodiments, the electrolyte can be added to the fermentation medium when the growth phase of the microorganism slows down. In some embodiments, the electrolyte can be added to the fermentation medium when the butanol production phase is finished. At least a portion of the fermentation medium containing | —Butanol is placed in contact with a first organic extractor immiscible in! water selected from the group consisting of C12 to C22 fatty alcohols, | fatty acids C12 to C> 22, esters of fatty acids C12 to C22, fatty aldehydes | | C12 to C22, fatty amides Ci2 to C ', and mixtures thereof, and optionally, | ii) a second water-immiscible organic extractor selected from | Group consisting of C alcohols; to C22, C carboxylic acids; to C> 7, esters | carboxylic acids C; to C22, C aldehydes; to C22, C fatty amides; to Cx and mixtures thereof, to form a two-phase mixture comprising an aqueous phase and an organic phase containing butanol. The organic phase | | | -. . —— s and sn cc
| | 58 containing butanol is then separated from the aqueous phase, as described | CV previously.
Subsequently, the butanol is recovered from the organic phase containing butanol, as previously described.
At least part of the aqueous phase returns to the fermentation medium.
In one embodiment, the fermentation medium additionally comprises ethanol, and the organic phase containing butanol can contain ethanol.
Isobutanol can be produced by extractive fermentation using a modified strain of Escherichia coli in combination with an oleyl alcohol as an organic extractor, as disclosed in US Patent Application 12/478389. The method produces a greater effective titer for isobutanol (i.e., 37 g / L)) compared to using conventional fermentation techniques (see Example 6 of US Patent Application 12/478389). For example, Atsumi et al. (Nature 451 (3): 86-90, 2008) described isobutanol titers of up to 22 g / L using fermentation with an Escherichia coli that was genetically modified to contain an isobutanol biosynthetic pathway.
The highest butanol titre obtained with the | extractive fermentation disclosed in US Patent Application 12/478389 resulted, | in part, the removal of the toxic butanol product from the fermentation medium, thus maintaining a level below the toxicity for the microorganism.
It is reasonable to assume that the present method of extractive fermentation employing the use of at least one electrolyte in a concentration in the fermentation medium that is at least sufficient to increase the partition coefficient of butanol in relation to that in the presence of the salt concentration of the basal medium. for fermentation as defined in | present, it would be used in a similar way and would provide similar results.
The butanol produced by the methods disclosed herein can have an effective titre greater than 22 g per liter of the fermentation medium. |
| 59 Alternatively, the butanol produced by the disclosed methods may have a | the effective titre of at least 25 g per liter of fermentation medium. s Alternatively, the butanol produced by the methods described in the present invention can have an effective titre of at least 30 g per liter of fermentation medium.
Alternatively, the butanol produced by the methods described in the present invention can have an effective titre of at least 37 g per liter of fermentation medium.
The present methods are described below in general with reference to Figure 1 to Figure 7. | 10 Referring now to Fig. 1, a schematic representation of an example of carrying out processes for the production and recovery of butanol using extractive fermentation in situ is shown.
An aqueous stream (10) of at least one source of fermentable carbon, optionally containing the electrolyte, is introduced into a fermenter (20), which contains at least one genetically modified (not shown) microorganism that produces butanol from a medium fermentation process comprising at least one source of fermentable carbon.
Optionally, the electrolyte can be added as a separate flow (not shown) to the fermenter.
A flow from the first extractor (12) and a flow from an optional second extractor (14) are introduced to a container (16), in which the first and second extractors are combined to form the combined extractor (18). Optionally, the electrolyte can be added (not shown) to the flow (18), the container (16), the flow of the first extractor (12), the flow of the second extractor (14), or a combination thereof.
A flow from the extractor (18) is introduced into the container (20), which contacts the fermentation medium with the extractor to form a mixture of two phases comprising an aqueous phase and an organic phase containing butanol.
A stream (26) comprising both the aqueous and organic phases is introduced into a container (38), in which the
| 60 separation of the aqueous and organic phases is carried out to produce an organic phase containing butanol (40) and an aqueous phase (42). Optionally, at least a portion of the aqueous phase (42) containing electrolyte is returned (not | shown) to the fermenter (20) or to another fermenter (not shown). - The point (s) of addition of the electrolyte in the process is (are) selected (s) in such a way that the concentration of electrolyte in the aqueous phase (42) is at least | enough to increase the partition coefficient of butanol in relation to | to that in the presence of the salt concentration of the basal medium for fermentation.
Referring now to Fig. 2, a schematic representation of an example of carrying out processes for the production and recovery of butanol using extractive fermentation in situ is shown.
An aqueous stream (10) of at least one source of fermentable carbon, optionally containing the electrolyte, is introduced into a fermenter (20), which contains at least one genetically modified (not shown) microorganism that produces butanol from a fermentation medium comprising at least one source of fermentable carbon.
Optionally, the electrolyte can be added as a separate flow (not shown) to the fermenter.
A flow from the first extractor (12) and a flow from the optional second extractor (14) are introduced separately into the fermenter (20), so that the contact between my fermentation with the extractor to form a mixture of two phases comprising an aqueous phase and an organic phase containing butanol occurs.
Optionally, the electrolyte can be added (not shown) to the flow (12), the flow (14), or a combination thereof.
A flow (26) comprising both the aqueous and organic phases is introduced into a container (38) in which the separation of the aqueous and organic phases is carried out to produce an organic phase containing butanol (40) and an aqueous phase (42). Optionally, at least a portion of the aqueous phase (42) containing electrolyte is returned (not shown) to the fermenter (20) or to another fermenter (not shown). The point (s) of addition of the electrolyte in the process is (are) selected (s) in such a way that the concentration of electrolyte in the phase. aqueous (42) is at least sufficient to increase the partition coefficient of butanol in relation to that in the presence of the salt concentration of the basal parafermentation medium.
Referring now to Fig. 3, a schematic representation of an example of carrying out processes for the production and recovery of butanol using extractive fermentation in situ is shown. An aqueous stream (10) of at least one source of fermentable carbon, optionally containing the electrolyte, is introduced into a first fermenter (20), which contains at least one genetically modified (not shown) microorganism that produces butanol from a fermentation medium comprising at least one source of fermentable carbon. Optionally, the electrolyte can be added as a separate flow (not shown) to the fermenter. A flow from the first extractor (12) is introduced to the fermenter (20), and a flow (22) comprising a mixture of the first extractor and the contents of the fermenter (20) is introduced into a second fermenter (24). A flow from the optional second extractor (14) is introduced into the second fermenter (24), so that the contact of the | 20 fermentation with the extractor to form a two-phase mixture | comprising an aqueous phase and an organic phase containing butanol. Optionally, the electrolyte can be added (not shown) to the flow (12), the flow (22), the flow (14), the container (24) or a combination thereof. A stream (26) comprising both the aqueous and organic phases is introduced into a container (38), in which the separation of the aqueous and organic phases is carried out to produce an organic phase containing butanol (40) and an aqueous phase (42) . Optionally, at least a portion of the aqueous phase (42) containing electrolyte is returned (not shown) to the fermenter (20) or to another fermenter (not shown). The point (s) of addition of the electrolyte in the Vs process is (are) selected (s) in such a way that the concentration of electrolyte in the. aqueous phase (42) is at least sufficient to increase the partition coefficient of butanol in relation to that in the presence of the salt concentration of the —basal medium for fermentation.
Referring now to Fig. 4, a schematic representation of an example of carrying out processes for the production and: butanol recovery is shown, in which the product extraction is carried out downstream of the fermenter, instead of “in situ”. An aqueous stream (110) of at least one source of fermentable carbon, optionally containing the electrolyte, is introduced into a fermenter (120), which contains at least one genetically modified (not shown) microorganism that produces butanol from a fermentation medium comprising at least one source of fermentable carbon. Optionally, the electrolyte can be added as a separate flow (not shown) to the fermenter. A flow from the first extractor (112) and a flow from an optional second extractor (114) are introduced to a container (116), in which the first and second extractors are combined to form the combined extractor (118). At least a portion, shown as a flow (122), of the fermentation medium in the fermenter (120) is introduced into the container (124). Optionally, the electrolyte can be added (not shown) to the flow (112), the flow (114), the container (116), the flow (118), the container (124) or a combination thereof. A flow from the extractor (118) is also introduced into the container (124), so that the fermentation medium comes into contact with the extractor to form a mixture of two phases - comprising an aqueous phase and an organic phase containing butanol. A stream (126) comprising both the aqueous and organic phases is introduced into a container (138), in which the separation of the aqueous and organic phases is carried out to produce an organic phase containing butanol (140) and an aqueous phase (142) . At least a portion of the aqueous phase (142) containing! electrolyte is returned to the fermenter (120), or optionally to another * fermenter (not shown). The point (s) of addition of the electrolyte in the process is (are) selected (s) in such a way that the concentration of electrolyte in the aqueous phase (142) is at least sufficient to increase the partition coefficient of the butanol in relation to to that in the presence of the salt concentration of the basal medium for fermentation.
Referring now to Fig. 5, a schematic representation of an example of carrying out processes for the production and recovery of butanol is shown in which the product is extracted downstream of the fermenter, instead of “in situ”. An aqueous stream (110) of at least one | fermentable carbon source, optionally containing the electrolyte, is introduced into a fermenter (120), which contains at least one genetically modified microorganism (not shown) which produces butanol from a fermentation medium comprising at least one fermentable carbon source.
Optionally, the electrolyte can be added as a separate flow (not shown) to the fermenter.
A flow from the first extractor (112) and a flow from the second extractor (114) are introduced separately into a container (124), in which the first and second extractors are combined to form the combined extractor.
Optionally, the electrolyte can be added (not shown) to the flow (112), the flow (114), the flow (122), the container (124) or combinations thereof.
At least a portion, displayed as a flow (122) of the fermentation medium in the fermenter (120) is also introduced into the container (124), so that the contact between the fermentation medium and the extractor occurs to form a two-phase mixture comprising an aqueous phase and an organic phase containing butanol.
A stream (126) comprising both the aqueous and organic phases is introduced into a container (138), in which the separation of the aqueous and organic phases is carried out to produce an organic phase containing butane! (140) and an aqueous phase ( 142). | VD At least a portion of the aqueous phase (142) containing electrolyte is returned to the fermenter (120), or optionally to another fermenter (not shown). The point (s) of addition of the electrolyte in the process is (are) '5 - selected (s) in such a way that the concentration of electrolyte in the aqueous phase | (142) is at least sufficient to increase the partition coefficient of butanol in relation to that in the presence of the salt concentration of the basal medium for fermentation.
Referring now to Fig. 6, a schematic representation of an example of carrying out processes for the production and recovery of butanol is shown, in which the product extraction is carried out downstream of the fermenter, instead of “in situ”. An aqueous stream (110) of at least one | fermentable carbon source, optionally containing the electrolyte, is introduced into a fermenter (120), which contains at least one genetically modified micro-organism (not shown) that produces butanol from a fermentation medium comprising at least one source of fermentable carbon.
Optionally, the electrolyte can be added as a separate flow (not shown) to the fermenter.
A flow from the first extractor (112) is introduced to the container (128), and at least a portion, displayed as flow (122) of the fermentation medium in the fermenter (120) is also introduced into the container (128). Optionally, the electrolyte can be added (not shown) to the flow (122), the flow (112), the container (128), or a combination thereof.
A flow (130) comprising mixing the first extractor and the contents of the fermenter (120) is introduced into a second container (132). Optionally, the electrolyte can be added (not shown) to the flow (130), the flow (114), the container (132), or a combination thereof.
A flow from the optional second extractor (114) is introduced into the second container (132), so that the middle contact runs.
65 of fermentation with the extractor to form a mixture of two CG phases comprising an aqueous phase and an organic phase containing butanol. One . flow (134) comprising both the aqueous and organic phases is introduced into a container (138), in which the separation of the aqueous and organic phases is carried out to produce an organic phase containing butanol (140) and an aqueous phase (142). At least a portion of the aqueous phase (142) containing electrolyte is returned to the fermenter (120), or optionally to another fermenter (not shown). The point (s) of addition of the electrolyte in the process is (are) selected in such a way that the concentration of electrolyte in the aqueous phase (142) is at least sufficient to increase the partition coefficient of the butane! L in relation to that in the presence of the salt concentration of the basal medium for fermentation.
The extraction processes described in the present invention can be performed in a batch mode or can be performed in a continuous mode where fresh extractor is added and the extractor used is pumped out so that the amount of extractor in the fermenter remains constant throughout the fermentation process. This continuous extraction of fermentation products and by-products can increase the rate, title and effective yield.
In another example of additional realization, it is also possible to operate the liquid-liquid extraction in a co-current way or, alternatively, in a counter-current way that contributes to the difference in the batch operation profile when a series of batch-type fermenters are used. In this scenario, the fermenters are filled with fermentable wort that provides at least one source of fermentable carbon and microorganism in a continuous form after another, while the cultivation is operating. Referring to Figure 7, since the Fermenter (F100) is filled with wort and micro-organisms, the wort and micro-
i 66 organisms advance to the Fermenter (F101) and then to the Fermenter VU (F102) and back to the Fermenter (F100) in a continuous loop. . The electrolyte can be added (not shown) to one or more fermenters, to the fermenter inlet flow, to the fermenter outflow, or to a combination of these.
Fermentation in any of the fermenters begins when the wort and microorganisms are present together and continues until fermentation is complete.
The time for filling must and microorganisms is equal to the number of fermenters divided by the total cycle time (filling, yeast, emptying and cleaning). If the total cycle time is 60 hours and there are 3 fermenters then the filling time is 20 hours.
If the total cycle time is 60 hours and there are 4 fermenters then the filling time is 15 hours.
Adaptive co-current extraction follows the fermentation profile assuming that the fermenter operating at a higher titer in the broth phase can use the extraction solvent flow richer in the butane concentration! and the fermenter operating at a lower titer in the broth phase will benefit from the poorer extraction solvent flow in the butanol concentration.
For example, referring again to Figure 7, consider the case where the Fermenter (F100) is at the beginning of the fermentation and operating a butane titer! relatively lower in the broth phase (B), the Fermenter (F101) is in the middle of fermentation and operating on a relatively moderate butanol titer in the broth phase, and the fermenter (F102) is near the end of the fermentation and operating in a butane title! relatively higher in the broth stage.
In this case, the poor extraction solvent (S), with minimal or no extracted butanol, can be fed into Fermenter F100, the flow of "outgoing solvent" (S ') from Fermenter F100 with an extracted butanol component can be fed in Fermenter F101 as an "inlet solvent" flow and the flow of
| The F101 outlet solvent can then be fed into the F102 GV fermentor as a solvent flow. The solvent outlet flow from (F102) can be. sent to be processed to recover the butanol present in the stream. The stream of processed solvent from which most of the butanol is removed can be returned to the system as a poor extraction solvent and would be the solvent in the Fermenter (F100) feed above. How fermentations proceed in an orderly manner valves | in the extraction solvent collector, they can be repositioned to feed the poorest extraction solvents to the fermenter operating with a lower butanol titer in the broth phase. For example, supposing that (a) Fermenter (F102) completes its fermentation and has been refilled and fermentation starts again, (b) Fermenter (F100) is in the middle of its fermentation and operating on a moderate butanol titer at the broth and (c) the Fermenter (F101) is near the end of its fermentation and operating on a higher butanol titer in the broth phase. In this scenario, the poorest extraction solvent would feed (F102), the extraction solvent out of (F102) would feed the Fermenter (F100) and the extraction solvent out of the Fermenter (F100) would feed the Fermenter (F101). The advantage of operating in this way can be to keep the broth phase in a butanol grade as low as possible for as long as possible to promote improved productivity. In addition, it may be possible to lower the temperature in the other fermenters that have progressed further in the fermentation that are operating with butane bonds! higher in the broth phase. The drop in temperature may allow for an improved tolerance to the higher titers of butane! at the broth stage.
ADVANTAGES OF THE METHODS OF THE PRESENT INVENTION The present extractive fermentation methods provide: butanol which is known to have an energy content similar to that of gasoline and which can be mixed with any fossil fuel.
Butanol is favored as a fuel or fuel additive. fuel, since it produces only CO, and little or no SOx and NOx when burned in a standard internal combustion engine.
In addition, the; 5 - butanol is less corrosive than ethanol, the preferred fuel as an additive fuel to date.
In addition to its usefulness as a biofuel or fuel additive, butane! produced according to the present methods has the potential to impact hydrogen distribution problems in the emerging fuel cell industry.
Fuel cells are currently affected by safety issues associated with the transport and distribution of hydrogen.
Butanol can be easily regenerated by its hydrogen content and can be distributed through existing fuel stations in the necessary purity for both cells and | 15 vehicles.
In addition, the present methods produce butanol from carbon sources derived from vegetables, avoiding the negative environmental impact associated with standard petrochemical processes for the production of butanol.
The advantages of the present methods include the feasibility of producing butanol at a rate, title and final effective yield that are significantly higher and more economical than the threshold levels of butanol obtained by a two-stage extractive fermentation process without the addition of hair at least one electrolyte in a concentration at least sufficient to increase the partition coefficient of butanol in relation to that presence of the salt concentration of the basal medium for fermentation.
The present method can also reduce the final amount of fresh or recycled extractor needed to achieve a desired level of butane production! from batch fermentation. EEE rim EE EEE A to E
| 69 |
EXAMPLES] The present invention is further defined in the following. Examples. It should be understood that these examples, while indicating preferred embodiments of the present invention, are given for illustrative purposes only. From the above discussion and these Examples, a person skilled in the art can determine the essential features of the present invention, and without departing from the spirit and scope of the present invention, you can make several changes and modifications to the invention to adapt it to different uses and conditions.
MATERIALS The following materials were used in the examples. All commercial reagents were used as received. All solvents were obtained from Sigma-Aldrich (St. Louis, MO) and were used without further purification. The oleyl alcohol used was of technical quality, which contained a mixture of oleyl alcohol (65%) and upper and lower fatty alcohols. Isobutanol (99.5% purity) was obtained from Sigma-Aldrich and was used without further purification. Sodium sulfate (Na2SO ,, CAS 7757-82-6, greater than 99% pure) was obtained from Sigma-Aldrich (St. Louis, MO). Sodium chloride (NaCl, CAS 7647-14-5, technical grade) was purchased from EMD Chemicals, Inc. (Gibbstown, NJ).
GENERAL METHODS The reading of the optical density to measure the concentration of cells of microorganisms was made using a spectrophotometer Thermo Electron Corporation Helios Alpha. The measurements were typically made using a wavelength of 600 nanometers. Glucose concentration in the culture broth was measured quickly using a “2700 Select Biochemistry Analyzer" analyzer (YSI Life Sciences, Yellow Springs, OH). Culture broth samples were
| 70 centrifuged at room temperature for 2 minutes at 13,200 rpm in tubes | 1.8 ml Eppendorf, and the aqueous supernatant analyzed for glucose concentration.
The analyzer performed a self-calibration with a known glucose standard before evaluating each set of fermentor samples; an external standard was also periodically analyzed to ensure the integrity of the culture broth tests.
The analyzer specifications for the analysis were as follows: Sample size: 15 ul Black chemical probe: dextrose White chemical probe: dextrose Isobutane concentrations! and glucose in the aqueous phase were measured by HPLC (Model Waters Alliance, Milford, MA or Agilent 1200 series, Santa Clara, CA) using a BioRad Aminex HPX-87H column, 7.8 mm x 300 mm, (Bio-Rad laboratories , Hercules, CA) with suitable guard columns, using 0.01 N aqueous sulfuric acid, isocratic as eluent.
The sample was passed through a 0.2 µm centrifuge filter (Nanosep MF modified nylon) in an HPLC flask.
The HPLC execution conditions were as follows: Injection Volume: 10 ul Flow rate: 0.60 mL / minute Execution time: 40 minutes Column temperature: 40 ºC Detector: refractive index Detector temperature: 35 ºC UV detection : 210 nm, 8 nm bandwidth After execution, the concentrations in the samples were determined from standard curves for each of the compounds.
The retention times were 32.6 and 9.1 minutes for isobutanol and glucose,
j | 71 | 'respectively. 'The isobutane! and ethanol in the organic extractor phase were measured - using gas chromatography (GC) as described below.
The following GC method was used to determine the amount of isobutanol and ethanol in the organic phase.
The gas chromatography method used a J&W Scientific DB-WAXETR column (50 m x 0.32 mm | D, 1 µm film) from Agilent Technologies (Santa Clara, CA). The carrier gas was helium at a flow rate of 4 mL / min measured at 150 ºC with a constant pressure; split type injector was from 1: 5 to 250 ºC; the oven temperature was 40ºC for 5 min, 40ºC to 230ºC to 10ºC / min, and 230ºC for 5min.
Detection using a flame ionization detector (FID) was used at 250 ºC, with 230 mL / min of helium gas composition.
Samples of culture broth were centrifuged before injection.
The injection volume was 1.0 ul.
Calibrated standard curves were generated for ethanol and isobutanol.
Under these conditions, the retention time of isobutane! was 9.9 minutes, and the retention time for ethanol was 8.7 minutes. CONSTRUCTION OF A CEPA OF E.
COLI HAVING DELETIONS OF THE PFLB, FRDB, LDHA AND ADHE GENES A suitable method for deleting the pf / B, frdB, IdhA, adhE genes from E. coli is provided in the present invention.
The Keio collection of E. coli strains (Baba et al., Mol.
Syst.
Biol., 2: 1-11, 2006) was used to produce eight of the knockouts.
The Keio collection (available from the NBRP at the National Institute of Genetics in Japan) is a library of gene knockouts created on the E. coli strain BW25113 by the method of Datsenko and Wanner (Datsenko, K.
A. & Wanner, B.
L., Proc.
Natl.
Acad.
Sci.
USA, 97 6640-6645, 2000). In the collection, each deleted gene was replaced by a kanamycin marker flanked by FRT that was removable by recombinant Flp.
There
72 E. coli strain carrying multiple knockouts was constructed by removing the kanamycin-knockout marker from the donor strain Keio by. transduction in bacteriophage P1 to a receptor strain. After each P1 transduction to produce a knockout, the canamiycin marker was removed by - recombinase Flp. This unlabeled strain acted as the new receptor strain for the next P1 transduction. One of the described knockouts was built directly on the strain using the method of Datsenko and Wanner! (supra), instead of using the P1 transduction. The E. coli 4KO strain was built on the Keio strain JWO0886 by P1y transductions, with P1 phage lysates prepared from three Keio strains. The Keio strains used are listed below: - JWO886: the kan marker is inserted in pflB - JW4114: the kan marker is inserted in frdB - JW1375: the kan marker is inserted in / dhA - JW1228: the kan marker is inserted in adhE [The sequences corresponding to the inactivated genes are: pflB (SEQ ID NO: 71), frdB (SEQ ID NO: 73), IdhA (SEQ ID NO: 77), adhE (SEQ ID NO: 75).] Removing the marker kanamycin flanked with FTR from the chromosome was performed by transforming the kanamycin-resistant strain with a pCP20, an ampicillin-resistant plasmid (Cherepanov and Wackernagel, supra). The transformants were seeded on LB plates containing 100 µg / ml ampicillin. Plasmid pCP20 carries yeast FLP recombinase under the control of the promoter | Opgea expression from this promoter is controlled by the repressor | temperature sensitive cl857 residing in the plasmid. The origin of replication of pCP20 is also sensitive to temperature. The removal of the kanamycin marker flanked with loxP from the chromosome was performed by transforming the 'kanamycin-resistant strain with an ampicillin-resistant plasmid pJW168 (Wild et al.,' Gene. 223: 55-66, 1998) carrying the Cre recombinase bacteriophage P1. Cre recombinase (Hoess, RH and Abremski, K. supra) mediates the excision of the kanamycin resistance gene via recombination at the / oxP sites. The origin of replication for pJW168 is that of temperature-sensitive pSC101. The transformants were seeded on LB plates containing 100 µg / ml of ampiuitoina.
The JWO886 strain (ApflB :: kan) was transformed with the plasmid pCP20e sown on the LB plates containing 100 µg / ml ampicillin at 30 ºC. The ampicillin-resistant transformants were selected, striated over the LB plates and cultured at 42 ºC. Isolated colonies were plated on plates with selective medium for ampicillin and kanamycin and LB plates. Colonies sensitive to canamycin and ampiillicin were screened by colony PCR with primers pflB CKkUp (SEQ ID NO: 78) and pflB CkDn (SEQ ID NO: 79). A 10 µl aliquot of the PCR reaction mixture was analyzed by gel electrophoresis. The expected PCR product of approximately 0.4 kb was observed confirming the removal of the marker and creating the strain "JW0886 without marking". This strain has a deletion of the pfliB gene. The strain "JWO0886 without labeling" was transduced with a P1u lysate from JW4114 (frdB :: kan) and streaked on LB plates containing 25 Vvg / mL kanamycin. The kanamycin-resistant transducers were selected by colony PCR with frdB CkUp (SEQ ID NO: 80) and frdB CkDn (SEQ ID NO: 81) primers. The clones that produced the expected PCR product of approximately 1.6 kb were made electrocompetent and transformed with pCP20 to remove the marker, as described above. The transformants were first distributed on LB plates containing 100 pg / mL ampicillin at 30 ºC and the ampicillin resistant transformants q — ssCcíDDÇÇD— = º = om q Qasas q q Ç.— 2 aan se ess —. — .— ese
| | Do | were selected and sown on LB plates and grown at 42 ºC.
As | 'isolated colonies were seeded on plates with selective medium for ampicillin and kanamycin and LB plates.
Colonies sensitive to kanamycin and ampicillin were screened by colony PCR with frdB CkUp primers (SEQ ID NO: 380) and frdB CkDn primers (SEQ ID NO: 81). The expected PCR product of approximately 0.4 kb was observed confirming the removal of the marker and | creating the double knockout strain "ApfIB frdB". | The double knockout strain was transduced with a P1,; lysate, from JW1375 (AIldhA :: kan) and seeded on LB plates containing 25 po / mL kanamycin.
The kanamycin-resistant transducers were selected by colony PCR with primers IdhA CKkUp (SEQ ID NO: 82) and / IdhA CkKDn (SEQ ID NO: 83). The clones that produced the expected 1.5 kb PCR product were made electrocompetent and transformed with pCP20 to remove the marker, as described above.
Transformants were seeded on LB plates containing 100 pg / mL ampicillin at 30 ° C, and ampicillin resistant transformants were seeded on LB plates and cultured at 42 ° C.
Isolated colonies were plated on plates with selective medium for ampicillin and kanamycin and LB plates.
The kanamycin-sensitive and ampoulein-sensitive colonies were screened by PCR with the primers IdhA CKkUp (SEQ ID NO: 82) and / (dhA CkDn (SEQ ID NO: 83) for a 0.3 kb product.
The clones that produced the expected PCR product of approximately 0.3 kb confirmed the removal of the marker and created the triple knockout strain designated "3Ko" (ApflB frdB IdhA). The “3KO” strain was transduced with a P1,; lysate, from —JW1228 (AadhE: kan) and seeded on LB plates containing 25 pg / ml kanamycin.
The kanamycin-resistant transducers were selected by colony PCR with adhE CkUp (SEQ ID NO: 84) and adhE CkDn (SEQ ID NO: 85) primers. The clones that produced the expected 1.6 kb PCR product were named 3KO adhE :: kan. The 3KO adhE :: kan strain was made 'electrocompetent and transformed with pCP20 to remove the marker. The | transformants were seeded on LB plates containing 100 µg / ml | ampiuitoina at 30 ºC. The ampicillin resistant transformants were streaked on LB plates and cultured at 42 ºC. The isolated colonies were plated on plates selective for ampicillin and kanamycin and LB plates. Colonies sensitive to kanamycin and sensitive to ampicillin were screened by PCR with the primers adhE CKUp (SEQ ID NO: 84) and adhE CKDn (SEQ ID NO: 85). The | clones that produced the expected PCR product of approximately 0.4 | 10 kb were called “4KO” (ApfiB frdB IdhA adhE).
CONSTRUCTION OF AN E. COLI PRODUCTION HOST (CEPA NGC1-031) CONTAINING A BIOSYNTHETIC PATH OF ISOBUTANOL AND DELETIONS OF THE GENES PFLB, FRDB, LDHA, AND ADHE A fragment of sadB-encoding DNA, a butanol — dehydrogenase (DNA: SEQ ID NO: 9; protein: SEQ ID NO: 10) from Achromobacter xylosoxidans was amplified from A. xylosoxidans genomic DNA using standard conditions. DNA was prepared using the “Gentra Puregene" kit (Gentra Systems, Inc., Minneapolis, MN; catalog number D-5500A), following the recommended protocol for gram-negative microorganisms. PCR amplification was done using the forward (reverse) and reverse (reverse) primers, N473 and N469 (SEQ ID NOs: 86 and 87), respectively, with the DNA polymerase “Phusion High Fidelity DNA Polymerase” (New England Biolabs, Beverly, MA). PCR was | cloned by TOPO-Blunt into pCR4 Blunt (Invitrogen) to produce | 25 —pCR4Blunt: sadB, which was transformed into E. coli Mach-1 cells. The plasmid was subsequently isolated from four clones, and the verified sequence.
The sadB coding region was then cloned into the vector pTrc99a
(Amann et al, Gene 69: 301-315, 1988.). PCR4Blunt :: sadB was digested with: EcoRI, releasing the sadB fragment, which was ligated with pTrc99a digested with EcoRI to generate pTrc99a :: sadB.
This plasmid was transformed into E. coli Mach-1 cells and the resulting transformants were named —Mach1i / pTrc99a :: sadB.
The enzyme activity expressed from the sadB gene in these cells was determined to be 3.5 mmol / min / mg of protein in free cell extracts when analyzed using isobutyraldehyde as a standard.
The sadB gene was then subcloned into a pTrc99A :: budB-ilvC- ilvD-kivD as described below.
PTrc99A :: budB-ilvC-ilvD-kivD is the expression vector pTrc-99a carrying an operon for isobutanol expression (described in Examples 9-14 of Published Patent Application US 20070092957). The first gene in the pTrc99A isobutane operon :: budB- ilvC-ilvD-kivD is the budB that encodes an Klebsiella pneumoniae ATCC 25.955 acetolactate synthase, followed by the E. coli acetohydroxy acid encoding hydroxide gene This is followed by the E. coli gene ilvD encoding E. coli acetohydroxy acid dehydratase and finally the kivD gene encoding the L. / lactis branched chain keto acid decarboxylase.
The coding region for sadB was amplified from pTrc99a :: sadB using primers NG695A (SEQ ID NO: 88) and N696A (SEQ ID —NO: 89) with DNA polymerase "Phusion High Fidelity DNA polymerase" (New England Biolabs , Beverly, MA) Amplification was performed with an initial denaturation at 98 ºC for 1 min, followed by 30 cycles of denaturation at 98 ºC for 10 seconds, annealing at 62 ºC for 30 seconds, extension at 72 ºC for 20 seconds and one final extension cycle at 72 ºC for 5 min, followed by a final temperature of 4 ºC.
Primer N695A contained an Avril restriction site for cloning and an RBS upstream of the ATG initiation codon of the sadB coding region.
Primer N696A contained an Xbal site for cloning. THE
| 77 1.1 kb PCR product was digested with Avril and Xbal (New England Biolabs, 'Beverly, MA) and gel purified using a' Q / Aquick Gel Extraction Kit 'extraction kit (Qiagen Inc., Valencia, CA )). The purified fragment was ligated with pTrc99A :: budB-ilvC-ilvD-kivD, which had been cut with the same restriction enzymes, using a T4 DNA ligase (New England Biolabs, Beverly, MA). The ligation mixture was incubated at 16 ºC overnight, and then transformed into competent E. coli Mach-1 cells (Invitrogen) according to the manufacturer's protocol. Transformants were obtained after growth on LB agar with 100 µg / ml ampicillin. Plasmid DNA from the transformants was prepared with the 'Q / Aprep Spin Miniprep Kit' kit (Qiagen Inc., Valencia, CA) according to the manufacturer's protocol. The resulting plasmid was called pTrc99A :: budB-ilvC-ilvD-kivD-sadB. The electrocompetent cells of the 4KO strains were prepared as described and transformed with pTrc99A :: budB-ilvC-ilvD-kivD-sadB ("PBCDDB"). The transformants were streaked on LB plates! containing 100 µg / ml ampicillin. The resulting strain carrying the plasmid pTrc99A :: budB-ilvC-ilvD-kivD-sadB with 4KO (called the NGCI-031 strain) was used for fermentation studies in the indicated Examples. EXAMPLE 1
EFFECT OF ELECTROLYTE CONCENTRATION ON THE PARTITIONING COEFFICIENT (Ke) The purpose of this example was to evaluate the effect of electrolyte concentrations in the fermentation medium on the isobutanol - partition coefficient (K,) when oleyl alcohol was used as the extracting agent. The basal fermentation medium (BFM) normally used in E. coli fermentations was used as the fermentation medium in this Example. The composition of the BFM is shown in Table 2. E E to the ear E the Rr a a a a
TABLE 2 'COMPOSITION OF THE GOOD as indicated (millimoles / L; mM) Em = = heptahydrate meme o neem os e - os [= hurt (MLL) a The oligoelement solution used in the above medium was prepared as follows. The ingredients listed below were - added in the order listed and the solution heated to 50 - 60 ° C until all components were completely dissolved. Ferric citrate was added slowly after other ingredients in the solution. The solution was sterilized by filtration using 0.2 micron filters.
EDTA (Ethylene diaminetetraacetic acid) 0.84 g / L Cobalt dichloride hexahydrate (cobalt chloride 6-hydrate) 0.25 g / L Mganese dichloride tetrahydrate (manganese chloride 4-hydrate) 1.59 / L ts The EEEE ——————— «.« - 32 —————————————————————————————————————— —— € —— <——————
Cupric chloride dihydrate 0.159 / L 'Boric acid (H; BO; 3) 0.30 g / L Sodium molybdate dihydrate 0.25 g / L Zinc acetate dihydrate 1.380 g / L Ferric citrate 10.0 g / LO initial level total salts (sum of monobasic potassium phosphate, dibasic ammonium phosphate, citric acid monohydrate, and magnesium sulfate heptahydrate) in the BFM as shown in Table 2 is calculated to be approximately 144.2 mM.
Since a biocatalyst E.coli was used in the Examples shown below, betaine hydrochloride (Sigma-Aldrich) at 0.31 g / L (2 mmoles / L) was added to the basal medium for fermentation, since it is described in the literature (A, Cosquer et al; 1999; Appl Environ Microbiol 65: 3304-3311) for improving E.coli's tolerance to salt.
The following experimental procedure was used to generate the data from Tables 3 and 4. In these Kp measurement experiments, a specified amount of electrolyte such as sodium sulfate (Na2SO4) or sodium chloride (NaCl) was added to the basal medium for fermentation. In 30 ml of BFM supplemented with sucrose, 10 ml of isobutanol extractor rich in alcohol - oleyl (OA) containing 168 g / L of isobutanol were added and mixed vigorously for 4 - 8 hours at 30 ºC with agitation at 250 rom on a shaker (Innova 4230, New Brunswick Scientific, Edison, NJ) to achieve a balance between the two phases. The aqueous and organic phases in each flask were separated by decantation.
The aqueous phase was centrifuged (2 minutes at 13,000 rpm with an Eppendorf model 5415R centrifuge) to remove the residual extractor phase and the supernatant analyzed by HPLC to quantify glucose and isobutanol.
The analysis of isobutane levels in the aqueous phase after 4 hours of stirring was similar to that obtained after 8 hours of mixing suggesting that the equilibrium between the two phases was achieved within 4 hours. The intention was to demonstrate that the mixing procedure for more than 4 hours does not alter the K ,. The partition coefficients (K,) for the distribution of isobutanol between the organic and aqueous phases were calculated from the known amount of isobutanol added to the flask and the isobutanol concentration data measured in the aqueous phase. The concentration of isobutanol in the extractor phase was determined by mass balance. The partition coefficient was determined as the ratio between the concentrations of isobutanol in the organic and aqueous phases, that is, K, = [isobutane]] organic rase / [isobutanol] aqueous tase. Each data point corresponds to a specified level of electrolyte as shown in Table 3 and Table 4 was repeated twice and K values were reported as the average of the two vials. TABLE 3 EFFECT OF THE CONCENTRATION OF SODIUM SULFATE (NA2SO, 4) ON K, DO
ISOBUTANOL Total concentration Amount of total amount of initial salts in the BEM Sodium Sulfate salts in the experiment (Table 2) added to the BEM moles / l Na2SO, (moles / l) moles / l (a) (b) (a) + ( b) | Log ss e a a sos Ls as es O e e LR es e EO) CC RN O a a Lo a ns E ao aa eos
| 81 TABLE 4 | EFFECT OF SODIUM CHLORIDE (NACL) CONCENTRATION ON K, DO |
ISOBUTANOL Total concentration Amount of total amount of initial salts in sodium chloride salts in the BFM experiment (Table 2) added to the BFM moles / l NaCl (motes / l) moles / l (a) (b) (a) + (b )
RL A CS EE Eos RR a o) E e a o a a as a and if o O a in | | Ns E A A A E es e in ss The results of Table 3 and Table 4 demonstrate that the supplementation of the aqueous fermentation medium with the electrolytes Na2SO, and NaCl! resulted in higher K, for isobutanol in a two-phase system with oleyl alcohol as the extractor phase. EXAMPLE 2 EFFECT OF ELECTROLYTE SUPPLEMENTATION ON E.COLI GROWTH RATE To assess the effect of electrolytes, such as Na2SO, on the growth properties of the biocatalyst, E. coli strain 4KO was grown in shaking flasks in BFM medium supplemented with 0.31 g / L of betaine hydrochloride and different levels of Na2SO, (0 - 284 g / L) at 30 ºC, 250 RPM on / nnova table shakers. From a frozen flask, 25 mL of culture for sowing was grown in Difco LB broth, Miller medium, purchased from BD Laboratories (Becton Dickinson and Company, Sparks, MD,
21152, USA) at 30 ° C, 200 RPM. 1 ml of this culture for sowing was added to shaking flasks containing 30 ml of BFM medium supplemented with 0.31 g / L of betaine hydrochloride and different levels of Na2SO4. The samples were taken at the defined time points to monitor the biomass growth measured by ODsoo.
The growth rates were calculated from the biomass time profiles by adjusting the exponential growth rate equations.
TABLE 5 EFFECT OF NA / SO, ON THE GROWTH RATE OF E.
COLI CEPA 4KO Total amount of Total concentration mo, Concentration of salts in the experiment Initial rate of salts in Na2SOs (mol / L) growth of E, BFM (Table 2) o. added to BEM moles / l moles coli / l (8) (à) nr r (A) p A + B Lo o a a | | | LO: as A | es es es es | LT es Lo a Crescimento | 0.14 1.33 1.47 ion; insignificant | Growth ! 0.14 "”; The growth rate data shown in Table 5 suggests that the biocatalyst can tolerate salt levels as high as about 0.67 M Na-SO ;, (total salt level 0.81 M ) with a 30% loss of growth rate compared to any control electrolyte. | However, there is a significant drop (more than about | - in oa A and e | o |
| 83 80%) in the growth rate in salt concentrations of 1M. The data in Table 3 show that in the concentration of Na; SO, at 0.67 M, the K, for butanol increases in two times in comparison with | any control of salt addition when oleyl alcohol is present as | 5 retractor. ! Thus, the overall final effect obtained by adding an electrolyte in! a 2-stage extractive fermentation using a microorganism | recombinant butanol producer can be unpredictable, since on the one hand the electrolytes can inhibit cell growth (Table 3), but on the other hand they can increase the partition coefficient of the butane product! toxic, which can mitigate the toxic effects on the microorganism. EXAMPLE 3 EFFECT OF ELECTROLYTE ADDITION ON RATE, TITLE, AND PRODUCTION INCOME
OF BUTANOL IN A TWO PHASE EXTRACTIVE FERMENTATION PROCESS The purpose of this example was to demonstrate the advantages of adding at least a sufficient amount of electrolyte in the aqueous phase of a two-stage extractive fermentation in which butanol is produced by a recombinant microorganism , a strain of Escherichia coli (NGCI-031) that contains an isobutanol biosynthetic pathway. Extractive fermentation uses oleyl alcohol as a water-immiscible organic extractor.
Escherichia coli strain NGCI-031 was constructed as described in the General Methods section above. All sowing cultures for inoculum preparation were grown in Luria-Bertani (LB) medium with ampicillin (100 mg / L) as the antibiotic of choice. The fermentation medium used was a semi-synthetic medium supplemented with 2 mmoles / L of betaine hydrochloride, the composition of which is shown in Table 6.
a ——— m ç—— sa a assJ ——> »- O O“ ———— <——.——. ————— ..— [——-. — ...—
| TABLE 6
'Composition of the means of fermentation
De Balch with cobalt - 1000X (composition eee | 3 | am [Sao ce mageo misses aa [emo camemtm os as [opspa cmo mild - | os | ss [see eg [sm age [aeee der og [oprro dstama sos o tm | [Only the mami eai and [Season weighs ss [ae º Obtained from BD Diagnostic Systems, Sparks, MD PO Obtained from Sigma-Aldrich
TABLE 7
| MODIFIED BALCH METAL TRACES - 1000X emu - o
|
|
| Hand sodium molybdate | only
Ingredients 1-11 in Table 6 were added to the water in the
| [85 concentration prescribed to make a final volume of 0.4 L in the fermenter. The: contents of the fermenter were sterilized in an autoclave. Components 12-14 were mixed, sterilized by filtration and then added to the fermenter, after the autoclaved medium was cooled. The final total volume of the fermentation medium (in the aqueous phase) was about 0.5 L after adding 50 ml of the inoculum seeding.
The electrolyte in the form of NazSO was added in concentrations of O g / L, 40 g / L or 60 g / l to the medium before sterilization. The solutions of ampiuitoina, thiamine hydrochloride, and glucose sterilized by filters were added to the fermentation medium, after sterilization, in a final concentration of 100 mg / L, 5 mg / L and 20 g / L, respectively. Fermentations were carried out using a 1 L autoclavable bioreactor, Bio Console ADI 1025 (Applikon, Inc, Netherlands), with a working volume of 900 mL. The temperature was maintained at 30 ºC throughout the fermentation and the pH was maintained at 6.8 using ammonium hydroxide. After inoculation of the sterile fermentation medium with the culture for seeding (2-10% vol), the fermenter was operated under aerobic conditions at 30% dissolved oxygen (DO) fixed with 0.3 vvm of air flow, while the agitation rate (rpm) was automatically controlled. When the desired optical density (ODgçoo) was reached (ie ODgsoo = 10), the culture was induced with the addition of 0.4-0.5 mM isopropyl beta-D-1-thiogalactopyranoside to overexpress the biosynthetic pathway of isobutanol. Four hours after induction, the fermentation conditions were changed to microaerophilic conditions, reducing the air flow to 0.13 slpm and setting the DO (dissolved oxygen) point at 3-5%.
The move to microaerophilic conditions initiated isobutanol production, minimizing the amount of carbon for biomass production, and thereby decoupling the formation of biomass from isobutanol production.
pr E - ——— UM e MONO From e e e O a a E O O
'86 The alcohol! oleyl (about 250 mL) was added during the phase: of isobutanol production to alleviate the inhibition problem due to the accumulation of isobutanol in the aqueous phase. Glucose was added as a bolus (50% weight of the stock solution) as needed to the fermenter to keep glucose levels between 20 g / L and 2 g / L.
Because the efficient production of isobutanol requires microaerobic conditions to allow redox equilibrium in the biosynthetic pathway, air was continuously supplied to the fermenter at 0.3 vvm. Prolonged aeration led to a significant removal of isobutanol from the aqueous phase of the fermenter.
To quantify the loss of isobutanol due to desorption, the exhaust gas from the fermenter was sent directly to a mass spectrometer (Prima dB mass spectrometer, Thermo Electron Corp. Madison, WI) to quantify the amount of isobutanol in the gas stream . The isobutanol peaks at 74 or 42 charge ratios were used to determine the amount of isobutanol present in the gas stream.
For isobutanol production, the effective title, effective rate and | effective yield of isobutanol production, all corrected by the loss of isobutane! due to removal, they are shown below as a table (Table 8) Isobutanol in the aqueous phase was measured using the HPLC method described above herein.
Isobutanol in the extractor phase in oleyl alcohol was measured using the GC method described above in the present. Glucose levels were monitored using HPLC and YSI, as previously described.
As can be seen from the results in Table 8, the use of electrolytes in an extractive fermentation for the production of isobutane! results in significantly higher effective title, effective rate, and effective yield compared to the case where no salt is added.
| 87 The isobutanol product, which is toxic to the host bacteria, is! 'continuously extracted into the oleyl alcohol phase, decreasing its concentration in the aqueous phase, thereby reducing its toxicity to the micro-organism.
In addition, an unexpected improvement in the effective rate, effective title, and effective yield is seen when salt is added to | quite.
The addition of salts, in principle, could not only have a deleterious effect on the metabolism of the butanol-producing biocatalyst, but could also alleviate the inhibitory effect of butanol, increasing the Kp of | 10 —butanol compared to any control without the addition of salt. | The final effect of adding salts to our two-phase extraction system favors increased butane production and recovery !. | TABLE 8 SALES EFFECT ON THE RATE, TITRATION AND YIELD OF PRODUCTION | BUTANOL IN EXAMPLE 3. Concentration | of hs “o Quantity K dictated« total salts [co and c pelcionare ao no Título Rate | Yield in the middle of:,,. phase. experiment | effective | effective effective | fermentation in OAJ [Conc: (Table 6+ | (g / L / h) | (g / L) (9/9) | Table 6 in Phase I Na7SO,)! (moles / l) Aq] j moles / l | Es and E Os | o) the om | om | as | The initial amount of salts in the fermentation medium (Table 6) was | of about 0.05 moles / L.
EXAMPLE 4 'EFFECT OF THE ADDITION OF ELECTROLYTE ON THE RATE, TITRATION AND YIELD OF PRODUCTION OF BUTANOL COUPLED WITH GAS EXTRACTION (DESORPTION) FROM
BUTANOL DURING FERMENTATION In order to assess the effect of adding electrolyte on butanol production during the aqueous fermentation phase without the addition of an oleyl alcohol extractor, Example 3 was repeated, except that oleyl alcohol was not added to any of the fermenters . In this example, desorption of butanol from the aqueous phase was prevalent due to the spraying of fermenting arches. The amount of butanol evaporated into the exhaust gas was quantified as in Example 3 using mass spectrometry. The effective rate, effective title, and effective yield, all corrected for the loss of butanol due to evaporation are shown below in Table 9. TABLE 9 SALES EFFECT ON THE RATE, TITLE AND YIELD OF THE PRODUCTION OF BUTANOL COUPLED WITH GAS EXTRACTION (DESORPTION) OF BUTANOL DURING FERMENTATION FOR EXAMPLE 4 Concentration Amount of Na2SO, total “Grams” salts added to no Rate Title Yield isobutanol effective experiment effective effective lost fermentation in (Table 6 + (g / L / h) (g / L) (9/9) due to Table 6 Na2SO4) removal (moles / l) moles / lo es De and ww Gs) [e es [e | and [og Tor De e] |) [Initial amount of salts in the fermentation medium (Table 9) was about 0.05 moles / L]. The results in table 9 show that the addition of electrolyte
| 89 in the aqueous phase, the effective rate, titre and yield of butanol production increases in the absence of oleyl alcohol by increasing the desorption rate of isobutanol. The grams of butanol extracted by desorption are almost twice as high in the presence of salt when compared to the case in which there was no addition of electrolyte.
Although examples of specific embodiments of the present invention have been described in the description above, it will be understood by those skilled in the art that the invention is susceptible to numerous modifications, substitutions and rearrangements without departing from the spirit or essential attributes of the invention. Reference should be made to the appended claims, rather than the aforementioned specification, as indicative of the scope of the invention.
| | | | |

权利要求:
Claims (1)
[1]
: CLAIMS; '1 METHOD FOR RECOVERING BUTANOL from a fermentation medium, characterized by the fact that it comprises: a) providing a fermentation medium comprising butanol, water, at least one electrolyte in a concentration at least sufficient to increase the partition coefficient of butanol in relation to that in the presence of the salt concentration of the basal medium for fermentation, and a genetically modified micro-organism that produces butanol from at least one source of fermentable carbon; b) put the fermentation medium in contact with i) a first water-immiscible organic extractor selected from the group consisting of fatty alcohols C12 to C> 2, fatty acids C; 2 to Cp, esters of fatty acids C12 to C22 , fatty aldehydes C12 to C22, fatty amides C17 to Cx and mixtures thereof, and optionally, ii) a second water-immiscible organic extractor selected from the group consisting of C fatty alcohols; to C272, C fatty acids; to C77, esters of C fatty acids; a Cz> 2, fatty aldehydes C; at C> 2, C fatty amides; to C27, and mixtures thereof to form a two-phase mixture comprising an aqueous phase and an organic phase containing butanol; and c) recovering the butanol from the organic phase containing butanol to produce recovered butanol.
2. METHOD, according to claim 1, characterized by the fact that a portion of the butanol is removed simultaneously from the fermentation medium by a process comprising the steps of: a) desorbing (stripping) the butanol from the fermentation medium with a gas to form a gas phase containing butanol; and b) recovering the butanol from the gas phase containing butanol.
Í '2
3. METHOD, according to claim 1, characterized: by the fact that the electrolyte is added to the fermentation medium, to the first extractor, to the optional second extractor, or to the combination thereof.
4. METHOD, according to claim 1, characterized by the fact that the electrolyte comprises a salt having a cation selected from the group consisting of lithium, sodium, potassium, rubidium, cesium, magnesium, calcium, strontium, barium, ammonium, phosphonium, and combinations thereof.
5. METHOD, according to claim 1, characterized by the fact that the electrolyte comprises a salt having an anion selected from the group consisting of sulfate, carbonate, acetate, citrate, lactate, phosphate, fluoride, chloride, bromide, iodide, and combinations thereof.
6. METHOD, according to claim 1, characterized by the fact that the electrolyte is selected from the group consisting of sodium sulfate, sodium chloride, and combinations thereof.
7. METHOD, according to claim 1, characterized by the fact that the genetically modified microorganism is selected from the group consisting of bacteria, cyanobacteria, filamentous fungi and yeasts.
8. METHOD, according to claim 7, characterized by the fact that the bacterium is selected from the group consisting of Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, - Pediococcus, - Alcaligenes, Klebsiella , —Paenibacillus, Arthrobacter, Corynebacterium and Brevibacterium.
9. METHOD, according to claim 7, characterized by the fact that yeast is selected from the group consisting of Pichia, Candida, Hansenula, Kluyveromyces, Issatchenkia and Saccharomyces.
| | 3 | | - | 10. METHOD, according to claim 1, characterized: by the fact that the first extractor is selected from the group that | consists of oleyl alcohol, berrenic alcohol, cetyl alcohol, alcohol! lauryl, myristyl alcohol, stearyl alcohol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, methyl oleate, lauric aldehyde, 1-dodecanol and a combination thereof.
11. METHOD, according to claim 1, characterized by the fact that the first extractor comprises oleyl alcohol.
12. METHOD, according to claim 1, characterized | by the fact that the second extractor is selected from the group consisting of 1-nonanol, 1-decanol, 1-undecanol, 2-undecanol, 1-nonanal, and a combination thereof.
13. METHOD, according to claim 1, characterized by the fact that butane! is 1-butanol.
14. METHOD, according to claim 1, characterized by the fact that butane! is 2-butanol.
15. - METHOD, according to claim 1, characterized by the fact that butanol is isobutanol.
16. METHOD, according to claim 1, characterized by the fact that the fermentation medium additionally comprises ethanol, and the organic phase containing butanol contains ethanol.
17. METHOD, according to claim 1, characterized by the fact that the genetically modified microorganism comprises a modification that inactivates a pathway that competes for the carbon flow.
18. - METHOD, according to claim 1, characterized by the fact that the genetically modified micro-organism does not produce acetone.
192. METHOD TO PRODUCE BUTANOL, characterized
| : 4 for the fact that you understand: ; a) supply a genetically modified microorganism that produces butanol from at least one source of fermentable carbon; b) growing the microorganism in a fermentation medium - biphasic comprising an aqueous phase ei) a first water-immiscible organic extractor selected from the group consisting of fatty alcohols C12 to C22, fatty acids C12 to Cz7, esters of acids fatty acids C12 to C22, fatty aldehydes C12 to C22, fatty amides C12 to C22, and mixtures thereof and, optionally, ii) a second water-immiscible organic extractor selected from the group consisting of C alcohols; to C> 2, C carboxylic acids; to C> 22, esters of C carboxylic acids; to C22, C aldehydes; to C22, C fatty amides; to C2o, and mixtures thereof, in which the biphasic fermentation medium additionally comprises at least one electrolyte, in a concentration at least sufficient to increase the partition coefficient of butane! in relation to that in the presence of the salt concentration of the basal medium for fermentation, for a time sufficient to allow the extraction of the butanol in the organic extractor to form an organic phase containing butanol; c) separating the organic phase containing butanol from the aqueous phase; and d) optionally recovering the butanol from the butanol-containing organic phase to produce butane! recovered.
20. METHOD, according to claim 19, characterized by the fact that the electrolyte is added to the aqueous phase during the growth phase of the microorganism, to the aqueous phase during the production phase of —butanol, to the aqueous phase when the butanol concentration in the aqueous phase is inhibitory to the first extractor, the optional second extractor, or a combination thereof.
21. METHOD according to claim 19, characterized gs EE a Err rmrrr rt ter rrr rr rrtraete As A EM by the fact that the electrolyte is obtained from a fermentation medium. 22. METHOD TO PRODUCE BUTANOL, characterized by the fact that it comprises: a) providing a genetically modified micro-organism 5 that produces butanol from at least one fermentable carbon source; b) growing the microorganism in a fermentation medium in which the microorganism produces butanol in the fermentation medium to produce a fermentation medium containing butanol; c) add at least one electrolyte to the fermentation medium to supply the electrolyte in a concentration at least sufficient to | to increase the partition coefficient of butanol in relation to that in the presence | the concentration of salt in the basal medium for fermentation; | d) put at least a portion of the medium in contact with | butanol-containing fermentation with i) a first water-immiscible organic extractor selected from the group consisting of fatty alcohols Ci7 to C22, fatty acids C17 to C727, fatty acid esters C12 to C22, fatty aldehydes C12 to C22, fatty starches C12 a C2 »> and mixtures thereof and, optionally, ii) a second water-immiscible organic extractor selected from the group consisting of C alcohols; Co, carboxylic acids C; to C77, esters of C carboxylic acids; to C22, C aldehydes; to C22, C fatty amides; to C727 and mixtures thereof, to form a two-phase mixture comprising an aqueous phase and an organic phase containing butanol; e) optionally, recovering the butanol from the - organic phase containing butanol; and f) optionally, returning at least a part of the aqueous phase to the fermentation medium.
23. - METHOD, according to claim 22, characterized NS Dn a à
'6 | by the fact that the electrolyte is added to the fermentation medium in step (c),. when the growth phase of the microorganism slows down.
24. METHOD, according to claim 22, characterized by the fact that the electrolyte is added to the fermentation medium in step (c), - when the butanol production phase is complete.
25. METHOD, according to one of claims 1, 19 or 22, characterized by the fact that said at least one source of fermentable carbon is present in the fermentation medium and comprises renewable carbon from agricultural raw materials, algae, cellulose, hemicellulose, lignocellulose, or any combination thereof.
26. COMPOSITION, characterized by the fact that it comprises: (a) a fermentation medium comprising butanol, water, at least one electrolyte in a concentration at least sufficient to increase the partition coefficient of butane! in the ionic medium, and a genetically modified microorganism that produces butanol from at least one source of fermentable carbon; (bj) a first water-immiscible organic extractor selected from the group consisting of fatty alcohols C12 to C22, fatty acids Ci to Cx, fatty acid esters C12 to C22, fatty aldehydes C12 to C22, fatty amides C12 to C27 and mixtures thereof; and (c) optionally a second water-immiscible organic extractor selected from the group consisting of C fatty alcohols; to C22, C fatty acids; to C> 7, esters of C fatty acids; to C22, fatty aldehydes C; aC, fatty amides C; to C2, and mixtures thereof; wherein said composition forms a mixture of two phases comprising an aqueous phase and an organic phase containing butanol in which the butanol can be separated from the fermentation medium of (a).
| U7 | The
T o + S -
S uu o | | * 2 | |
| | | | the bx en -. co o
QN QN
DD = 4 o> »| 2 '| o + | | S | s | Me | <| | = o | : a> | | «O | | o |
SI 2
| 4/7 and $ o | X
H = | The
SI e = | | | Yv = o s u | e x es = = SI | o = =
| | 517 es | | Ss ”= v
SD | 4 es
S 2 | | | E:: 1 11 IÀNI0TI1dMMM MW l CCO PI A RO ENS OE
P: | 6/7 | : | o | 2 | | if | 3 | | and the | 8 = R u | º | | - | 2 aee
PP Sa Ar Te TFO OM 7I7 | o (e) Ss of z oz mn
N> - Ta on = 2 do u os dn | = on TC 3

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法律状态:
2021-02-09| B08F| Application dismissed because of non-payment of annual fees [chapter 8.6 patent gazette]|Free format text: REFERENTE AS 9A E 10A ANUIDADES. |

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2021-05-25| B08K| Patent lapsed as no evidence of payment of the annual fee has been furnished to inpi [chapter 8.11 patent gazette]|Free format text: EM VIRTUDE DO ARQUIVAMENTO PUBLICADO NA RPI 2614 DE 09-02-2021 E CONSIDERANDO AUSENCIA DE MANIFESTACAO DENTRO DOS PRAZOS LEGAIS, INFORMO QUE CABE SER MANTIDO O ARQUIVAMENTO DO PEDIDO DE PATENTE, CONFORME O DISPOSTO NO ARTIGO 12, DA RESOLUCAO 113/2013. |

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2021-12-14| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US26351909P| true| 2009-11-23|2009-11-23|

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US61/263,519|2009-11-23|

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PCT/US2010/057791|WO2011063391A1|2009-11-23|2010-11-23|Method for producing butanol using extractive fermentation with electrolyte addition|

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