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    • 专利摘要:
    The present invention relates to a genetically modified fungal microorganism for the production of rambinone, said microorganism having the following characteristics: - the ability to produce rambinone from tyrosine; and a low capacity or inability to degrade tyrosine to tyrosol, p-hydroxyphenyl acetaldehyde and / or p-hydroxyphenyl acetate, and its use for the production of rambinone.
    公开号:FR3052170A1
    申请号:FR1655089
    申请日:2016-06-03
    公开日:2017-12-08
    发明作者:Alexander Matthias Farwick；Thomas Desfougeres；Georges Pignede；Anais Roussel；Isabelle Mouly；Brieuc Morvan
    申请人:Lesaffre et Cie SA；
    IPC主号:
    专利说明:

    FRAMBINONE PRODUCTION BY RECOMBINANT FUNGAL MICROORGANISM
    FIELD OF THE INVENTION
    The present invention provides methods for efficiently producing rambinone from tyrosine using fungal microorganisms and adapted culture conditions.
    STATE OF THE ART
    The flavor of raspberry (Rubus idaeus) is linked to more than 200 compounds, but frambinone, a natural phenolic compound, is the compound that has the greatest impact, defining its characteristic taste (Klesk et al., 2004, J Agric., Food Chem 52, 5155-61, Larsen et al., 1991, Acta Agric., Scand 41, 447-54). It can also be found in other fruits and vegetables, including peach, apple and rhubarb (Beekwilder et al., 2007, Biotechnol, J. 2, 1270-79). Because raspberries are only present in small quantities in raspberries (1-4 mg per kg of fruit), natural rambinone is of great value (Larsen et al., 1991). As its natural availability is limited, its biotech production is highly desirable.
    Frambinone (CAS No. 5471-51-2), also known as raspberry ketone or 4- (4-hydroxyphenyl) butan-2-one, has the following structure:
    Frambinone can be used in a wide range of applications, including agri-food and cosmetics (as a flavor), agriculture (as bait / lure for insects) and even in the field health (as a slimming product) or medicine (as an inhibitor of melanogenesis).
    Frambinone can be obtained from the aromatic amino acid L-tyrosine as an initial substrate via a 4-step biosynthetic pathway (Figure 1, Beekwilder et al., 2007, Biotechnol, J. 2, 1270-79). tyrosine is de-aminated by a tyrosine ammonia lyase (TAL, EC 4.3.1.23) to form coumaric acid. Catalysted by a 4-coumarate: CoA ligase (4CL, EC 6.2.1.12), a Coenzyme A (CoA) molecule is grafted onto coumaric acid. Coumaroyl-CoA is then converted by benzalacetone synthase (BAS, EC 2.3.1.212) to 4-hydroxybenzalacetone. This reaction is a decarboxylative condensation and uses a malonyl-CoA unit as cosubstrate. The final step is the reduction of 4-hydroxy benzalacetone to frambinone by benzalacetone reductase (BAR, EC 1.3.1.x).
    An alternative substrate for frambinone production is coumaric acid, an intermediate of the pathway described above (noted (2) in Figure 1). However, given the price of the substrates used for bioconversion, industrial applications are more profitable when tyrosine is implemented rather than coumaric acid.
    L-phenylalanine (denoted (7) in Figure 1) can also be used as a substrate since it can be converted to coumaric acid via cinnamic acid (denoted (8) in Figure 1). Indeed, frambinone formation in plants uses the general route of phenylpropanoids, which begins with phenylalanine (Borejsza-Wysocki and Hrazdina, 1994, Phytochemistry 35, 623-28.). The first step is phenylalanine ammonia lyase catalyzed deamination (PAL, EC 4.3.1.24). The cinnamic acid product is then hydroxylated by a cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) to form coumaric acid which is converted as described above. C4H is a cytochrome P450 and is associated with the endoplasmic reticulum membrane. Its expression seems problematic and the full activity also requires an auxiliary enzyme (cytochrome P450 reductase, CPR) (Bassard et al., 2012, Plant Cell 24, 4465-82, Schuckel et al., 2012, ChemBioChem 13, 2758-63; Winkel, 2004, Rev. Plant Biol 55, 85-107).
    The biosynthesis of two other compounds of biotechnological interest, resveratrol and naringenin, show numerous similarities with the proposed route of frambinone (Jeandet et al., 2012, J. Biomed, Biotechnol, 2012, 1-14, Lussier et al. , 2012, Comput Biotechnol Struct, J. 3, 1-11). TAL and 4CL are used to convert tyrosine and coumaric acid to coumaroyl-CoA. Stilbene synthase (STS) or chalcone synthase (CHS) then catalyzes the subsequent condensation with three malonyl-CoA units to form resveratrol or naringenin chalcone, respectively.
    In view of the interest for an alternative production to the chemical synthesis of rambinone, it has been attempted to transpose these synthetic routes into recombinant microorganisms, in particular Escherichia coli and Saccharomyces cerevisiae.
    In particular, GB 2 416 769 describes the possibility of producing rambinone with the aid of a microorganism (in particular bacteria and yeasts) containing a 4CL and BAS coding sequence, at least one being of heterologous source. It may further comprise a sequence encoding BAR, C4H, PAL and / or CHS, the BAR coding sequence being advantageously endogenous. In the examples, this document reports: the cloning of the raspberry CHS gene; cloning of the tobacco 4CL gene; cloning of the BAS gene of rhubarb; - the transformation of E. coli with the Raspberry BAR gene; the production of benzalacetone and frambinone (0.2 μg in 50 ml) from coumaric acid in E. coli transformed with BAS and 4Cl; the production of rambinone from benzalacetone in E. coli having endogenous BAR activity.
    Based on the assumption that CHS has BAS activity, GB 2,416,770 describes the possibility of producing benzalacetone and frambinone using a microorganism comprising a 4CL (eg tobacco) coding sequence and CHS. (eg raspberry or petunia), at least one being of heterologous source. It may further comprise a coding sequence BAR (eg raspberry), C4H and PAL. In the examples, this document reports: the production of benzalacetone and naringenin from coumaric acid in E. coli transformed with CHS and 4CL; the construction of a CHS mutated protein (CHS *) presumed to have a higher BAS activity; the production of benzalacetone and frambinone (14.2 / 0.3 μg in 50 ml) from coumaric acid in E. coli having endogenous BAR activity and transformed with CHS / CHS * and 4CL; - the production of benzalacetone from coumaric acid in S. cerevisiae (with endogenous BAR activity) transformed with CHS and 4CL. No value is given in relation to frambinone. In addition, the mutant CHS * protein appears to be ineffective.
    Similarly, Beekwilder et al. (2007, Biotechnol, J. 2, 1270-79) reports only the successful production of rambinone from coumaric acid in E. coli transformed with CHS and 4CL but with a yield of 0.3 mg / L. No convincing data are reported for production in yeast. Recently, Lee et al. (2016, Microb Cell Factories 15. doi: 10.1186 / sl2934-016-0446-2) demonstrated the synthesis of frambinone from coumaric acid by expressing a 4CL and BAS gene in S. cerevisiae (up to 8 mg / L). In addition, the expression of PAL / TAL and C4H allowed de novo production of frambinone (up to 4 mg / L).
    There is, however, a clear need to develop new technical solutions for efficient production of rambinone.
    DESCRIPTION OF THE INVENTION Definitions
    The definitions below correspond to the meaning generally used in the context of the invention and are to be taken into account, unless another definition is explicitly indicated.
    Within the meaning of the invention, the articles "a" and "an" are used to refer to one or more (for example at least one) units of the grammatical object of the article. For example, "an element" refers to at least one element, that is, one or more elements.
    The terms "approximately" or "approximately", used with reference to a measurable value such as quantity, duration, and other similar values, shall be understood to include measurement uncertainties of ± 20% or ± 10%, preferably ± 5%, still more preferably ± 1%, and particularly preferably ± 0.1% of the specified value.
    Intervals: Throughout the present description, the various features of the invention may be presented as a range of values. It should be understood that the description of values in the form of an interval is only intended to make the reading simpler and should not be interpreted as a rigid limitation of the scope of the invention. Accordingly, the description of a range of values should be considered as specifically disclosing all possible intermediate ranges as well as each of the values within that range. For example, the description of a range from 1 to 6 should be considered as specifically describing each of the ranges it includes, such as ranges from 1 to 3, 1 to 4, 1 to 5, 2 to 4, from 2 to 6, from 3 to 6, etc., as well as each of the values in this range, for example, 1, 2, 2,7, 3, 4, 5, 5,3 and 6. is worth regardless of the range of the interval.
    The term "isolated" should be understood in the context of the invention as synonymous with removed or extracted from its environment or natural state. For example, an isolated nucleic acid or peptide is a nucleic acid or a peptide extracted from the natural environment in which it is usually found, whether it is a plant or a live animal, for example. Thus, a nucleic acid or a peptide naturally present in a living animal is not a nucleic acid or an isolated peptide within the meaning of the invention, whereas the same nucleic acid or peptide, partially or completely separated from the other elements present in its natural context is "isolated" within the meaning of the invention. An isolated nucleic acid or peptide may exist in a substantially purified form, or may exist in a non-native environment such as, for example, a host cell.
    In the context of the invention, the following abbreviations are used for the most common nucleic acid bases. "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.
    Unless otherwise indicated, within the meaning of the invention, a "nucleotide sequence encoding an amino acid sequence" refers to all the nucleotide sequences that encode the amino acid sequence, including degenerate nucleotide sequences allowing for obtain said amino acid sequence. The nucleotide sequence that encodes a protein or RNA or cDNA may optionally include introns.
    The terms "coding" or "coding for", "code" or "code for" refer to the property inherent in the specific nucleotide sequences in a polynucleotide, such as a gene, cDNA or mRNA, to serve as a template. for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (eg, rRNA, tRNA and mRNA), or a defined sequence of amino acids, and the resulting biological properties . Thus, a gene encodes a protein if the transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and which is generally described in the sequence and database listings, the non-coding strand, used as a template for the transcription of a gene or cDNA, may be referred to as coding for the protein or other product of that gene or cDNA.
    The term "polynucleotide" as used in the context of the invention is defined as a chain of nucleotides. In addition, the nucleic acids are nucleotide polymers. Thus, the terms nucleic acids and polynucleotides as used in the context of the invention are interchangeable. It is well known in the field of molecular biology and genetic engineering that nucleic acids are polynucleotides, which can be hydrolyzed to monomers. Nucleotides in monomeric form can be hydrolyzed to nucleosides. As used in the context of the invention, the term polynucleotide refers, without limitation, to any type of nucleic acid molecule, that is to say nucleic acid molecules obtainable by any means available in the art, including by recombinant means, namely the cloning of nucleic acid sequences from a recombinant library or the genome of a cell, using standard cloning technologies such as PCR, or by synthesis.
    Within the meaning of the invention, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound consisting of amino acid residues covalently linked by peptide bonds. A protein contains by definition at least two amino acids, without limitation as to the maximum number of amino acids. The polypeptides interchangeably include several peptides and / or proteins, which themselves comprise two or more amino acids connected to each other by peptide bonds. As used herein, the term refers to both short chains, which are also commonly referred to in the art as peptides, oligopeptides and oligomers for example, and longer chains, which are generally designated in the art as proteins, of which there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, among others . The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
    The terms "homologous" and "identical" refer to sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in each of the two compared sequences is occupied by the same amino acid base or subunit monomer (for example, when a position in each of the two DNA molecules is occupied by an adenine), then the molecules are homologous or identical for this position. The percentage of identity between two sequences is a function of the number of corresponding positions shared by the two sequences, and corresponds to this number divided by the number of positions compared and multiplied by 100. For example, if 6 out of 10 positions in two sequences matched are identical, so the two sequences are 60% identical. In general, the comparison is made by aligning the two sequences so as to give maximum homology / identity.
    A "vector" within the meaning of the invention is a molecular construct which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid into a cell. Many vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" designates, for example, an autonomously replicating plasmid or a virus. The term should also be understood to include non-plasmid or non-viral compounds that facilitate the transfer of nucleic acids into cells, such as, for example, polylysine compounds, liposomes, and the like.
    The term "expression vector" denotes a vector comprising a recombinant polynucleotide, which comprises expression control sequences operably linked to a nucleotide sequence to be expressed. An expression vector comprises, in particular, expression elements acting in cis; other elements for expression that may be provided by the host cell or by an in vitro expression system. Expression vectors within the meaning of the invention include all those known in the art, such as cosmids, plasmids (for example naked or contained in liposomes) and viruses (for example lentiviruses, retroviruses, adenovirus and adeno-associated viruses) that incorporate the recombinant polynucleotide.
    The term "promoter" as used herein is defined as a DNA sequence recognized by the cell synthesis machinery, or introduced synthetic machinery, necessary to initiate the specific transcription of a polynucleotide sequence.
    Within the meaning of the invention, the term "promoter / regulatory sequence" denotes a nucleic acid sequence necessary for the expression of the polynucleotide operably linked to the promoter / regulatory sequence. In some cases, this sequence may be the promoter base sequence, while in other cases this sequence may also include an activator sequence and other regulatory elements useful for polynucleotide expression. The promoter / regulatory sequence may be, for example, a sequence allowing the expression of the polynucleotide which is specific for a tissue, that is to say preferably occurring in this tissue.
    Within the meaning of the invention, a "constitutive" promoter is a nucleotide sequence which, when operably linked to a polynucleotide, leads to expression of the polynucleotide in most or all physiological conditions of the cell.
    Within the meaning of the invention, an "inducible" promoter is a nucleotide sequence which, when operably linked to a polynucleotide, leads to expression of the polynucleotide only when a promoter inducer is present in the cell.
    DETAILED DESCRIPTION OF THE INVENTION
    The present invention relates to a genetically modified fungal microorganism for the production of rambinone, said microorganism having the following characteristics: the ability to produce rambinone from tyrosine; and a low capacity or inability to degrade tyrosine to tyrosol, p-hydroxyphenyl acetaldehyde and / or p-hydroxyphenyl acetate.
    In the context of the invention, the expression "genetically modified microorganism" means that the microorganism according to the invention is not found in nature and is modified by introduction of new genetic elements and / or by deletion or modification of the genetic elements. endogenous microorganisms. Such a microorganism may be subjected to selection pressure by combining site-directed mutagenesis and culturing in the selection medium.
    In the remainder of the presentation and for reasons of simplification, the term "genetic element" is used in a manner equivalent to "gene" or "sequence". It is therefore a nucleic acid sequence, which can have any type of functionality. It may, for example, correspond to a coding sequence (in particular coding an enzyme of the synthesis or degradation pathway of interest), or a regulatory sequence, in particular a promoter or a terminator. In particular when it is a coding sequence, it can be optimized, that is to say modified to integrate the preferred codons of the host, in this case the fungal microorganism, in which this sequence is to express. According to another preferred embodiment, only the coding sequence of the gene of interest or ORF (for "Open Reading Frame") is isolated and implemented.
    According to a first aspect, the novel genetic elements introduced into the microorganism targeted by the invention are so-called exogenous or heterologous genetic elements, which may be of a synthetic nature or from other organisms (or sources). In particular, a microorganism can express exogenous or heterologous genes if they are introduced into said microorganism with all the elements allowing their expression in the host microorganism.
    According to another embodiment, the endogenous genes may be modified to modulate their expression and / or their activity, for example by introducing mutations into the coding sequences in order to modify the gene product or by modifying the regulatory sequences, for example by introducing heterologous sequences in addition to or instead of the endogenous regulatory sequences. The modulation of the endogenous genes may result in overexpression or an increase in the activity of the endogenous gene product or, conversely, a decrease in the expression or activity thereof.
    In addition, supernumerary or additional copies of an endogenous gene may also be introduced into the microorganism, thereby increasing the level of expression and thus the activity of the product encoded by the gene.
    Techniques for introducing DNA into a host (or transformation) are well known to those skilled in the art and include permeabilization membranes by applying an electric field (electroporation), thermally (application of a thermal shock) or chemically, for example using lithium acetate.
    The introduced genetic elements can be integrated into the genome of the host, in particular by homologous recombination or chromosomal integration, advantageously using integrative cassettes, or expressed extrachromosomally using plasmids or vectors. Different types of plasmids, advantageously self-replicating, are well known to those skilled in the art, which differ in particular by their origin of replication, their promoter (inducible or constitutive), their marker (for example a resistance to an antibiotic or the ability to grow in a selective medium) and the number of copies per cell.
    Advantageously, in a fungal microorganism, the chromosomal integration of genes, in particular expression cassettes bearing heterologous genes or supernumerary copies of endogenous genes, is done by the so-called modular cassette integration technique ("modular cassette integration"). technical "). According to a particular embodiment, the integration of the gene or genes is at the HO locus. In the case of integration of several cassettes, these are chosen to have ends having homologous sequences, called recombination regions (RR), allowing homologous recombination and integration in the desired order and at the desired position. different cassettes. Advantageously, one of the cassettes called a "marker cassette" encodes a marker, for example an antibiotic resistance or the ability to grow in a selective medium, to select or identify microorganisms in which the chromosomal integration actually took place. Advantageously, an expression cassette comprises the coding or ORF part of a gene of interest, in particular enzymes involved in the raspinone biosynthesis pathway from tyrosine, placed under the control of regulatory sequences, advantageously at the level of minus a promoter and a terminator, which may be the native regulatory sequences of this gene or heterologous sequences chosen for their functionality and / or efficacy in the host microorganism.
    As regards the inactivation of endogenous genes, this can be carried out by introducing, for example by homologous recombination at the level of the target gene, a cassette, either at the level of the regulatory regions thus inhibiting the expression of the gene, or at the level of the coding sequence resulting in inactivation of the gene product. According to a particular embodiment, the cassette is a marker cassette, advantageously comprising a dominant marker gene under the control of a promoter and a terminator. Even more advantageously, said cassette comprises at its 5 'and 3' ends regions homologous to the 5 'and 3' regions of the targeted gene, for example 5 'corresponding to the promoter of the target gene and corresponding in 3' to the terminator of the targeted gene. In addition, the cassette may contain loxP sites for excision of this genome cassette through the action of the recombinase cre.
    In the context of the invention, suitable markers are genes conferring resistance to antibiotics, which are then introduced into the culture medium of the genetically modified microorganism to ensure the selection and maintenance of the genetic modification. Many markers are available to those skilled in the art, for example: the kanMX4 gene conferring resistance to geneticin (or G418); the hphNT1 gene conferring resistance to hygromycin B; the bsd gene conferring resistance to blasticidin; the ble gene conferring resistance to phleomycin.
    The proper introduction and functionality of the desired genetic modifications can be verified by any technique known to those skilled in the art, including: selection by virtue of the marker (s) present in the expression cassette or in the vector ; targeting of the introduced genetic element, for example by sequencing, by PCR ("Polymerase Chain Reaction") or by hybridization ("Southern blot" or "Northern blot"); targeting the product of the targeted gene, for example by immunological detection ("Western blot") or by measuring the activity, for example enzymatic, associated.
    As already stated, important elements for controlling gene expression are the promoters, placed upstream of the coding sequence whose expression is governed by the promoter. Thus, the genes can be expressed using inducible or constitutive variable force promoters. According to a particular embodiment, the promoters used in the context of the invention are constitutive promoters. These promoters may be homologous or heterologous. In the context of the invention, promoters commonly used by those skilled in the art are, for example: the promoter of the S. cerevisiae TDH3 gene, for example that of sequence SEQ ID NO: 1; the promoter of the S. cerevisiae PFK2 gene, for example that of sequence SEQ ID NO: 4; the promoter of the PGI1 gene of S. cerevisiae, for example that of sequence SEQ ID NO: 7; the promoter of the S. cerevisiae PMA1 gene, for example that of sequence SEQ ID NO: 10; the promoter of the S. cerevisiae PYK1 gene, for example that of sequence SEQ ID NO: 13; the promoter of the TEF1 gene of Ashbya gossypii, for example that of sequence SEQ ID NO: 15 or N cerevisiae. Other important elements for controlling gene expression are the terminator sequences, also called terminators, placed downstream of the coding sequence to be expressed. Again, they may be homologous terminators, from the microorganism in question, or heterologous, namely artificial sequences or terminators from a source other than the host microorganism. Many terminator sequences are available and known to those skilled in the art, such as, for example: the terminator of the Saccharomyces cerevisiae CYC1 gene, for example that of sequence SEQ ID NO: 3; the terminator of the S. cerevisiae PFK2 gene, for example that of sequence SEQ ID NO: 6;
    the terminator of the PGI1 gene of S. cerevisiae, for example that of sequence SEQ ID NO: 9; the terminator of the S. cerevisiae ZWF1 gene, for example that of sequence SEQ ID NO: 12; the terminator of the S. cerevisiae PYK1 gene, for example that of sequence SEQ ID NO: 14; the terminator of the TEF1 gene of Ashbya gossypii, for example that of sequence SEQ ID NO: 17.
    In the context of the invention, the term "fungal microorganism" advantageously designates a yeast or a fungus.
    In the context of the invention, the term "fungal microorganism" is understood as a "fungal microorganism strain". Indeed, and advantageously, the genetically modified fungal microorganism, object of the present invention, is obtained from an isolated strain and at least partially characterized. As an illustration and in connection with yeasts, "yeast" means a commercial product obtained through the implementation of a method for producing a yeast strain. Thus, yeasts having different characteristics can be obtained from the same strain, these differences being related to the production method used.
    More specifically, the invention relates to a yeast strain, in other words a strain belonging to the ascomycete phyla or basidiomycetes. Advantageously, the strain belongs to the genus Saccharomycetales, even more advantageously to the families of Debaryomycetaceae, Dipodascaceae or Saccharomycetaceae. According to a preferred embodiment, the strain belongs to the genera Yarrowia, Debaryomyces, Arxula, Scheffersomyces, Geotrichum, Pichia or Saccharomyces. It may for example be species Yarrowia lipolytica, Debaryomyces hansenii or Saccharomyces cerevisiae.
    According to a particular embodiment, the strain used for the construction of a strain according to the invention or for the implementation of a method according to the invention is a so-called industrial strain, as opposed to a so-called strain of laboratory. The so-called industrial yeast strains are those capable of being produced using industrial substrates as a carbon source. By way of example, said carbon source may be sugar cane molasses or beetroot.
    According to an advantageous embodiment, the microorganism is chosen in connection with at least one of the following characteristics: an at least partial capacity to produce rambinone from tyrosine; and / or - a low capacity or inability to degrade tyrosine to tyrosol, p-hydroxyphenyl acetaldehyde and / or p-hydroxyphenyl acetate.
    Concerning the first characteristic, the microorganism that will be genetically modified may have at least one endogenous enzymatic activity involved in the conversion of tyrosine to rambinone. According to a particular embodiment, it is capable of converting 4-hydroxy benzalacetone to rambinone and thus naturally has a BAR activity, as detailed below.
    The microorganism may be chosen for other characteristics of interest, particularly in view of its application in biotechnology (knowledge of the genome, tools available for genetic manipulation, etc.), its metabolism (especially respiratory and lipidic promoting production malonyl-CoA or acetyl-CoA, co-substrates of the synthetic route of rambinone), or conditions of its industrial exploitation (aerobic growth, tolerance to stress and toxic compounds, etc.).
    According to a particular embodiment, a microorganism of interest is chosen from the following list: Beauveria bassiana, Candida boidinii, Galactomyces candidum (Geotrichum candidum), Kloeckera saturnus, Kodamaea ohmeri (Pichia ohmeri), Komagataella pastoris (Pichia pastoris), Mucor nederlandicus (Mucor subtilissimus), Pichia membranifaciens, Schwanniomyces etchellsii (Pichia etchellsii), Torulaspora delbrueckii (Saccharomyces fermentait), Wickerhamomyces anomalus (Hansenula anomala), Yarrowia lipolytica (Candida lipolytica), Saccharomyces cerevisiae, Debaryomyces hansenii. Advantageously, it is Saccharomyces cerevisiae, Yarrowia lipolytica or Debaryomyces hansenii.
    Fungal microorganisms of particular interest are, for example, Yarrowia lipolytica or Debaryomyces hansenii.
    A microorganism according to the invention is intended to produce frambinone from tyrosine. Advantageously, such a microorganism has an improved ability to produce rambinone from tyrosine, especially with respect to the strains already described or with respect to this same microorganism that has not been genetically modified.
    It is known to produce rambinone from other substrates but from an economic point of view in particular, tyrosine is of obvious interest. In addition, it has been shown that the addition of tyrosine in the culture medium, advantageously a fermentation medium, was possible in terms of solubility and did not exhibit any known toxicity. Suitably and as will be seen in more detail in connection with the process according to the invention, the production of frambinone is from exogenous tyrosine to the microorganism according to the invention, preferably by addition in its culture medium. Typically, the concentration of tyrosine in the culture medium is greater than or equal to 50 mg / l, or even greater than or equal to 100, 150, 200, 250, 300, 350, 400 or even 450 mg / l. In addition, it is advantageously less than or equal to 1 g / L, or even less than or equal to 950, 900, 850, 800, 750, 700, 650, 600, 550, 500 or even 450 mg / L.
    According to another aspect, the invention also relates to the use of a microorganism as defined in the context of the present application for the production of rambinone from tyrosine. In the context of the invention, it is expected that the levels of production of rambinone by a microorganism that have not yet been reached, advantageously a concentration in the culture medium of the microorganism greater than 4 mg / L, more advantageously greater than or equal to 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or even 20, 25 or 30 mg / L. At these concentrations and under the conditions of the process according to the invention, the rambinone remains soluble and shows no toxicity with respect to the microorganisms according to the invention.
    According to a first characteristic, a microorganism according to the invention or used in a process according to the invention is genetically modified to exhibit an ability to produce or synthesize rambinone from tyrosine, or even to have an improved ability to produce or synthesize rambinone from tyrosine.
    According to the invention, the microorganism can naturally have an ability to synthesize frambinone from tyrosine and the genetic modifications made are intended to improve this capacity. Alternatively, the microorganism does not naturally have the capacity to synthesize frambinone from tyrosine and the genetic modifications made are intended to confer this capacity.
    As a reminder and as illustrated in FIG. 1, a route of synthesis of rambinone from tyrosine involves 4 enzymatic activities, namely: a tyrosine ammonia lyase activity (EC 4.3.1.23), denoted TAL, capable of deamminating tyrosine to form coumaric acid; a 4-coumarate: CoA ligase activity (EC 6.2.1.12), denoted 4CL, capable of catalyzing the grafting of a Coenzyme A (CoA) molecule on coumaric acid to form coumaroyl-CoA; a benzalacetone synthase activity (EC 2.3.1.212), denoted BAS, capable of converting coumaroyl-CoA to 4-hydroxybenzalacetone in the presence of malonyl-CoA as a co-substrate; a benzalacetone reductase activity (EC 1.3.1.x), labeled BAR, for reducing 4-hydroxybenzalacetone to frambinone
    According to an advantageous embodiment, the microorganism according to the invention is genetically modified to ensure or improve the production of rambinone from tyrosol. Advantageously, the genetic modifications made make it possible to increase at least one of the four enzymatic activities mentioned above. It may for example be: mutations in the coding part of the gene making it possible to obtain an enzyme having the desired activity or having an improved activity, particularly in terms of specificity and affinity of the substrate; modification of the regulatory sequences making it possible to increase the level of expression of an endogenous gene encoding an enzyme exhibiting the desired activity; - Providing a copy (s) sum (s) of an endogenous gene encoding an enzyme having the desired activity, having possibly been mutated to improve the activity; the provision of one or more copies of at least one heterologous gene (artificial or derived from another source organism) encoding an enzyme having the desired activity, placed under the control of appropriate regulatory sequences to produce said enzyme .
    Concerning TAL, the enzymes of the family of lyases having a predominant activity for tyrosine (tyrosine ammonia lyase, EC 4.3.1.23) are rare. Often, it has an affinity for the phenylalanine substrate at least equal to or even greater (phenylalanine / tyrosine ammonia lyase, PAL / TAL, EC 4.3.1.25).
    In certain cases, it may be desirable to choose a TAL enzyme which has no or very little PAL activity so as to avoid the accumulation of the cinnamic acid intermediate which may have some toxicity. In the case where the selected TAL enzyme has a notorious PAL activity, it may be advantageous to ensure that the microorganism further has a C4H and / or CPR activity, and possibly to modify it to introduce sequences coding for these enzymatic activities. useful for the conversion of cinnamic acid into coumaric acid. Alternatively, it may be envisaged to mutate the TAL coding sequence to reduce the possibly associated PAL activity. In addition, a cinnamic acid buildup may exert a negative feedback on TAL so that a balanced expression of 4CL may be required. It should be noted that enzymatic tests to evaluate the TAL and PAL activities are well known to those skilled in the art and are described, for example, in the documents Berner et al. (J. Bacteriol., 2006, 188, 2666-73), Kyndt et al. (FEBS Lett., 2002 512, 240 ^ 14) and Rosier et al. (Plant Physiol., 1997, 113, 175-79).
    Genes of interest that can be used in the context of the invention are listed in Table 1 below:
    According to a particular embodiment, the TAL protein encoded by the gene introduced into the microorganism according to the invention has the sequence SEQ ID NO: 18, or a protein sequence exhibiting at least 70%, or even 80, 85, 90, 95 or even 99% homology or identity with the sequence SEQ ID NO: 18 and having a TAL activity.
    According to another particular embodiment, the sequence coding for the TAL protein, introduced into the microorganism according to the invention, has the sequence SEQ ID NO: 5, or a sequence presenting at least 60%, or even 70, 80, 85, 90 , 95 or even 99% identity with the sequence SEQ ID NO: 5. Its expression can be placed under the control of the regulatory sequences SEQ ID NO: 4 and / or SEQ ID NO: 6.
    Concerning 4CL, it is a priori an enzyme not present in the fungal microorganisms targeted by the present invention. Thus, and advantageously, at least one copy of at least one heterologous gene is introduced. Note that enzymatic tests to evaluate the 4CL activity are well known to those skilled in the art and are for example described in the documents Ehlting et al. (Plant J., 1999, 19, 9-20), Knobloch and Hahlbrock (Arch Biochem Biophys., 1977, 184, 237-48) and Lee and Douglas (Plant Physiol., 1996, 112, 193-205). .
    The majority of 4-coumarate: CoA ligases (4CL; EC6.2.1.12) are found in plants. Genes of interest that can be used in the context of the invention are listed in Table 2 below:
    According to a particular embodiment, the 4CL protein encoded by the gene introduced into the microorganism according to the invention has the sequence SEQ ID NO: 19, or a protein sequence exhibiting at least 70%, or even 80, 85, 90, 95 or even 99% homology or identity with the sequence SEQ ID NO: 19 and having a 4CL activity.
    According to another particular embodiment, the sequence encoding the 4CL protein, introduced into the microorganism according to the invention, has the sequence SEQ ID NO: 8, or a sequence exhibiting at least 60%, or even 70, 80, 85, 90 , 95 or even 99% identity with the sequence SEQ ID NO: 8. Its expression can be placed under the control of the regulatory sequences SEQ ID NO: 7 and / or SEQ ID NO: 9. Note that the accumulation of coumaryl-CoA generated by 4CL can cause the undesirable formation of phloretic acid and inhibit TAL activity. It is therefore important that the microorganism according to the invention has a BAS activity (catalyzing the next step in the route of synthesis of rambinone) adapted.
    Concerning BAS, it is a priori an enzyme not present in the fungal microorganisms targeted by the present invention. Thus, and advantageously, at least one copy of at least one heterologous gene is introduced. It should be noted that enzymatic tests for evaluating the BAS activity are well known to those skilled in the art and are for example described in the documents Abe et al. (Eur J. Biochem., 2001, 268, 3354-59 and Morita et al., 2010). Morita et al. (Acad Sci., 2010, 107, 669-673).
    Benzalacetone synthases (BAS, EC2.3.1.212) are part of the family of PKS (PolyKetone Synthase), which also includes chalcone synthases (CHS, for example involved in the synthesis of naringenin) and stilbene synthases (STS; involved for example in the synthesis of resveratrol). These enzymes accept coumaroyl-CoA and other substrates, and catalyze condensation with malonyl-CoA. Malonyl-CoA is an intermediate in the synthesis of fatty acids and its formation requires ΓΑΤΡ. While the CHS and STS add three malonyl-CoA units, BAS adds only one. However, the described BAS enzymes also have CHS activity. Genes of interest that can be used in the context of the invention are listed in Table 3 below:
    According to a particular embodiment, the BAS protein encoded by the gene introduced into the microorganism according to the invention has the sequence SEQ ID NO: 20, or a protein sequence exhibiting at least 70%, or even 80, 85, 90, 95 or even 99% homology or identity with the sequence SEQ ID NO: 20 and having a BAS activity.
    According to another particular embodiment, the sequence coding for the BAS protein, introduced into the microorganism according to the invention, has the sequence SEQ ID NO: 11, or a sequence presenting at least 60%, or even 70, 80, 85, 90 , 95 or even 99% identity with the sequence SEQ ID NO: 11. Its expression may be placed under the control of the regulatory sequences, in particular a promoter of sequence SEQ ID NO: 10 or SEQ ID NO: 13 and / or a terminator of sequence SEQ ID NO: 12 or SEQ ID NO: 14.
    According to another particular embodiment, at least two BAS coding sequences, for example two copies of the same sequence, are introduced into the microorganism according to the invention. Note that they can be placed under the control of different regulatory sequences.
    The final step in the synthesis pathway of rambinone is the reduction of the α, β double bond to p-hydroxy benzalacetone, which requires NADPH, catalyzed by benzalacetone reductase (BAR, EC 1.3.1.x). Only two enzymes with this activity have been identified to date. However, some microorganisms have been reported to have endogenous BAR activity such as E. coli and S. cerevisiae (Beckwilder et al., 2007, Biotechnol, J. 2, 1270-79). The first enzyme is described in GB 2 416 769: a 309 amino acid protein was isolated from raspberry protein fractions having BAR activity. It has homology with isoflavone reductases (EC 1.3.1.45) and is capable of converting p-hydroxy benzalacetone to rambinone in in vitro assays with purified enzyme. In 2011, Koeduka et al. (Biochem Biophys Res Commun 412, 104-108) have identified a ketone / zingerone synthase of R. idaeus that has BAR activity (RiRZS1, Uniprot G1FCG0). The purified protein effectively converts p-hydroxy benzalacetone to rambinone in an enzyme test. It should be noted that enzymatic tests for evaluating the BAR activity are well known to those skilled in the art and are for example described in the document Koeduka et al. (Biochem Biophys Res Commun, 2011, 412, 104-108).
    According to a particular embodiment, the BAR protein is encoded by a gene introduced into the microorganism according to the invention and it has the sequence SEQ ID NO: 21, or a protein sequence exhibiting at least 70%, or even 80, 85, 90 , 95 or even 99% homology or identity with the sequence SEQ ID NO: 21 and having a BAR activity.
    According to another particular embodiment, the sequence encoding the BAR protein, introduced into the microorganism according to the invention, has the sequence SEQ ID NO: 2, or a sequence exhibiting at least 60%, or even 70, 80, 85, 90 , 95 or even 99% identity with the sequence SEQ ID NO: 2. Its expression may be placed under the control of the regulatory sequences, in particular sequences SEQ ID NO: 1 and / or SEQ ID NO: 3.
    According to a particular embodiment, the fungal microorganism according to the invention naturally lacks at least one enzymatic activity among 4CL and BAS, or even 2.
    Advantageously, the fungal microorganism according to the invention comprises at least one heterologous sequence encoding the enzyme 4-coumarate: CoA ligase (4CL) or benzalacetone synthase (BAS), advantageously the 4CL and BAS enzymes.
    According to a preferred embodiment, said sequence encodes a 4CL enzyme having the sequence SEQ ID NO: 19, or a protein sequence exhibiting at least 70%, even 80, 85, 90, 95 or even 99% homology or homology. identity with the sequence SEQ ID NO: 19 and having a 4CL activity. Preferably, this sequence comprises the sequence SEQ ID NO: 8, or a sequence exhibiting at least 60%, even 70, 80, 85, 90, 95 or even 99% identity with the sequence SEQ ID NO: 8. It may also comprise the sequence SEQ ID NO: 7, advantageously located upstream of the 4CL coding sequence, and / or the sequence SEQ ID NO: 9, advantageously located downstream of the 4CL coding sequence.
    According to another preferred embodiment, said sequence encodes a BAS enzyme having the sequence SEQ ID NO: 20, or a protein sequence exhibiting at least 70%, even 80, 85, 90, 95 or even 99% homology or identity with SEQ ID NO: 20 and having BAS activity. Preferably, this sequence comprises the sequence SEQ ID NO: 11, or a sequence exhibiting at least 60%, even 70, 80, 85, 90, 95 or even 99% identity with the sequence SEQ ID NO: 11. It may also comprise the sequence SEQ ID NO: 10 or SEQ ID NO: 13, advantageously located upstream of the BAS coding sequence, and / or the sequence SEQ ID NO: 12 or SEQ ID NO: 14, advantageously located downstream of the coding sequence BAS.
    According to a particular embodiment, the fungal microorganism according to the invention comprises at least two heterologous sequences encoding the enzyme benzalacetone synthase (BAS), advantageously from the same source, still more advantageously of the same coding sequence but possibly placed under the control different regulatory sequences.
    According to another embodiment, the fungal microorganisms targeted by the present invention do not exhibit at least one of the following activities: TAL, 4CL, BAS and / or BAR.
    In another aspect, the fungal microorganism according to the invention comprises at least one heterologous or supernumerary sequence encoding the enzyme tyrosine ammonia lyase (TAL) or benzalacetone reductase (BAR), advantageously the TAL and BAR enzymes.
    According to a preferred embodiment, said sequence encodes a TAL enzyme having the sequence SEQ ID NO: 18, or a protein sequence exhibiting at least 70%, even 80, 85, 90, 95 or even 99% homology or homology. identity with the sequence SEQ ID NO: 18 and having a TAL activity. Preferably, this sequence comprises the sequence SEQ ID NO: 5, or a sequence exhibiting at least 60%, even 70, 80, 85, 90, 95 or even 99% identity with the sequence SEQ ID NO: 5. It may also comprise the sequence SEQ ID NO: 4, advantageously located upstream of the TAL coding sequence, and / or the sequence SEQ ID NO: 6, advantageously located downstream of the TAL coding sequence.
    According to a preferred embodiment, said sequence encodes a BAR enzyme having the sequence SEQ ID NO: 21, or a protein sequence exhibiting at least 70%, even 80, 85, 90, 95 or even 99% homology or homology. identity with the sequence SEQ ID NO: 21 and having BAR activity. Preferably, this sequence comprises the sequence SEQ ID NO: 2, or a sequence exhibiting at least 60%, or even 70, 80, 85, 90, 95 or even 99% identity with the sequence SEQ ID NO: 2. It may also comprise the sequence SEQ ID NO: 1, advantageously located upstream of the BAR coding sequence, and / or the sequence SEQ ID NO: 3, advantageously located downstream of the BAR coding sequence.
    According to a particular embodiment, the fungal microorganisms targeted by the present invention comprise at least one heterologous sequence encoding the enzymes 4-coumarate: CoA ligase (4CL) and benzalacetone synthase (BAS), as well as at least one heterologous or supernumerary sequence encoding the enzyme tyrosine ammonia lyase (TAL) and benzalacetone reductase (BAR).
    A particular strain of Saccharomyces cerevisiae having these characteristics, and therefore a conversion route from tyrosine to functional rambinone adapted to the targeted applications, is the industrial strain RK4, deposited with the CNCM (National Collection of Cultures of Microorganisms, Pasteur Institute, 25 rue du Docteur Roux, Paris 75724 Cedex 15) dated June 1st, 2016, under the number 1-5101. This was obtained by chromosomal integration, at the HO locus of the strain deposited with the CNCM on September 4, 2008 under the number 1-4071, of expression cassettes encoding these 4 enzymes, as described below. below (Examples of realization).
    In relation to the synthetic route of frambinone, the fungal microorganisms according to the invention may undergo other genetic modifications, such as, for example: any means for introducing or increasing the capacity of the microorganism to synthesize the frambinone from phenylalanine, for example via the introduction of a gene encoding a PAL enzyme. As already stated, PAL enzymes are close to the TAL enzymes described above. Optionally, it can be implemented a TAL enzyme also having a PAL activity. any means for introducing or increasing the capacity of the microorganism to convert cinnamic acid to coumaric acid, for example by introducing a gene encoding a C4H enzyme, optionally in combination with a gene encoding a CPR enzyme; . any means making it possible to increase the capacity of the microorganism to produce malonyl-CoA, for example as reported in Y. lipolytica (Qiao et al., 2015, Metab.Eng .: 29: 56-65) or by the overproduction of Acetyl-CoA carboxylase ACC1 in S. cerevisiae (Shin et al., 2012, Enzyme Microb Technol 51, 211-216).
    Thus, and according to a particular embodiment, the microorganism used in the context of the invention comprises at least one heterologous or supernumerary sequence coding for the enzyme phenylalanine ammonia lyase (PAL) or cinnamate 4-hydroxylase (C4H), advantageously the PAL and C4H enzymes.
    According to a second advantageous characteristic, a fungal microorganism targeted by the invention is affected in its tyrosine degradation pathway as shown in FIG. 2. According to this pathway, called the tyrosine tyrosine degradation pathway in the As a result of the disclosure, tyrosine is converted to p-hydroxyphenyl acetaldehyde, which may persist as it is or may be converted to either tyrosol or p-hydroxyphenyl acetate. Thus and advantageously, a fungal microorganism targeted by the invention has a low capacity or an inability to degrade or convert tyrosine to tyrosol, p-hydroxyphenyl acetaldehyde and / or p-hydroxyphenyl acetate.
    It has been demonstrated, in the context of the present application, that the predominant degradation pathway of tyrosine in the fungal microorganisms of interest was the Tyrosine tyrosine degradation pathway, as illustrated in FIG.
    This ability can be evaluated as described in the "Examples of Achievement" section below, by growing fungal microorganisms potentially of interest for the production of raspinone, genetically modified or not, in the presence of tyrosine and following the production of tyrosol, for example by HPLC. Typically, a microorganism may be of interest if under the conditions described in the experimental part, namely under aerobic fermentation conditions carried out in advantageously mineral medium, for example composed of 1.7 g / l of YNB (Difco ™), 5 g / L of ammonium sulfate, 2.7 g / L of potassium phosphate and 20 g / L of dextrose, and containing tyrosine, advantageously up to 300 mg / L, the amount of tyrosol produced is less than or equal to at 150 mg / l, advantageously less than or equal to 100 mg / l, or even 50, 40, 30, 20 mg / l or even 10 mg / l.
    Alternatively, and as can be seen in FIG. 2, strains of fungal microorganisms of interest can be selected on the basis of their hydroxyphenyl pyruvate decarboxylase (HPPDC) activity, a method of measurement of which is detailed in the embodiment examples below.
    Advantageously, the HPPDC activity in the microorganism according to the invention is less than or equal to 2 × 10 -6 kat per g of protein, advantageously less than or equal to 1 × 10 -6 kat per g of protein, or even to 5 × 10.7 kat per gram of protein. In the context of the invention, it refers to the proteins extracted from the microorganism.
    Thus and according to the invention, the microorganism is selected for its low or no capacity to degrade tyrosine, evaluated for example according to one of the two methods mentioned above. As already stated, this selection of adapted microorganisms can be carried out before or after the genetic modification of said microorganism.
    According to a first embodiment, the microorganism is selected for its natural ability to degrade weakly or not at all tyrosine in tyrosol, in p-hydroxyphenyl acetaldehyde and / or in p-hydroxyphenyl acetate
    Alternatively, a microorganism of interest, in particular for its capacity to synthesize rambinone, is subjected to genetic modifications so as to reduce or even eliminate its ability to degrade tyrosine to tyrosol, p-hydroxyphenyl acetaldehyde and / or p-hydroxyphenyl acetate
    As illustrated in FIG. 2, this can be achieved by inhibiting or inactivating one of the steps ensuring the transformation of tyrosine into tyrosol, p-hydroxyphenyl acetaldehyde and / or p-hydroxyphenyl acetate, in particular the deamination step. , decarboxylation, or even reduction.
    As already stated, various means are available to those skilled in the art for effecting this inhibition or inactivation by genetic modification of the microorganism, in particular the chromosomal insertion of exogenous genetic elements or at the level of the regulatory regions so as to interfere with the expression of the target gene, either at the level of the coding sequence so as to prevent the production of the gene product or to cause the production of a truncated and / or inactive protein. It is also possible to mutate the target gene at the level of critical sequences for its expression or activity.
    According to a particular embodiment, the target gene is inactivated using a cassette capable of expressing a marker (Goldstein and McCusker, 1999, Yeast Chichester Engl, 15, 1541-53, Güldener et al., 2002, Nucleic Acids Res 30, e23, Güldener et al., 1996, Nucleic Acids Res 24, 2519-24, Janke et al., 2004, Yeast 21, 947-62, Sauer, 1987, Mol Cell Biol. 7, 2087-96), at the ends of which the 5 'and 3' regions of the target gene are inserted to allow homologous recombination and to replace the coding portion of the gene with the marker expression cassette.
    According to an advantageous embodiment, the fungal microorganism according to the invention is inactivated at the level of the activity involved in the decarboxylation of hydroxyphenyl pyruvate. In particular, with respect to S. cerevisiae, at least 3 genes have been described as being involved in this activity, namely ARO10, PDC5 and PDC6 (Hazelwood et al., 2008; Appl. Environ. Microbiol. 74, 2259-66). Kneen et al., 2011, FEBS J. 278, 1842-53, Vuralhan et al., 2005, Appl., Environ Microbiol 71, 3276-84, Vuralhan et al., 2003, Appl., Environ Microbiol. , 4534-41). Advantageously, at least one of the genes encoding phenylpyruvate decarboxylase ARO10, pyruvate decarboxylase PDC5 and pyruvate decarboxylase PDC6 is inactivated. According to one particular embodiment, the genes encoding phenylpyruvate decarboxylase ARO10, pyruvate decarboxylase PDC5 and pyruvate decarboxylase PDC6 are inactivated.
    Alternatively, the deaminase (s) involved in the first step of the tyrosine degradation pathway are inactivated. In connection with S. cerevisiae, it may be Aro8 deaminase and / or Aro9 deaminase, or their counterparts in other fungal microorganisms.
    Similarly, the alcohol (s) dehydrogenase (denoted DHA) involved in the third step of tyrosine tyrosine degradation pathway may be targeted.
    Thus, and in a particular aspect, the present invention is directed to a fungal microorganism comprising at least one mutation or deletion in at least one of the genes encoding the following enzymes: Aro8 deaminase, Aro9 deaminase, ArolO decarboxylase, Pdc5 decarboxylase, Pdc6 decarboxylase, alcohol dehydrogenase ( ADH). In the context of the invention, said mutations and / or deletion result in a reduction or even a suppression of the capacity of the microorganism to degrade tyrosine to tyrosol, p-hydroxyphenyl acetaldehyde and / or p-hydroxyphenyl acetate. Advantageously, said microorganism also has an ability to synthesize frambinone from tyrosine, possibly through the introduction of genetic modifications as described above.
    In another aspect, the present invention relates to the use of a fungal microorganism having the ability to produce raspinone from tyrosine and a low capacity or inability to degrade tyrosine to tyrosol, p-hydroxyphenyl acetaldehyde and / or or p-hydroxyphenyl acetate, advantageously as described above, for the production of rambinone, advantageously by aerobic fermentation.
    In other words, the invention also relates to a process for producing rambinone comprising culturing a fungal microorganism having the ability to produce raspinone from tyrosine and a low capacity or inability to degrade tyrosine to tyrosol , p-hydroxyphenyl acetaldehyde and / or p-hydroxyphenyl acetate, advantageously as described above, in a medium comprising tyrosine. According to one particular embodiment, the tyrosine is added to the culture medium at a concentration of between 50 and 450 or 500 mg / l, for example of the order of 300 mg / l. Optionally, the culture medium may also be supplemented with coumaric acid and / or phenylalanine.
    Alternatively, the microorganism used in the context of the invention naturally has a certain capacity to degrade tyrosine tyrosol, p-hydroxyphenyl acetaldehyde and / or p-hydroxyphenyl acetate, but this pathway of degradation is inhibited by the addition of a repressor of this pathway, for example an inhibitor of one of the enzymes involved in this pathway.
    Thus and according to another aspect, the invention is directed to a process for producing rambinone comprising culturing a fungal microorganism having the capacity to produce rambinone from tyrosine in a medium comprising tyrosine and at least one repressor of the tyrosine degradation pathway to tyrosol, p-hydroxyphenylacetaldehyde and / or p-hydroxyphenylacetate. Optionally, it is a genetically modified microorganism to confer or increase the ability of said microorganism to produce rambinone from tyrosine, as described above. According to a particular embodiment, the microorganism used is the industrial strain Saccharomyces cerevisiae RK4, deposited with the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris Cedex 15) dated 1st of June 2016 under the number 1-5101.
    According to a particular embodiment, the tyrosine degradation repressor tyrosol, p-hydroxyphenyl acetaldehyde and / or p-hydroxyphenyl acetate is chosen from glutamate, glutamine or one of their derivatives, advantageously glutamate . According to a particular embodiment, this repressor, in particular glutamate, is added to the culture medium at a concentration greater than or equal to 0.5 g / L, or even 1, 2 or 3 g / L, for example order of 2 g / L.
    In the context of the invention, the fungal microorganism is cultured under conditions favoring the production of rambinone. Particularly suitable conditions of culture are the following: aerobically; in a growth medium allowing fermentation, advantageously a mineral medium, for example composed of 1.7 g / l of YNB (Difco ™), 5 g / l of ammonium sulphate, 2.7 g / l of potassium phosphate and 20 g / L dextrose; - for a duration ranging from several hours to several days; at a pH of between 5 and 7, for example equal to 6; at a temperature between 25 ° C and 32 ° C, for example equal to 30 ° C.
    As already stated and under these conditions, it has been observed that frambinone production levels have never been reached, the culture medium having a frambinone concentration advantageously greater than 4 mg / l, more advantageously greater than or equal to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or even 20, 25 or 30 mg / L.
    In a manner known to those skilled in the art, the rambinone thus produced can be isolated from the culture medium. The invention will be described in more detail in the exemplary embodiments which follow in support of the appended figures. These examples are provided solely to illustrate the invention and have no limiting scope.
    It is considered that without more precision, the skilled person will be able, in view of the description and examples of implementation implement and use the microorganisms according to the invention and the claimed methods.
    LEGENDS OF FIGURES
    Figure 1: Route of biosynthesis of rambinone (1) L-tyrosine (2) p-coumaric acid (3) coumaroyl-CoA (4) p-hydroxy benzalacetone (5) frambinone (6) malonyl-CoA (7) phenylalanine (4) 8) cinnamic acid TAL: tyrosine ammonia lyase, 4Cl: 4-coumarate-CoA ligase, BAS: benzalacetone synthase, BAR: benzalacetone reductase, PAL: phenylalanine ammonia lyase, C4H: cinnamate 4 hydroxylase
    Figure 2: Tyrosine degradation pathway
    It consists of deamination (Aro8 / 9) and decarboxylation (Arol0 / Pdc5 / Pdc6) steps, resulting in the formation of p-hydroxyphenyl acetaldehyde. This can be reduced using an alcohol dehydrogenase (ADH) tyrosol or oxidized p-hydroxyphenyl acetate. A possible inactivation of the way is indicated by a cross. Figure 3: Diagram of the modified HO locus in the genome of S. cerevisiae strain RK4
    Five gene expression cassettes and a marker cassette were integrated using different overlapping recombination regions (RR1-5) between the cassettes for in vivo assembly.
    Figure 4: Production of rambinone by S. cerevisiae strain RK4 after 7 days
    The concentration of frambinone (in mg / L) was determined after 7 days of culture depending on the substrate (tyrosine synthesized by the cell from glucose and ammonium sulphate (called de novo), tyrosine or coumaric acid added in the culture medium).
    Figure 5: Tyrosol production by S. cerevisiae strain RK4 after 7 days
    The concentration of tyrosol (in mg / L) was determined after 7 days of culture depending on the substrate (tyrosine synthesized by the cell from glucose and ammonium sulphate (so-called de novo), tyrosine or coumaric acid added in the culture medium).
    Figure 6: HDPPC activity in cell extracts (strain S. cerevisiae RK4) after 16 hours of fermentation in a synthetic medium containing tyrosine with or without glutamate The hydroxy-phenylpyruvate activity (HDPPC) (expressed in pkat per g of protein) was determined in the cell extract after 16 hours of fermentation in a synthetic medium containing 0.3 g / L of tyrosine and optionally 2 g / L of glutamate.
    EXAMPLES OF REALIZATION
    The present invention will be further illustrated in connection with a genetically engineered strain of Saccharomyces cerevisiae for expressing 4 heterologous genes encoding the integrated TAL, 4CL, BAS and BAR enzymes at its HO locus and effectively producing rambinone from tyrosine. in a medium supplemented with glutamate. However, this example is in no way limiting. 1 / Material and methods Generation of expression cassettes and recombinant strain
    To synthesize rambinone from tyrosine, it was chosen to express four heterologous genes in Saccharomyces cerevisiae as shown in Table 4 below:
    Table 4: Heterologous Genes Used to Establish a Synthetic Route of Frambinone in S. cerevisiae
    The coding sequences were "codon-optimized" for expression in S. cerevisiae. The corresponding sequences are shown in Table 4 above.
    They have been cloned between individual promoters and terminators to ensure their expression. Five gene expression cassettes were constructed (Table 5 below) and, in addition to a marker cassette, were integrated into the genome of the S. cerevisiae industrial strain deposited with the CNCM on September 4th, 2008. under number 1-4071, at the HO locus using the modular cassette integration technique (Figure 3). The resulting strain, called RK4, was deposited with the CNCM (National Collection of Cultures of Microorganisms, Pasteur Institute, 25 rue du Docteur Roux, Paris 75724 Cedex 15) dated June 1, 2016, under the number 1-5101.
    Table 5: Gene Expression and Marker Cassettes
    * S. cerevisiae with the exception of TEF1 (Ashbya gossypii)
    Ri = Rubus idaeus Rg = Rhodotorula glutinis At = Arabidopsis thaliana Rp = Rheum palmatum
    HPLC measurement of intermediates and products of the way
    Frambinone, tyrosol and other intermediates of the pathway were analyzed and quantified by HPLC. The HPLC device and its parameters are summarized in Table 6 below and allow the separation of rambinone and tyrosol from other compounds. For quantification, a calibration was performed using standard solutions between 0.1 and 100 mg / L. Samples of the yeast cultures were centrifuged (> 15,000xg, 10 min) and the supernatant was filtered through a 0.45 μm filter prior to injection into the HPLC.
    Table 6: HPLC Device and Parameters
    Enzymatic detection of hydroxyphenyl pvruvate decarboxylase (HPPDC) activity To quantify the enzymatic activity of the decarboxylation of hydroxyphenyl pyruvate (HPPDC activity), an enzymatic test coupled with a crude cell extract was developed. The cell extract was prepared from a culture on the night (16h) of the strain of interest in a synthetic medium composed of 1.7 g / L of YNB (Difco ™), 5 g / L of sodium sulfate. ammonium, 2.7 g / L potassium phosphate and 20 g / L dextrose. The medium was further supplemented with 300 mg / L L-tyrosine and optionally different nitrogen sources (eg L-glutamate). Cells were harvested by centrifugation (5000xg, 4 min, 4 ° C) and washed twice in wash buffer (10 mM potassium phosphate, 2 mM EDTA, pH 6.8). The cell pellet was taken up in extraction buffer (100 mM potassium phosphate, 2 mM magnesium chloride, 1 mM DTT, 1x cOmplete ™ proteinase inhibitors, pH 6.8) and the cells were broken with a "FastPrep disruptor" press (with 0.45 mm diameter glass beads, four cycles of 30 s at 6 m / s and 1 min on ice). Cell debris was removed by centrifugation and the supernatant was used as a crude cell extract. The protein concentration was determined using the "Uptima BC Assay Protein Quantification Kit" according to the manufacturer's instructions. The enzymatic assay was performed as described by Kneen et al. (2011, FEBS J. 278, 1842-53), with minor modifications. The test couples the HPPDC reaction (decarboxylation of hydroxyphenyl pyruvate (HPP) to hydroxyphenyl acetaldehyde) with a second reaction (oxidation of hydroxyphenyl acetaldehyde to hydroxyphenyl ethanol / Tyrosol) catalyzed by the auxiliary enzyme alcohol dehydrogenase (ADH). ADH activity leads to a reduction of NADH to NAD + which can be monitored by decreasing absorption at 340 nm in a spectrophotometer. The reaction mixture (1 ml) contained 100 mM potassium phosphate, 1 mM magnesium chloride, 0.5 mM thiamine pyrophosphate, 0.1 mM NADH, 1 U horse liver ADH, 4 mM HPP, and 1 mM crude cell extract equivalent to approximately 200 μg of total protein. The reactions were measured at 32 ° C and pH 6.8. The reaction was initiated by adding the HPP substrate. II / Results 1 / Characterization of the constructed strain:
    Production of rambinone and other metabolites from tyrosine or coumaric acid as a substrate
    The fermentation tests were carried out with the strain RK4 cultivated in a mineral medium, composed of 1.7 g / l of YNB (Difco ™), 5 g / l of ammonium sulphate, 2.7 g / l of potassium phosphate and g / L dextrose. Optionally, the medium may contain 300 mg / L of tyrosine or 100 mg / L of coumaric acid. As proposed by Lee et al. (2016, Microb Cell Factories 15. doi: 10.1186 / sl2934-016-0446-2), fermentation tests were conducted under aerobic conditions to optimize frambinone production.
    As shown in Figure 4, the constructed strain can synthesize about 6 mg / L of frambinone from tyrosine. When coumaric acid is used as a substrate, the concentration of rambinone in media is approximately 14 mg / L. These results are in agreement with those of the prior art and confirm that the production of rambinone from aromatic amino acids is less efficient than from coumaric acid. However, taking into account the price of the substrate used for bioconversion, industrial applications are more beneficial when tyrosine is used rather than coumaric acid.
    A final point of these results concerns the de novo synthesis of rambinone by the constructed strain. It should be noted that the concentration of rambinone is more or less the same as that observed in the presence of tyrosine. The hypothesis has been made that this is probably related to the regulation of tyrosine biosynthesis by extracellular tyrosine but also to the diversion of this amino acid by a degradation pathway. 2 / Characterization of the constructed strain: Degradation of tyrosine tyrosol
    Tyrosine degradation to tyrosol is a well-known degradation pathway (Figure 2) (Hazelwood et al., 2008, Appl., Environ Microbiol 74, 2259-66). The first step is a transamination of tyrosine leading to the formation of hydroxyphenyl pyruvate. This compound is then decarboxylated to hydroxyphenyl acetaldehyde (EC: 4.1.1.80). Finally, the aldehyde function is reduced to form a hydroxylated molecule called tyrosol (EC: 1.1.1.90).
    The production of tyrosol during the fermentation of strain RK4 was followed. Figure 5 reveals the tyrosol concentrations in the various media after 7 days of fermentation. The results confirm that most of the tyrosine provided is used for tyrosol production. 3 / Inhibition of tyrosine degradation to Tyrosol
    To reduce the production of tyrosol, it has been decided to inhibit the enzymatic activity involved in the decarboxylation of hydroxyphenyl pyruvate by adding to the fermentation medium compounds which decrease the activity of hydroxyphenyl pyruvate decarboxylase (HPPDC ).
    It was chosen to add glutamate in the media to reduce HPPDC activity. To be sure that the addition of this additional amino acid results in a reduction in enzyme activity, the enzyme test was carried out as described above. Figure 6 shows the activity in the cell extract after culture in the fermentation medium containing tyrosine with or without 2 g / L glutamate. The results confirm the decrease in HPPDC activity in fermenting cells in the presence of glutamate. 4 / Impact of inhibition of tyrosine degradation on frambinone production
    In addition, the production of tyrosol and frambinone was followed under both conditions. The observed tyrosol concentrations demonstrate that reduction of HPPDC activity also reduces tyrosol formation: a 27% reduction in tyrosol concentration was observed in response to glutamate, consistent with the reduction of HPPDC activity. At the same time, frambinone production increased by 40%. These data strongly suggest that reduction of HPPDC activity reduces tyrosol production, thus making tyrosine more available for the rambinone pathway. III / Conclusions:
    In conclusion, the introduction of the rambinone pathway as shown in Figure 1 is sufficient for S. cerevisiae production of rambinone from tyrosine. However, when tyrosine is added to the fermentation medium, the bioconversion is not very efficient and leads to the production of a large amount of co-products, essentially tyrosol as reported above. To reduce the "diversion" of the substrate, the HPPDC activity involved in the tyrosine degradation to tyrosol has been successfully reduced by the addition of an inhibitor of said enzymatic activity. As reported, this has increased the production of rambinone. Alternatively, other strategies can be implemented such as the selection of a microbial strain with very low HPPDC activity, based on a natural genetic background or via the introduction of other genetic modifications such as the deletion of the genes encoding the genes. HPPDC enzymes.
    权利要求:
    Claims (13)
    [1" id="c-fr-0001]
    A genetically modified fungal microorganism for the production of rambinone, said microorganism having the following characteristics: the ability to produce rambinone from tyrosine; and a low capacity or inability to degrade tyrosine to tyrosol, p-hydroxyphenyl acetaldehyde and / or p-hydroxyphenyl acetate.
    [2" id="c-fr-0002]
    2. Microorganism according to claim 1, characterized in that the microorganism is chosen from ascomycetes or basidiomycetes, advantageously of the genus Saccharomy this species, even more advantageously from the family of Debaryomycetaceae, Dipodascaceae or Saccharomycetaceae, preferably of the genus Saccharomycetaceae. Yarrowia, Debaryomyces, Arxula, Scheffersomyces, Geotrichum, Pichia or Saccharomyces, preferentially of the species Yarrowia lipolytica, Debaryomyces hansenii or Saccharomyces cerevisiae.
    [3" id="c-fr-0003]
    3. Microorganism according to one of the preceding claims characterized in that the microorganism comprises at least one heterologous sequence encoding the enzyme 4-coumarate: CoA ligase (4CL) or benzalacetone synthase (BAS), preferably 4CL and BAS enzymes.
    [4" id="c-fr-0004]
    4. Microorganism according to claim 3 characterized in that it further comprises at least one heterologous or supernumerary sequence encoding the enzyme tyrosine ammonia lyase (TAL) or benzalacetone reductase (BAR), preferably TAL and BAR enzymes.
    [5" id="c-fr-0005]
    5. Microorganism according to claim 3 or 4 characterized in that it further comprises at least one heterologous or supernumerary sequence encoding the enzyme phenylalanine ammonia lyase (PAL) or cinnamate 4-hydroxylase (C4H), preferably PAL and C4H enzymes. .
    [6" id="c-fr-0006]
    6. Microorganism according to one of the preceding claims characterized in that it has a hydroxyphenyl pyruvate decarboxylase activity (HPPDC) less than or equal to 2 x 10-6 kat per g of protein, preferably less than or equal to 5 x 10'7 kat per g of protein.
    [7" id="c-fr-0007]
    7. Microorganism according to claim 6, characterized in that it comprises at least one mutation or deletion in at least one of the genes encoding the following enzymes: Aro8 deaminase, Aro9 deaminase, ArolO decarboxylase, Pdc5 decarboxylase, Pdc6 decarboxylase, alcohol dehydrogenase ( ADH), advantageously ArolO, Pdc5 and / or Pdc6.
    [8" id="c-fr-0008]
    8. Use of a fungal microorganism according to one of claims 1 to 7 for the production of frambinone, preferably by aerobic fermentation.
    [9" id="c-fr-0009]
    9. A process for producing rambinone comprising culturing a fungal microorganism according to one of claims 1 to 7 in a medium comprising tyrosine.
    [10" id="c-fr-0010]
    A process for producing rambinone comprising culturing a fungal microorganism having an ability to produce rambinone from tyrosine in a medium comprising tyrosine and a tyrosine tyrosine degradation repressor in p tyrosine hydroxyphenyl acetaldehyde and / or p-hydroxyphenyl acetate.
    [11" id="c-fr-0011]
    11. Process for producing rambinone according to claim 10, characterized in that the repressor is glutamate.
    [12" id="c-fr-0012]
    12. Process for producing rambinone according to claim 10 or 11, characterized in that the microorganism has the characteristics described in claims 2 to 5.
    [13" id="c-fr-0013]
    13. Process for producing rambinone according to one of claims 10 to 12 characterized in that the microorganism is the industrial strain Saccharomyces cerevisiae RK4 deposited with the CNCM (National Collection of Cultures of Microorganisms, Institut Pasteur, 25 rue du Docteur Roux , 75724 Paris Cedex 15) dated June 1st, 2016, under the number 1-5101.
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    同族专利:
    公开号 | 公开日
    WO2017207950A1|2017-12-07|
    US20190309330A1|2019-10-10|
    US20200392545A1|2020-12-17|
    US10793880B2|2020-10-06|
    EP3464556A1|2019-04-10|
    FR3052170B1|2021-01-22|
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    GB2416769A|2004-07-28|2006-02-08|Danisco|Biosynthesis of raspberry ketone|
    EP2957629A1|2014-06-18|2015-12-23|Rhodia Opérations|Improved production of vanilloids by fermentation|
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    GB2416770A|2004-07-28|2006-02-08|Danisco|Synthesis of benzalacetone/raspberry ketone by chalcone synthase|EP3714056A1|2017-11-20|2020-09-30|Axxence Holding B.V.|Production of a flavour compound in a host cell|
    CN108753852B|2018-06-22|2021-10-26|江南大学|Method for preparing raspberry ketone by biological method|
    CN111748534A|2020-08-10|2020-10-09|天津中医药大学|SmC4H protein and construction and expression method thereof|
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1655089A|FR3052170B1|2016-06-03|2016-06-03|PRODUCTION OF FRAMBINONE BY A RECOMBINANT FUNGAL MICROORGANISM|FR1655089A| FR3052170B1|2016-06-03|2016-06-03|PRODUCTION OF FRAMBINONE BY A RECOMBINANT FUNGAL MICROORGANISM|
    EP17732985.1A| EP3464556A1|2016-06-03|2017-06-02|Production of frambinone by a recombinant fungal microorganism|
    PCT/FR2017/051407| WO2017207950A1|2016-06-03|2017-06-02|Production of frambinone by a recombinant fungal microorganism|
    US16/302,527| US10793880B2|2016-06-03|2017-06-02|Production of frambinone by a recombinant fungal microorganism|
    US17/004,863| US20200392545A1|2016-06-03|2020-08-27|Production of frambinone by a recombinant fungal microorganism|
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