process for the production of l-ornithine by fermentation

专利摘要:
METHOD FOR THE PRODUCTION OF L-ORNITINE BY FERMENTATION. The present invention relates to a method for the production of L-ornithine by fermentation using a microorganism, which is characterized by an increased export of the amino acid.
公开号:BR112012024799B1
申请号:R112012024799-9
申请日:2011-03-24
公开日:2020-11-17
发明作者:Wilfried Claes；Robert Gerstmeier
申请人:Evonik Operations Gmbh；
IPC主号:

专利说明:

Prior art
[001] The present invention refers to L-ornithine is known for its stimulatory action in relation to liver function and is often used as an ingredient in medicines and in sports nutrition.
[002] Nowadays, L-ornithine is prepared by several processes. One method is fermentative preparation with the aid of microorganisms. Another method is the alkaline hydrolysis of arginine, for example, with barium hydroxide (CN 1594282 A). Another method is the biotransformation of arginine by immobilized microorganisms that have an arginase activity (KR589121B1). A method of preparing L-ornithine from L-citrulline has also been described in the patent literature (JP 42007767 B4).
[003] Microorganisms that are differentiated by excreting L-ornithine in the culture medium have been described in the literature. Examples of said microorganisms are bacteria of the genera Corynebacterium, Brevibacterium, Bacillus (JP 43010996 B4, JP 57041912 B), Escherichia (US 3668072 A), Providencia (JP 03195494) or Arthrobacter (US 3574061).
[004] L-ornithine-producing microorganisms are often differentiated in that they are auxotrophic for the amino acids L-arginine or L-citrulline (described for Brevibacterium, Bacillus, Corynebacterium in EP 392708 B1 and KR 161147 B1 and for Escherichia in US 366072 THE). In addition, microorganisms have been described that are resistant to 2-thiazol-alanine, sulfaguanidine or 2-fluoropyruvate (Japanese publication open to public inspection No. 61-1119194). EP 0393708 B1 describes L-ornithine producers that are differentiated by less resistance to ornitol and mycophenolic acid. Said properties can also be in a combined form.
[005] The release of basic amino acids like L-lysine, L-arginine and L-ornithine through passive diffusion from the cell is very weak (Bellmann et al. (Microbiology 2001; 147: 1765-74)). This has been well described for lysine, by way of example. Vrlijc et al. (Journal of Bacteriology 1995; 177 (14): 4021-7) studied a plurality of export-deficient Corynebacterium glutamicum mutants. For a mutant, an intracellular concentration of 174 mM L-lysine was measured, while a value of only 0.7 mM was measured extracellularly.
[006] Vrlijc et al. (Molecular Microbiology 1996; 22 (5): 815-26 and Journal of Molecular Microbiology and Biotechnology 1999; 1: 327-336) and EP 0868527 B1 identified and described a new exporter as an exporter of L-lysine (LysE). A defined mutant for defined LysE was no longer able to transport L-lysine out of the cell. The polypeptide encoded by the lysE gene is 233 amino acids or amino acid residues in length and is represented in SEQ ID No. 2. After overexpression of the lysE gene in a lysine producer, an increase in L-lysine excretion was observed.
[007] Von Bellmann et al. (Microbiology 2001; 147: 1765-74) characterized the LysE exporter in more detail with regard to the transport of various basic amino acids in C. glutamicum. The authors demonstrated that the transporter specifically exports the amino acids L-lysine and L-arginine outside the cell. The authors further investigated whether LysE also exports L-ornithine outside the cell. For this purpose, first, a strain of C. glutamicum auxotrophic for L-arginine called ATCC13032 :: argF was prepared.
[008] The strain was grown in 50 ml (batch culture) of a minimal medium called CGXII, which contained 40 g / L of glucose. After a 24-hour incubation period, 60 mM of L-ornithine, corresponding to 7.9 g / L, were measured. Intracellularly, a concentration of L-ornithine of approximately 200 mM was measured in the cells of said strain over an incubation period of approximately 70 minutes. In order to clarify whether LysE also transports L-ornithine out of the cell, the 13032 :: argF strain was transformed with the replicative plasmid pEC7lysE. This measure was aimed at providing the strain with increased LysE activity, thus allowing the strain to transport L-ornithine into the medium at a higher rate of export. However, the said measure did not increase the export rate of L-ornithine. The same export rate (0.6 nmol min1 (mg dry mass) ’1) was determined for the control strain (13032 :: argF) and in the transformant (13032 :: argF, carrying pEC7lysE). From this, the authors concluded that L-ornithine is not exported by the LysE exporter. They further concluded that there must be another unknown export function (export protein) for L-ornithine in Corynebacterium glutamicum (Bellmann et al., 2001, page 1771, figure 5b and page 1772, lines 21 to 28).
[009] A variant LysE (see SEQ ID No. 4) has been identified in C. glutamicum R, which differs from the amino acid sequence of the LysE exporter of strain ATCC 13032, shown in SEQ ID No. 2, by a termination N prolonged by three amino acid residues. The sequence of said amino acid residues is: methionine, valine, isoleucine (MVI). This LysE polypeptide of the R strain was described in EP 1266966 B1 as a variant that differs from wild protein in the formation of a loop region, or more specifically, can no longer form said loop and is therefore capable of carrying out export enhanced L-lysine and L-arginine.
[0010] Another variant of LysE has been described by Gunji and Yasueda (Journal of Biotechnology 127, 2006, 1-13). The authors were interested in the formation of L-lysine by the obligatorily methyltotrophic bacteria, Methylophilus methylotrophus. They transformed M.methylotrophus with a plasmid called pSE that contained the lysE ATCC13869 gene from C. glutamicum in order to improve lysine formation by M. methylotrophus. However, the authors found that they were able to establish only one mutated form of the lysE gene (lysE24) in a stable form in M. methylotrophus. The open reading phase of the lysE gene was displaced in the lysE24 allele due to the insertion of a thymine residue, resulting in the termination of the reading phase after 432 bp. The truncated reading frame encodes a LysE protein that is shorter by 92 amino acid residues at the C-terminus than the wild-type LysE protein from C. glutamicum ATCC13869. It has 141 amino acid residues in length. In addition, the last 6 C-terminal amino acids of the truncated protein (residues 135 to 141) differ from the amino acids in the amino acid sequence of wild LysE. A strain of M. methylotrophus carrying the modified LysE allele in a plasmid (pSE24) was tested for lysine formation. For this, the strain was tested in 0.3 L of a minimum medium called SEIIc in the form of a batch culture fed for 50 hours. The authors observed that the transformer also formed small amounts (0.07 mM corresponding to 11.8 mg / L) of L-ornithine, in addition to 0.55 mM of L-lysine and 0.19 mM of L -arginine. As explained by the authors, this observed formation of L-ornithine is caused by an altered substrate specificity of the mutated transporter or possibly by a set of altered intracellular L-arginine of the strain. EP 1266966 B1 (inventors: Gunji and Yasueda) describe the positive action of the LysE24 transporter on the excretion of L-lysine and L-arginine. Object of the invention
[0011] It is an object of the invention to provide a new process for the fermentative preparation of L-ornithine. Description of the Invention
[0012] The invention relates to a process for the preparation of L-ornithine, characterized by the fact that the following steps are performed: a) fermentation of a bacterium that excretes L-ornithine selected from the group consisting of Corynebacterium, Bacillus, Streptomyces, Arthrobacter and Enterobacteriaceae that overexpress a polynucleotide that encodes a polypeptide that has the activity of an L-ornithine exporter and whose amino acid sequence is at least (>) 35%,> 40%,> 50%,> 55% , £ 60%,> 65%,> 70%,> 75%,> 80%,> 85%,> 90%,> 92%,> 94%,> 96%,> 97%,> 98%,> 99% or 100%, preferably> 70%, particularly, preferably,> 90%, very particularly, preferably £ 96% and, most preferably, 100%, identical to the amino acid sequence of SEQ ID No 2, in a medium, b) accumulation of said L-ornithine in said medium, in which a fermentation broth is obtained, c) in which the plasmid pEC7lysE, deposited in DSM23239, is not used for overexpression, d) and in that the with length of the encoded polypeptide, where appropriate, is> 146 to <286 amino acids or amino acid residues.
[0013] Preference is given to select group length ranges consisting of> 171 to <286,> 196 to <261,> 203 to <258,> 218 to <243,> 228 to <236, and £ 228 to < 233 amino acids or amino acid residues.
[0014] Particular preference is given to ranges of length> 203 to <258,> 218 to <243,> 228 to <236, and> 228 to £ 233, and very particular preference is given to length ranges of £ 228 to < 236 and> 228 to <233.
[0015] When L-ornithine is mentioned later in this document, the term also includes its salts, for example, L-ornithine monohydrochloride or L-ornithine sulfate.
[0016] A process according to the invention makes use of bacteria selected from the group consisting of the genera Corynebacterium, Bacillus, Streptomyces, Arthrobacter and the Enterobacteriaceae family.
[0017] Within the genus Corynebacterium, preference is given to strains based on the following species: Corynebacterium efficiens, for example, the DSM44549 strain, Corynebacterium glutamicum, for example, the ATCC13032 or R strain, and Corynebacterium ammoniagenes, for example, strain ATCC6871, with particular preference being given to the species Corynebacterium glutamicum.
[0018] Some representatives of the species Corynebacterium glutamicum are also known in the prior art under other names. They include, for example: the ATCC13870 strain, called Corynebacterium acetoacidophilum, the DSM20137 strain, called Corynebacterium lilium, the ATCC17965 strain, called Corynebacterium melas- secola, the ATCC14067 strain, called Brevibacterium flavum8, the ATCC869 strain, CE69 strain, CEVAC69, ce69 called Brevibacterium lactofermentum, and the strain ATCC14020, called Brevibacterium divarica- tum.
[0019] The term "Micrococcus glutamicus" for Corynebacterium glutamicum has also been used. Some representatives of the species Corynebacterium efficiens have also been called in the prior art Corynebacterium thermoaminogenes, for example, the FERM strain BP-1539.
[0020] Within the genus Bacillus, preference is given to the species Bacillus subtilis.
[0021] Within the genus Arthrobacter, preference is given to the species Arthrobacter citreus.
[0022] Within the Enterobacteriacae family, preference is given to the genera Escherichia, Erwinia, Providencia, Pantoea and Serratia. Particular preference is given to the genres Escherichia and Serratia. Particular preference is given to the species Escherichia coli in the genus Escherichia, to the species Serratia marcescens in the genus Serratia, and to the species Providencia rettgeri in the genus Providencia.
[0023] The bacteria or strains (initial strains) used for the overexpression measures of the L-ornithine exporter already preferably have the ability to excrete L-ornithine in the nutrient medium around them and accumulate it there. The phrase "to produce" is also used for this later in this document. More specifically, the strains used for said overexpression measures have the ability to concentrate or accumulate in the nutrient medium> 0.1 g / L,> 0.3 g / L,> 1 g / L,> 3 g / L, £ 10 g / L of L-ornithine. The initial strains are preferably strains that have been prepared by mutagenesis and selection, recombinant DNA technologies or a combination of both methods.
[0024] It is obvious, and there is no need for further explanation, that a suitable bacterium for the measurements of the invention can also be obtained by first overexpressing a polynucleotide that encodes a polypeptide that has the activity of an exporter of L-ornithine and whose amino acid sequence is at least (>) 35% identical to SEQ ID No. 2, with the length of the codified polypeptides, where appropriate, being within the length ranges described above, in a wild strain such as, for example , in the strain of Corynebacterium glutamicum ATCC 13032 or in the strain ATCC 14067, and subsequently, causing said bacteria, by additional genetic means described in the prior art, to produce L-ornithine. Transforming a wild strain, such as the strain ATCC13032, ATCC14067, ATCC13869 or ATCC17965, only with the mentioned polynucleotide does not result in a process according to the invention.
[0025] Examples of strains of the species Corynebacterium glutamicum that excrete or produce L-ornithine are: Brevibacterium lactofermentum FERM-BP 2344, and Corynebacterium glutamicum FERM-BP 2345 described in US 5188947.
[0026] An example of a strain of the species Arthrobacter citreus that excretes or produces L-ornithine is: Arthrobacter citreus FERM-BP 2342 described in US 5188947.
[0027] An example of a strain of the species Bacillus subtilis that excretes or produces L-ornithine is: Bacillus subtilis BOR-32 (FERM-P 3647) described in JP 57041912.
[0028] An example of a strain of the species Providencia rettgeri that excretes or produces L-ornithine is: Providencia rettgeri ARGA6 (FERM P-11147) described in JP 03195494.
[0029] An example of a strain of the species Escherichia coli that excretes or produces L-ornithine is: Escherichia coli B-19-19 (ATCC 21104) described in US 3668072.
[0030] L-ornithine-producing bacteria are typically auxotrophic for the amino acids L-citrulline or L-arginine. As an alternative, L-ornithine-producing bacteria that are bradytrophic to L-citrulline or L-arginine can also be contemplated. Definitions of the terms auxotrophic and bradytrophic can be found, for example, on page 9 of WO 01/09286. Bradytrophs are also called "leaky" mutants in the art. The bradytrophic bacteria used are, in particular, those in which the activity of the gene products ArgF (ornithine carbamoyl transferase), ArgG (argininosuccinate synthase) or ArgH (argininosuccinate lyase) is greater than (>) zero but equal to or less than (< ) 10 percent, preferably> zero and <1%, compared to activity in the wild.
[0031] The prior art has presented polynucleotides that are called the lysE gene and that encode proteins or polypeptides that have the activity of an L-lysine exporter. These polypeptides are also known by the abbreviation LysE.
[0032] An exporter is a protein that resides in the cell membrane of a cell and that carries a metabolite, for example, L-lysine or L-ornithine, from the cytoplasm of said cell out into the surrounding environment. If the energy needed for this is provided in the form of adenosine triphosphate (ATP), this is called export or primary active transport. This is called export or secondary active transport if said energy is supplied in the form of an ion gradient, for example, sodium ions (Jeremy M. Berg, John L. Tymoczko and L. Stryer; Biochemie [Biochemistry], 5th edition, pages 378-384, Spektrum Akademischer Verlag [editor], Heidelberg, Germany, 2003). Instructions for determining the export activity of L-ornithine can be found in Bellmann et al. (Microbiology 2001; 147: 1765-74).
[0033] During the course of the work leading to the present invention, it was observed that lysine exporters of the genera Corynebacterium, preferably Corynebacterium glutamicum, and Micrococcus, preferably Micrococcus luteus, have the activity of an exporter of L-ornithine in addition to the export activity of L-lysine.
[0034] The measurements of the invention use genes that encode polypeptides that have export activity for L-ornithine and whose amino acid sequence is at least (>) 35%,> 40%,> 50%,> 55%,> 60%,> 65%,> 70%,> 75%,> 80%,> 85%,> 90%,> 92%,> 94%,> 96%,> 97%,> 98%,> 99% or 100%, preferably> 70%, particularly preferably> 90%, very particularly preferably> 96% and most preferably> 100%, identical to the amino acid sequence of SEQ ID No. 2 , with the length of the encoded polypeptide, where appropriate, being within the length ranges described above.
[0035] Examples of suitable L-ornithine exporters are the lysine exporters or the LysE polypeptides of Corynebacterium glutamicum ATCC13032 (SEQ ID No. 2), Corynebacterium glutamicum R (SEQ ID No. 4), Corynebacterium glutamicum ATCC14067 (SEQ ID No. No. 5), Corynebacterium glutamicum ATCC13869 (SEQ ID No. 7), Corynebacterium efficiens YS-314 (SEQ ID No. 9), Corynebacterium diphteriae NCTC 13129 (SEQ ID No. 10), Corynebacterium striatum ATCC6940 (SEQ ID No. 11) ), Corynebacterium aurimucosum ATCC700975 (SEQ ID No. 12), Corynebacterium matruchotii ATCC33806 (SEQ ID No. 13), Corynebacterium pseudogenitalium ATCC33035 (SEQ ID No. 14), Corynebacterium accolens ATCC49725 (SEQ ID No. 18, 15 (SEQ ID No. 16), Micrococcus luteus NCTC2665 (SEQ ID No. 17), Corynebacterium tubuculostearicum SK141 (SEQ ID No. 18) and Corynebacterium matruchotii ATCC14266 (SEQ ID No. 19). SEQ ID No. 18 and SEQ ID No. 19 are also called in the ArgO polypeptide technique.
[0036] The nucleotide sequence of the lysE genes of Corynebacterium glutamicum ATCC14067 and Corynebacterium glutamicum ATCC13869 was determined in this study (SEQ ID No. 6 and SEQ ID No. 8). The amino acid sequences of the LysE polypeptide of Corynebacterium glutamicum ATCC14067 and Corynebacterium glutamicum ATCC13869 are shown in SEQ ID No. 5 and 7. They are identical to the amino acid sequence of C. glutamicum ATCC13032 Ly-sE, shown in SEQ ID No. 2.
[0037] Table 1 shows the accession numbers for the LysE polypeptides of various representatives of the genus Corynebacterium and Micrococcus luteus, which were taken from the databases of the National Center for Biotechnology Information (NCBI, Bethesda, MD, USA). In addition, Table 1 refers to the amino acid sequences of the LysE polypeptide that are shown in the sequence listing. Finally, Table 1 indicates the length (number of amino acids) of the encoded LysE polypeptide. TABLE 1


[0038] Figure 1 represents a multi-sequence alignment of the amino acid sequences of the LysE polypeptides from the bacteria mentioned in table 1. The alignments of the amino acid sequences shown in figure 1 were produced by the Clone Manager 9 Professional Edition program (Scientific & Educational Software 600 Pinner Weald Way Ste 202 Cary NC 27513 USA). The reference molecule used for the alignment was the LysE polypeptide (LysE) from ATCC13032. For the classification matrix, the "Blosum 62" setting (see: Jeremy M. Berg, John L. Tymoczko and L. Stryer; Biochemie, 5th edition, pages 194 to 197, Spektrum Akademischer Verlag, Heidelberg, Germany, 2003)) was chose.
[0039] It is also possible, when appropriate, to use the programs described in the prior art, such as, for example, the ClustalX program (Thompson, JD, Gibson, TJ, Plewniak, F., Jeanmougin, F. and Higgins, DG (1997 The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25: 4876-4882).
[0040] Amino acid residues 4-236 of the Corynebacterium glutamicum R polypeptide R (see SEQ ID No. 4) correspond to the LysE amino acid sequence of C. glutamicum ATCC13032 shown in SEQ ID No. 2. The C. polypeptide glutamicum R has an additional sequence of three amino acid residues at the N-terminus (methionine-valine-isoleucine). These additional residues are produced when the start codon located 9 base pairs further upstream of the lysE gene is used as an alternative to the start codon of the lysE gene in C. glutamicum ATCC13032 (see SEQ ID No. 1).
[0041] The amino acid sequence of the LysE polypeptide of C. efficiens YS-314 is 71%, and that of C. diphteriae NCTC 13129 is 44%, that of Corynebacterium striatum ATCC6940 is 44%, that of Corynebacterium aurimucosum ATCC700975 is 42% , that of Corynebacterium matruchotii ATCC33806 is 43%, that of Corynebacterium pseudogenitalium ATCC33035 is 43%, that of Corynebacterium accolens ATCC49725 is 43%, that of Corynebacterium glucuronalyticum ATCC 51867 is 36%, that of Micrococcus lute amino acids of C. glutamicum ATCC13032 shown in SEQ ID No. 2. In addition, the amino acid sequence of the C. tubuculostearicum SK141 ArgO polypeptide is 43% identical to the amino acid sequence of SEQ ID No. 2. In addition, the amino acid sequence of amino acids of the C. matruchotii ATCC14266 ArgO polypeptide is 44% identical to the amino acid sequence of SEQ ID No. 2. The percentages of identity were produced by generating a global sequence alignment with the aid of the Clone Manager 9 program using the Blum Sum 62 settings (see figure 2).
[0042] The lysE genes, that is, the polynucleotides that encode the polypeptides that have the activity of an L-ornithine exporter, can be isolated from the organisms with the aid of the polymerase chain reaction (PCR) with the use of primers appropriate. Instructions can be found, among others, in the "PCR" laboratory manual written by Newton and Graham (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994), and in WO 2006/100211 on pages 14 to 17.
[0043] Particular preference is given to the use, in a process according to the invention, of genes encoding polypeptides that have L-ornithine export activity and whose amino acid sequence includes one or more of the selected characteristics of the group consisting of a) amino acid sequence according to SEQ ID No. 2 or SEQ ID No. 4, b) amino acid sequence according to SEQ ID No. 2, including one or more, up to 25, 20, 15, 10, 5, 4, 3, 2, or 1, amino acid deletion (s), c) amino acid sequence according to SEQ ID No. 2, including one or more, up to 25, 20, 15, 10, 5, 4, 3, 2, or 1, amino acid insertion (s), and d) amino acid sequence according to SEQ ID No. 2, including one or more, up to 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 5, 4, 3, 2, or 1, preferably up to 5, 4, 3, 2, or 1, exchange (s) (replacement (s) )) of amino acids. e) amino acid sequence according to SEQ ID No. 2, which includes one or more, up to 25, 20, 15, 10, 5, 4, 3, 2, or 1, preferably up to 5, 4, 3 , 2, or 1, addition (s) of amino acids at the N termination and / or the C termination.
[0044] Where appropriate, preference is given to amino acid substitutions conservatives. In the case of aromatic amino acids, conservative substitutions are those in which the amino acids phenylalanine, tryptophan and tyrosine are substituted for each other. In the case of hydrophobic amino acids, conservative substitutions are those in which the amino acids leucine, isoleucine and valine are substituted for each other. In the case of polar amino acids, conservative substitutions are those in which the amino acids glutamine and asparagine are replaced by each other. In the case of basic amino acids, conservative substitutions are those in which the amino acids arginine, lysine and histidine are replaced by each other. In the case of acidic amino acids, conservative substitutions are those in which the amino acids aspartic acid and glutamic acid are replaced by each other. In the case of amino acids containing hydroxyl groups, conservative substitutions are those in which the amino acids serine and threonine are substituted for each other.
[0045] It is also possible to use polynucleotides that hybridize under stringent conditions to the nucleotide sequence complementary to SEQ ID No. 1, preferably to the coding region of SEQ ID No. 1, and that encode a polypeptide that has export activity of L-ornithine, with the amino acid sequence of the encoded protein being> 70% identical to the amino acid sequence of SEQ ID No. 2 and the length of the encoded polypeptide, where appropriate, being within the length ranges described above.
[0046] Instructions regarding the hybridization of nucleic acids and polynucleotides, respectively, can be found by those skilled in the art, among others, in the manual "The DIG System Users Guide for Filter Hybridization" by Boehringer Mannheim GmbH (Mannheim, Germany, 1993) and in Liebl etal. (International Journal of Systematic Bacteriology 41: 255-260 (1991)). Hybridization occurs under stringent conditions, that is, only hybrids in which the probe, that is, a polynucleotide comprising the nucleotide sequence complementary to SEQ ID No. 1, preferably the coding region of SEQ ID No. 1, and the target sequence, i.e., the polynucleotides treated with or identified by said probe, are at least 70% identical. It is known that the stringency of hybridization, including the washing steps, are influenced or determined by varying the buffer composition, temperature and salt concentration. The hybridization reaction is generally performed with relatively low stringency compared to the washing steps (Hybaid Hybridisation Guide, Hybaid Limited, Teddington, UK, 1996).
[0047] For example, a 5x SSC buffer at a temperature of approximately 50 ° C to 68 ° C can be used for the hybridization reaction. Here, probes can also hybridize with polynucleotides that are less than 70% identical to the nucleotide sequence of the probe employed. Such hybrids are less stable and are washed away under stringent conditions. This can be achieved, for example, by reducing the salt concentration to SSC 2x or SSC 1xâ “and, where appropriate, subsequently to SSC 0.5x (The DIG System User's Guide for Filter Hybridisation, Boehringer Mannheim, Mannheim, Germany, 1995 ), with a temperature of approximately 50 ° C to 68 ° C, approximately 52'C to 68 ° C, approximately 54 ° C to 68 ° C, approximately 56 ° C to 68'C, approximately 58 ° C to 68 ° C approximately 60 ° C to 68 ° C, approximately 62 ° C to 68 ° C, approximately 64 ° C to 68 ° C, approximately 66 ° C to 68 ° C being adjusted. Preference is given to temperature ranges of approximately 64 ° C to 68 ° C or approximately 66 ° C to 68 ° C. It is optionally possible to lower the salt concentration to a concentration that corresponds to SSC 0.2x or SSC 0.1x. The SSC buffer optionally contains sodium dodecyl sulfate (SDS) at a concentration of 0.1%. By gradually increasing the hybridization temperature in the steps of approximately 1 to 2 ° C from 50 ° C to 68 ° C, it is possible to isolate the polynucleotide fragments that are at least 70%, at least 80%, at least 90%, at least at least 92%, at least 94%, at least 96%, at least 97%, at least 98%, or at least 99%, when appropriate 100%, identical to the sequence or complementary sequence of the probe employed and encoding a polypeptide that has export activity of L-ornithine. Additional instructions regarding hybridization can be obtained on the market in the form of kits (eg DIG Easy Hyb from Roche Diagnostics GmbH, Mannheim, Germany, catalog number 1603558).
[0048] For the measurements of the invention, a polynucleotide that encodes a protein that has L-ornithine export activity is overexpressed in an initial or original bacterium or strain that produces L-ornithine, with the amino acid sequence of the encoded protein being> 35% identical to the amino acid sequence of SEQ ID No. 2 and the length of the encoded polypeptide, where appropriate, being within the ranges described above.
[0049] Overexpression generally means an increase in the intracellular concentration or activity of a ribonucleic acid, protein (polypeptide) or enzyme compared to the initial strain (original strain) or wild strain, if the latter is the initial strain . An initial strain (original strain) means a strain in which the measure leading to overexpression has been performed.
[0050] The terms protein and polypeptide are considered interchangeable.
[0051] For overexpression, preference is given to recombinant overexpression methods. They include any methods in which a microorganism is prepared using a DNA molecule supplied in vitro. Examples of these DNA molecules include promoters, expression cassettes, genes, alleles, coding regions, etc. They are transferred to the desired microorganism by transformation, conjugation, transduction or similar methods.
[0052] Overexpression measures increase the activity or concentration of the corresponding polypeptide generally by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, from preferably, up to 1000%, 2000%, 4000%, 10000% or 20000%, based on the level of activity or concentration of said polypeptide in the strain before the measurement results in overexpression.
[0053] When strains of the species Corynebacterium glutamicum are used, the export activity of L-ornithine in strain ATCC13032 or ATCC14067 or ATCC13869 or ATCC17965, when appropriate, is an appropriate reference point for determining overexpression. When strains based on or derived from ATCC13032 are used, said strain ATCC13032 is an appropriate reference point. An example of this is the strain prepared during the course of the work that leads to the present invention, ATCC13032_Delta_argFRGH / pVWEx1_lysE, which is based on the ATCC13032 strain. When using strains based on or derived from ATCC14067, said strain ATCC14067 is an appropriate reference point. When strains based on or derived from ATCC13869 are used, said strain ATCC13869 is an appropriate reference point. Suitable additional reference points are produced accordingly.
[0054] When Escherichia coli strains are used, preferably Escherichia coli K12 strain, the export activity of L-ornithine in the MG1655 strain, when appropriate, is an appropriate reference point for determining overexpression.
[0055] Overexpression is achieved by a multiplicity of methods available in the prior art.
[0056] They include increasing the number of copies and modifying the nucleotide sequences that direct or control the expression of the gene. The transcription of a gene is controlled, among others, by the promoter and, optionally, by proteins that overexpress (repressor proteins) or promote (activator proteins) transcription. The translation of the formed RNA is controlled, among others, by the ribosome binding site and the start codon. Polynucleotides or DNA molecules that include a promoter and a ribosome binding site and, optionally, a start codon are also called an expression cassette.
[0057] Said methods also include the use of variants of polypeptides or enzymes, which have an increased catalytic activity.
[0058] The number of copies can be increased through plasmids that replicate in the bacterial cytoplasm. For this, several plasmids are described in the prior art for very different groups of microorganisms, whose plasmids can be used to establish the desired increase in the number of copies of the gene. Suitable plasmids for the genus Escherichia are described, for example, in the manual Molecular Biology, Labfax (Ed .: T.A. Brown, Bios Scientific, Oxford, UK, 1991). Plasmids suitable for the genus Corynebacterium are described, for example, in Tauch et al. (Journal of Biotechnology 104 (1-3), 27-40, (2003)) or in Stansen et al. (Applied and Environmental Microbiology 71, 5920-5928 (2005)).
[0059] The use of plasmid pEC7lysE, deposited in DSM 23239, to increase the number of copies in strains of Corynebacterium glutamicum is excluded from the measures leading to the present invention The nucleotide sequence of plasmid pEC7lysE has been determined and is shown in SEQ ID No 29.
[0060] The number of copies can be further increased by at least one (1) copy by introducing more copies into the bacterium's chromosome. Suitable methods for the genus Corynebacterium, preferably Corynebacterium glutamicum, are described, for example, in patent documents WO 03/014330, WO 03/040373 and WO 04/069996. WO 03/014330 describes methods for tandem duplication of genes at the locus of the native gene. WO 03/040373 describes methods for incorporating a second or third copy of a gene into more gene loci, with the particular gene locus not being essential for the growth or production of the specific amino acid, L-ornithine, in the case of the present invention. Examples of loci suitable genes for incorporating a second or additional copy of the lysE gene in a process according to the invention are the odh, sucA, dapA, dapB, ddh, lysA, argR, argF, argG and argH genes. WO 04/069996 (see tables 12 and 13) describes intergenic regions of C. glutamicum and genes encoding phages or phage components, which are suitable for incorporating additional copies of the lysE gene.
[0061] Examples of methods suitable for the genus Escherichia are the incorporation of a copy of the gene into the phage att site (Yu and Court, Gene 223, 77-81 (1998)), chromosomal amplification with the aid of Mu phage, as described in EP 0 332 448, or the methods of gene replacement with the aid of conditional replication plasmids, as described by Hamilton et al. (Journal of Bacteri-ology 174, 4617-4622 (1989)) or Link et al. (Journal of Bacteriology 179, 6228-6237 (1997)).
[0062] Gene expression can be further increased by the use of a strong promoter that is functionally linked to the gene to be expressed. Preference is given to using a promoter that is stronger than the natural promoter, that is, the one present in the wild or original strain. For this, the prior art has several methods available.
[0063] Promoters and expression systems suitable for the genus Corynebacterium can be found, among others, in EP 0 629 699 A2, US 2007/0259408 A1 (gap promoter), WO 2006/069711, EP 1 881 076 A1, WO 2008/088158, WO 2009/025470 (butA promoter, pyk promoter), US 6,861,246 (MC20 and MA16 variants of the dapA promoter), and EP 1 918 378 A1 (sod promoter), and in overviews such as the "Handbook of Corynebacterium glutamicum "(Eds .: Lothar Eggeling and Michael Bott, CRC Press, Boca Raton, US (2005)), or the book" Corynebacteria, Genomics and Molecular Biology "(Ed .: Andreas Burkovski, Caister Academic Press, Norfolk, UK ( 2008)). Examples of promoters that allow controlled expression, that is, inducible or repressible, are described, for example, in Tsuchiya and Morinaga (Bio / Technology 6, 428-430 (1988)).
[0064] Promoters suitable for the genus Escherichia have been known for some time. They include, among others, the classic promoters lac promoter, trp promoter, hybrid promoters tac and trc, promoters PL and PR of phage I. Similarly, it is possible to use promoters of phage T7, promoters "gear-box ", the nar promoter or the promoters of the rrsG, rnpB, csrA, csrB, ompA, fusA, pepQ, rplX or rpsG genes. Controlled expression is permitted, for example, by the phage I CI857-PR or CI857-PL system (Götting et al., BioTechniques 24, 362-366 (1998)). Overviews can be found in Makriides (Microbiological Reviews 60 (3), 512-538 (1996)) or in the manual "Escherichia coli and Salmonella, Cellular and Molecular Biology" (FC Neidhardt (Editor-in-Chief), ASM Press, Washington , USA (1996)).
Such expression promoters or cassettes are typically employed at a distance of 1 to 1000, preferably 1 to 500, nucleotides upstream of the first nucleotide of the start codon of the coding region of the gene. At a distance of 1 it means that the promoter or the expression cassette is positioned immediately in front of the first base of the start codon of the coding region.
[0066] To increase the expression of the lysE gene in C. glutamicum, preference is given to inserting suitable promoters such as, for example, the C. glutamicum sod promoter (see SEQ ID No. 1 of EP 1918 378 A1) or gap promoter of C. glutamicum (see SEQ ID No. 3 of US 2007/0259408) between positions 930 and 990 of SEQ ID No. 1.
[0067] When using expression cassettes containing a promoter and a ribosome binding site (RBS), such as the expression unit of the C. glutamicum sod gene (see SEQ ID No. 2 of EP 1918 378 A1) or expression unit of the C. glutamicum gap gene, described in US 2007/0259408 and shown in SEQ ID No. 28 (and called there PgapRBS), for example, they are inserted, in the case of C. glutamicum, preferably, between positions 930 and 1001, particularly preferably between positions 1000 and 1001, of SEQ ID No. 1. An example of a suitable ribosome binding site in such an expression cassette is the 5'-agaaaggagg nucleotide sequence -3 'specified by Amador (Microbiology 145, 915-924 (1999)).
[0068] It is also possible to place a plurality of promoters upstream of the desired gene or functionally link them to the gene to be expressed and, in this way, obtain increase in expression. This is described, for example, in WO 2006/069711.
[0069] The structure of the Corynebacterium glutamicum and Escherichia coli promoters is well known. Therefore, it is possible to increase the strength of a promoter by modifying its sequence through one or more substitution (s) and / or one or more insertion (s) and / or one or more nucleotide deletion (s). Examples of this can be found, among others, in "Herder Lexikon der Biologie" (Herder's Encyclopedia of Biology) (Spektrum Akademischer Cher Verlag, Heidelberg, Germany (1994)).
Consequently, a suitable measure to overexpress the lysE gene is to modify or mutate the promoter of said lysE gene.
[0071] The structure of the Corynebacterium glutamicum and Escherichia coli ribosome binding sites is also well known and is described, for example, in Amador (Microbiology 145, 915-924 (1999)), and in textbooks and textbooks of genetics, for example, "Gene und Klonne" [Genes and Clones] (Winnacker, Verlag Chemie, Weinheim, Germany (1990)) or "Molecular Genetics of Bacteria" (Dale and Park, Wiley and Sons Ltd., Chichester, UK (2004)). Well-expressed genes, that is, the most important structural genes in an organism, have a good ribosome binding site (Amador, Microbiology 145, 915-924 (1999)), that is, the latter is very similar to or corresponds to consensus sequence. Highly expressed genes have been shown in the literature to have a strong ribosome binding site (Karlin and Mrázek, Journal of Bacteriology 2000; 182 (18): 5238-50). Consequently, the efficiency of translating a gene or mRNA can be obtained by adjusting the ribosome binding site.
[0072] It is also possible to increase the efficiency of translation by adjusting the use of codons in the genes to be expressed (for example, Najafabiad etal., Nucleic Acids Research 2009, 37 (21): 7014-7023).
[0073] Overexpression can also be achieved by increasing the expression of activating proteins or by reducing or shutting down the expression of repressor proteins.
[0074] The activating protein LysG to express lysE has been described by Bellmann et al. (Microbiology 2001; 147: 1765-74) and is here called "positive regulator". The LysG amino acid sequence of Corynebacterium glutamicum ATCC13032 is shown in SEQ ID No. 30. In a global sequence alignment, the amino acid sequence of the LysG polypeptide of Corynebacterium diphteriae NCTC13129 is 62%, the amino acid sequence of the LysG polypeptide of Cory efficiens YS-314 is 81%, and the amino acid sequence of the LysG polypeptide from Corynebacterium glutamicum R is 94%, identical to that of SEQ ID No. 30.
[0075] For activator proteins, preference is given to a polypeptide that is> (at least) 55%, preferably> 80%, particularly preferably> 90%,> 92% or> 94%, very particularly, preferably> 99% and, most preferably, 100%, identical to the amino acid sequence shown in SEQ ID No. 30.
[0076] The aforementioned overexpression measures, selected preferably from the group consisting of increasing the number of copies, using a strong promoter, mutating the promoter, using a suitable expression cassette and overexpressing an activating protein, can be combined in an appropriate way. In this way, it is possible, for example, to combine the use of a suitable promoter with an increase in the number of copies, or overexpression of an activator protein with the use of a suitable promoter or a suitable expression cassette.
[0077] It is also possible, in addition to measures related to the polylucleotide that encodes a protein that has L-ornithine export activity, to attenuate the individual biosynthesis genes.
[0078] To improve the production of L-ornithine, it is convenient, when appropriate, to additionally attenuate one or more of the selected genes from the group consisting of a) odhA gene encoding the E1 alpha-ketoglutarate dehydrogenase subunit (EC 1.2 .4.2), b) sucA gene encoding dihydrolipoamide succinyl transferase (EC 2.3.1.61), c) dapA gene encoding dihydrodipicolinate synthase (DapA, EC 4.2.1.52), d) dapB gene encoding dihydrodipicolinate synthase (DapB , EC 1.3.1.26), e) ddh gene that encodes a meso-diaminopimelate dehydrogenase (Ddh, EC 1.4.1.16), f) lysA gene that encodes a diaminopimelate decarboxylase (LysA, EC 4.1.1.20), g) gene argR encoding a L-arginine biosynthesis repressor (ArgR), h) argF gene encoding an ornithine carbamoyl transferase (ArgF, EC 2.1.3.3), i) argG gene encoding an argininosuccinate synthase (ArgG, EC 6.3 .4.5), j) argH gene encoding an argininosuccinate lyase (ASAL) (ArgH, EC 4.3.2.1), k) lysC gene encoding is an aspartate kinase (LysC, EC 2.7.2.4), and l) asd gene that encodes a semialdehyde aspartate dehygenase (Asd, EC 1.2.1.11).
[0079] Preference is given to the attenuation of one or more of the selected genes from the group consisting of lysA, odhA, argR, argF, argG and argH. Particular preference is given to the attenuation of one or more of the selected genes from the group consisting of lysA, odhA and argF. Very particular preference is given to the attenuation of the lysA and / or argF genes.
[0080] The term "attenuation", in this context, describes the reduction or shutdown of the intracellular activity of one or more enzymes (proteins) in a bacterium, which are encoded by the corresponding DNA, using, for example, a weak promoter or a gene or allele that encodes a corresponding enzyme that has a low activity, or by inactivating the corresponding gene or enzyme (protein) and, optionally, a combination of these measures.
[0081] An overview of known promoters of various forces in Corynebacterium glutamicum can be found in Pátek et al. (Journal of Biotechnology 104, 311-323 (2003)). Other weak promoters are described in communication 512057 in the December 2006 Research Disclosure magazine (pages 1616 to 1618).
[0082] Mutations that can be considered to generate an attenuation are transitions, transversions, insertions and deletions of at least one (1) base pair or nucleotide in the coding region of the gene in question. Depending on the effect of the amino acid substitution caused by a mutation on the activity of the protein or enzyme, the mutations are called missense mutations (meaning exchange) or nonsense mutations (meaningless) ".
[0083] The missense mutation results in an exchange of a given amino acid in one protein for another, said exchange being, in particular, a non-conservative amino acid substitution. This impairs the functionality or activity of the protein and reduces it to a value of> 0 to 75%,> 0 to 50%,> 0 to 25%,> 0 to 10% or> 0 to 5%.
[0084] The nonsense mutation results in a stop codon in the coding region of the gene and, therefore, in the early termination of the translation and, consequently, in a shutdown. Insertions or deletions of at least one base pair in a gene lead to frameshift mutations (phase shift) resulting in wrong amino acids being incorporated or translation being completed early. If the mutation results in a stop codon in the coding region, this also leads to an early termination of the translation. Measures for generating a nonsense mutation are preferably performed on the 5'-terminal part of the coding region, which encodes the N-terminus of the polypeptide. If the overall length of a polypeptide (measured by the number of chemically linked L-amino acids) is called 100%, then - within the scope of the present invention - the N termination of the polypeptide includes that part of the amino acid sequence that, by calculation from the starting amino acid, L-formyl-methionine, onwards, contains 80% of the L-amino acids downstream.
[0085] In vivo mutagenesis methods are described, for example, in the Manual of Methods for General Bacteriology (Gerhard et al. (Eds.), American Society for Microbiology, Washington, DC, USA, 1981) or in Tosaka et al. (Agricultural and Biological Chemistry 42 (4), 745-752 (1978)) or in Konicek etal. (Folia Microbiologica 33, 337-343 (1988)).
[0086] Suitable in vitro mutagenesis methods are, among others, hydroxylamine treatment according to Miller (Miller, JH: A Short Course in Bacterial Genetics. A Laboratory Manual and Handbook for Escherichia coli and OxyRated Bacteria, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1992), the use of mutagenic oligonucleotides (TA Brown: Gentechnologie für Einsteiger [Genetic Engineering for Beginners], Spektrum Akademischer Verlag, Heidelberg, 1993 and RM Horton: PCR-Mediated Recombination and Mutagenesis, Molecular Biotechnology 3, 93-99 (1995)), and the use of a polymerase chain reaction using a DNA polymerase with a high error rate. An example of such a DNA polymerase is the DNA polymerase Mutazyme (GeneMorph PCR Mutagenesis Kit, No. 600550) from Stratagene (LaJolla, CA, USA).
[0087] Additional instructions and overviews on the generation of mutations in vivo or in vitro can be found in the prior art in known textbooks on genetics and molecular biology, such as the textbook authored by Knippers ("Molekulare Genetik", 6th edition, Georg Thieme Verlag, Stuttgart, Germany, 1995), Winnacker's ("Gene and Klone", VCH Verlagsgesellschaft, Weinheim, Germany, 1990) or Hagemann's ("Allgemeine Genetik" [General Genetics], Gus-tav Fischer Verlag, Stuttgart, 1986), for example.
[0088] With the aid of the known process of replacing a gene or allele, the foundations of which are described in Schwarzer and Pühler (Bio / Technology 9, 84-87 (1991)), it is possible to transfer a mutation prepared in vitro, or a polynucleotide containing the desired mutation, into the chromosome. Von Schãfer et al. (Gene 145, 69-73 (1994)) employed this method in order to incorporate a deletion in the homon-thrB operon of C. glutamicum. Von Nakagawa et al. (EP 1108790) and Ohnishi et al. (Applied Microbiology and Biotechnology 58 (2), 217-223 (2002)) used this method to incorporate several mutations, starting from the isolated alleles, in the chromosome of C. glutamicum.
[0089] A method for the targeted reduction of gene expression is to place the gene to be attenuated under the control of a promoter that can be induced by adding measured quantities of IPTG (isopropyl bD-thiogalactopyranoside), such as, for example , the trc promoter or the tac promoter. Vectors are suitable for this purpose, for example, the Escherichia coli expression vector pXK99E (WO 0226787; deposited according to the Budapest Treaty on July 31, 2001 in DH5alpha / pXK99E as DSM14440 with the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ, Brunswick, Germany)), pEKEx2 (NCBI accession number AY585307) or pVWEx2 (Wendisch, doctoral thesis, Berichte des Forschungszen- trums Jülich, Jül-3397, ISSN 0994-2952, Jülich, Germany (1997)) , which allows the cloned gene to be expressed in an IPTG-dependent manner in Corynebacterium glutamicum.
[0090] This method was employed, for example, in patent document WO 02266787 for regulated expression of the deaD gene by integrating the pXK99EdeaD vector into the genome of Corynebacterium glutamicum, and by Simic et al. (Applied and Environmental Microbiology 68: 3321-3327 (2002)) for regulated expression of the glyA gene through integration of the pK18mobglyA 'vector in Corynebacterium glutamicum.
[0091] Another method to specifically reduce gene expression is the antisense technique that involves applying short oligodeoxynucleotides or vectors to the target cells to synthesize longer antisense RNA. There, antisense RNA can bind to complementary sections of specific mRNAs and reduce their stability or block their translation capacity. An example of this can be found by the person skilled in the art in Srivastava et al. (Applied Environmental Microbiology 2000 Oct; 66 (10): 4366-4371).
[0092] The stretching rate is influenced by the use of codons. Gene expression can be attenuated by the use of codons for t-RNAs that are rare in the original strain. This is described in detail in WO 2008049781 and WO 2009133063. For example, replacing an ATG start codon with the less common GTG or TTG codons can impair translation, since the AUG codon is two to three times more effective than codons GUG and UUG, for example (Khudyakov et al., FEBS Letters 232 (2): 369-71 (1988); Reddy et al., Proceedings of the National Academy of Sciences of the USA 82 (17): 5656-60 (1985)).
[0093] It is also possible, in addition to the measures related to the polynucleotide that encodes a protein that has L-ornithine export activity, to enhance individual biosynthesis genes.
[0094] To improve the production of L-ornithine, it is convenient, when appropriate, to further enhance the enzymatic activity of one or more of the proteins selected from the group consisting of a) glutamate dehydrogenase (EC 1.4.1.3) encoded by the gene gdh, b) glutamate N-acetyltransferase (EC 2.3.1.35 and EC 2.3.1.1) encoded by the argJ gene, c) acetyl glutamate kinase (EC 2.7.2.8) encoded by the argB gene, d) N-acetyl-gamma-glutamyl- phosphate reductase (EC 1.2.1.38) encoded by the argC gene, e) acetylornithine aminotransferase (EC 2.6.1.11), encoded by the argD gene, f) glucose-specific component EIIB (PtsG) (EC 2.7.1.69) of the absorption system glucose, encoded by the ptsG gene, g) sucrose-specific EIIB (PtsS) component (EC 2.7.1.69) of the saccharose absorption system, encoded by the ptsS gene, h) glucose-6-phosphate 1-dehydrogenase (EC 1.1.1.49 ) encoded by the zwf gene, i) glucose-6-phosphate isomerase (EC 5.3.1.9) encoded by the pgi gene, j) phosphofructokinase (EC 2.7.1 .11) encoded by the pfkA gene, k) fructose-bisphosphate aldolase (EC 4.1.2.13) encoded by the fda gene, l) glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.59) encoded by the gap, m) phosphoglycerate kinase gene (EC 2.7.2.3) encoded by the pgk gene, n) pyruvate kinase (EC 2.7.1.40) encoded by the pyk gene, o) pyruvate dehydrogenase E1 subunit (EC 1.2.4.1), encoded by the aceE gene, p) phosphoenolpyruvate carboxylase (EC 4.1 .1.31) encoded by the ppc gene, q) pyruvate carboxylase (EC 6.4.1.1), encoded by the pyc, r) aconitase gene (EC 4.2.1.3) encoded by the acn, es) isocitrate dehydrogenase gene (EC 1.1.1.42) encoded by icd gene.
[0095] The term intensification includes measures of overexpression and the use of variants that have increased catalytic activity compared to wild-type protein.
[0096] Particular preference is given to the intensification of one or more of the enzymes selected from the group consisting of glutamate dehydrogenase, glutamate N-acetyltransferase and acetylglutamate kinase.
[0097] The additional mitigation measures mentioned can be combined with the additional intensification measures.
[0098] Instructions for handling DNA, digesting and binding DNA, transforming and selecting transformants can be found, among others, in the well-known manual by Sambrook et al. "Molecular Cloning: A Laboratory Manual, Second Edition (Cold Spring Harbor Laboratory Press, 1989).
[0099] The extent of expression or overexpression can be determined by measuring the amount or concentration of mRNA transcribed from the gene, by determining the amount or concentration of the polypeptide and by determining the level of enzyme activity.
[00100] The amount of mRNA can be determined, among others, by using the methods "Northern blotting" and quantitative RT-PCR. In quantitative RT-PCR, the polymerase chain reaction is preceded by reverse transcription. The LightCycler ™ system from Roche Diagnostics (Boehringer Mannheim GmbH, Roche Molecular Biochemicals, Mannheim, Germany) can be used for this purpose, as described in Jungwirth et al. (FEMS Microbiology Letters 281, 190-197 (2008)), for example. The protein concentration can be determined by uni or two-dimensional fractionation of protein in gel and subsequent optical identification of the protein concentration in the gel using an appropriate evaluation program. A common method for preparing protein gels for corineform bacteria and for identifying proteins is the procedure described by Hermann et al. (Electrophoresis, 22: 1712-23 (2001)). Protein concentration can also be determined by Western-blot hybridization using an antibody that is specific for the protein to be detected (Sambrook et al., Molecular cloning: a laboratory manual, 2aEd. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989) and subsequent optical evaluation using an appropriate program for determining the concentration (Lohaus and Meyer (1998) Biospektrum 5: 32-39; Lottspeich, Angewandte Chemie 321: 2630- 2647 (1999) ).
[00101] The bacteria produced can be cultivated continuously - as described, for example, in WO 05/021772 - or discontinuously in a batch process (batch culture) or in a fed batch process or repeated fed batch process (described in US 6,562,601 for example) for the purpose of producing L-ornithine. A summary of a general nature on known cultivation methods is available in Chmiel's textbook (Bioprozesstecknik [Bioprocess Technology] 1. Einführung in die Bioverfahrenstechnik [Introduction to Bioprocess Engineering] (Gustav Fischer Verlag, Stuttgart, 1991)), or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and Peripheral Equipment] (Vieweg Verlag, Brunswick / Wiesbaden, Germany 1994).
[00102] The culture medium or fermentation medium to be used must be in an adequate form to satisfy the demands of the particular strains. The American Society for Bacteriology (Manual of Methods for General Bacteriology) (Washington D.C., USA, 1981) contains descriptions of culture media for various microorganisms. The terms growth medium, culture medium and fermentation medium or medium are interchangeable.
[00103] It is possible to use, as a carbon source, sugars and carbohydrates such as, for example, glucose, sucrose, lactose, fructose, maltose, molasses, solutions containing sucrose from the processing of sugar beet or sugar cane, starch, hydrolyzate starch and cellulose, oils and fats such as soybean oil, sunflower oil, peanut oil and coconut fat, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as for example, glycerol, methanol and ethanol, and organic acids such as, for example, acetic acid or lactic acid.
[00104] With sugars, preference is given to glucose, fructose, sucrose, mixtures of glucose and fructose, and mixtures of glucose, fructose and sucrose. When appropriate, sucrose is particularly preferred.
[00105] With alcohols, preference is given to glycerol.
[00106] It is possible to use, as nitrogen source, organic compounds containing nitrogen such as peptones, yeast extract, meat extract, malt extract, corn liquor, soy flour and urea, or inorganic compounds such as ammonium sulfate, chloride ammonium, ammonium phosphate, ammonium carbonate and ammonium nitrate. Nitrogen sources can be used individually or as a mixture.
[00107] It is possible to use phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts as a source of phosphorus.
[00108] The culture medium must also comprise salts, for example in the form of metal chlorides or sulphates, such as sodium, potassium, magnesium, calcium and iron, such as magnesium sulphate or iron sulphate, for example, that are necessary for growth. Finally, essential growth factors such as amino acids, for example, homoserine and vitamins, for example, thiamine, biotin or pantothenic acid, can be used in addition to the substances mentioned above.
[00109] The mentioned starting materials can be added to the culture in the form of a single batch or be fed in an appropriate way during cultivation.
[00110] The pH of the culture can be controlled by the use of basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds such as phosphoric acid or sulfuric acid, in an appropriate way. The pH is generally adjusted to a value of 6.0 to 8.5, preferably 6.5 to 8. To control foaming, it is possible to employ defoamers, such as polyglycolic fatty acid esters. To maintain the stability of plasmids, it is possible to add suitable selective substances, such as antibiotics, to the medium. Fermentation is preferably carried out under aerobic conditions. In order to maintain these conditions, oxygen or gas mixtures containing oxygen, such as air, are introduced into the culture. It is also possible to use liquids enriched with hydrogen peroxide. Fermentation is carried out, when appropriate, at high pressure, for example at an elevated pressure of 0.03 to 0.2 MPa. The temperature of the culture is usually from 20 ° C to 45 ° C and preferably from 25 ° C to 40 ° C, particularly preferably from 30 ° C to 37 ° C. In batch processes, preference is given to continuing the culture until an amount of the desired L-ornithine sufficient to be recovered has formed. This goal is usually achieved within 10 hours to 160 hours. With continuous processes, longer cultivation times are possible. Bacterial activity results in a concentration or increase in the concentration (accumulation) of L-ornithine in the fermentation medium.
[00111] Examples of suitable fermentation media can be found, inter alia, in JP 43010996 B4 (for B. subtilis), US 3668072 A (for E. coli) and JP 57041912 B (for B. flavum).
[00112] Where appropriate, the volume of the fermentation medium in a process according to the invention is> 0.5 I,> 1 I,> 5 I,> 10 I,> 50 I, 100 I, 500 I, £ 1000 I, preferably> 11, particularly preferably> 10 1, most particularly, preferably> 100 I and most preferably £ 1000 I.
[00113] To determine the concentration at one or more point (s) of time during the course of fermentation, L-ornithine can be analyzed by separating L-amino acids through ion exchange chromatography, preferably cations, with subsequent post-column derivatization using ninhydrin, as described in Spackman et al. (Analytical Chemistry 30: 1190-1206 (1958)). It is also possible to use ortho-phthalodialdehyde instead of ninhydrin for post-column derivatization. A general article on ion exchange chromatography can be found in Pickering (LC.GC (Magazine of Chromatographic Science) 7 (6), 484-487 (1989)).
[00114] It is also possible to perform a precolumn derivatization, for example, using ortho-phthalodialdehyde or phenyl isothiocyanate, and fractionate the resulting amino acid derivatives by reverse phase chromatography (RP), preferably in the form of liquid chromatography. high efficiency (HPLC). Such a method is described, for example, in Lindroth et al. (Analytical Chemistry 51: 1167-1174 (1979)). Detection is performed photometrically (absorbance, fluorescence).
[00115] A review related to amino acid analysis can be found, among others, in the textbook "Bioanalytik" by Lottspeich and Zorbas (Spektrum Akademischer Verlag, Heidelberg, Germany 1998).
[00116] The performance of fermentation processes or processes according to the invention, with respect to one or more of the parameters selected from the group consisting of concentration of L-ornithine (L-ornithine formed by volume), yield of L-ornithine ( L- ornithine formed by consumed carbon source), formation of L-ornithine (L-ornithine formed by volume and time), and specific formation of L-ornithine (L-ornithine formed by dry cell or dry biomass and time, or L-ornithine formed by cellular protein and time), or other process parameters and combinations thereof, is increased by at least 0.5%, at least 1%, at least 1.5% or at least 2 %, based on fermentation processes or processes that use bacteria that contain a non-overexpressed protein that has L-ornithine export activity or that have not been subjected to an overexpression measure.
[00117] The fermentation measures result in a fermentation broth that contains the desired L-ornithine.
[00118] A product containing L-ornithine is then supplied or produced or recovered in liquid or solid form.
[00119] A fermentation broth means a fermentation medium or growth medium in which a microorganism has been cultivated for a certain time and at a certain temperature. The fermentation medium or the means used during fermentation comprise / comprise all substances or components that guarantee the production of said L-ornithine and, typically, propagation and viability.
[00120] When the fermentation is complete, the resulting fermentation broth therefore comprises a) the bacterial biomass (cell mass) produced due to the spread of the bacterial cells, b) the L-ornithine formed during the course of the fermentation, c) the organic by-products formed during the course of fermentation, and d) the constituents of the fermentation medium employed or the starting materials, for example vitamins such as biotin or salts such as magnesium sulphate, which were not consumed in the fermentation.
[00121] Organic by-products include substances that are produced by the bacteria used in fermentation in addition to L-ornithine and are optionally excreted. They also include sugars like trehalose, for example.
[00122] The fermentation broth is removed from the culture vial or fermentation tank, optionally collected, and used to provide a product containing L-ornithine in liquid or solid form. The phrase "recover the product containing L-ornithine" is also used for this. In the simplest case, the fermentation broth containing L-ornithine itself, which has been removed from the fermentation tank, constitutes the recovered product.
[00123] One or more of the selected measures from the group consisting of a) partial (> 0% to <80%) to complete (100%) or virtually complete (> 80%, £ 90%, £ 95%, £ 96%, £ 97%,> 98% or> 99% to <100%) of water, b) partial removal (> 0% to <80%) to complete (100%) or virtually complete (> 80%, £ 90%, 95%, £ 96%,> 97%,> 98% or> 99% to <100%) of the biomass that is optionally inactivated before removal, c) partial removal (> 0% to <80%) complete (100%) or virtually complete (> 80%, 90%, £ 95%, 96%,> 97%,> 98%,> 99%,> 99.3% or £ 99.7% at <100 %) of organic by-products formed during the course of fermentation, and d) partial (> 0%) to complete (100%) or virtually complete (> 80%, £ 90%,> 95%,> 96%,> 97% ,> 98%,> 99%,> 99.3% or> 99.7% to <100%) of the constituents of the fermentation medium used or of the starting materials, which were not consumed in the fermentation, from the broth of fermentation reaches concentration or purification of L-ornithine. Products that have a desired L-ornithine content are isolated in this way.
[00124] Partial (> 0% to <80%) to complete (100%) or virtually complete (> 80% to <100%) water removal (measure a)) is also called drying.
[00125] In a process variant, the complete or virtually complete removal of water, biomass, organic by-products and uneaten constituents from the fermentation medium employed results in pure L-ornithine product forms (> 80%, in or> 90% by weight) or high purity (> 95% by weight> 97% by weight or 99% by weight). Various technical instructions for measurements according to a), b), c) or d) are available in the prior art.
[00126] In the case of the amino acid L-ornithine or its salts, three essentially different products have been described in the prior art.
[00127] One group describes L-ornithine HCL, from which L-ornithine is purified from the fermentation solution, after removal of the cells through an ion exchanger, and then crystallized through crystallization as L monochloride -ornithine and recrystallization as L-ornithine monochloride (US 2988489). The L-ornithine HCL obtained in that case has a purity of more than> 90%, preferably more than 95%, particularly preferably more than 98%, and most particularly preferably more than 99%.
[00128] An additional process is described in patent application EP 1995322. This involves applying the biomass-containing fermentation solution to the top of a weakly acid ion exchanger with a particle diameter of> 300 pm and purifying the L-ornithine by this stage. Selecting a suitable particle diameter prevents the biomass from blocking the resin. The cell removal efficiency was 99%.
[00129] The purified L-ornithine can then be used to prepare various salts of L-ornithine, for example, mono- or di-L-ornithine a-ketoglutarate, L-ornithine L-aspartate, etc.
[00130] EP 0477 991, for example, describes a process for preparing L-ornithine L-aspartate. This involves adding a water-soluble solvent to an aqueous solution of L-ornithine and L-aspartate in order to arrive at a solution that is at least 90% saturated or supersaturated. Said solution is heated under reflux until the formation of crystals has ended. A water-miscible solvent is then added continuously under reflux until the salt crystals form. The crystals can be removed, for example, by centrifugation and are subsequently dried under vacuum. The purity of the product is typically greater than 98.5%.
[00131] JP 46003194 describes a process for preparing L-ornithine L-ketoglutarate. This involves, for example, converting ornithine HCL into the free base by adsorption to an acid ion exchanger and eluting with aqueous ammonia, adding α-ketoglutarate and evaporating the solution under vacuum until the product crystallizes.
[00132] The plasmid pEC7lysE was deposited as the strain DH5alpha / pEC7lysE (DM2204) of Escherichia coli according to the Budapest Treaty with the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ, Brunswick, Germany) under the number of DSM access 23239 on January 15, 2010. EXAMPLES Example 1 Cloning and sequencing of the lysE gene of Corynebacterium qlutamicum ATCC 13032
[00133] The lysE gene of the ATCC13032 strain was cloned into the E. coli / C transport and expression vector. glutamicum pVWExl (Peters-Wendisch etal., J. Mol. Microbiol. Biotechnol. (2001) 3 (2): 295-300).
[00134] The cloning was performed in two stages. First, a polymerase chain reaction (PCR) amplified the Corynebacterium glutamicum gene ATCC13032 through the following oligonucleotide primers derived from SEQ ID No. 1. Said oligonucleotide included additional restriction cleavage sites at its 5 'end (underlined) : EcoRV for lysE lp and Avrll or Sspl for ly- sE_2.p). lysE_1 .p: 5'-iTCGATATCATGGAAATCTTCATTACAGGl-3 '(see SEQ ID No. 22) lysE_2.p: S'-fTGCCTAGGTCAATATTTGGGCGAAGGCCACC G] -3' (see SEQ ID No. 23)
[00135] The PCR reaction was performed in the presence of 200 pM deoxynucleotide triphosphates (dATP, dCTP, dGTP, dTTP), 0.5 pM of each of the corresponding oligonucleotides, 100 ng of Corynebacterium glutamicum chromosome DNA ATCC13032, 1 / 5 volume of 5x HF reaction buffer and 0.02 U / pl Phusion® DNA polymerase "Hot Start" (Biozym Scientific GmbH, D-31840 Hess. Oldentorf) in a thermocycler (Mastercycler, Eppendorf AG, Hamburg) under the following conditions: 98 ° C for 1 minute; 30 cycles' (98 ° C, 20 s; 63 ° C, 20 s; 72 ° C, 40 s); 72 ° C for 6 min.
[00136] The 761 bp lysE PCR fragment (see SEQ ID No. 3) was cloned into pVWExI as described below:
[00137] Vector preparation: 1 pg of plasmid DNA from pVWExI was cleaved in the enzyme-specific buffer system containing 10 units of the Pstl enzyme by incubation at 37 ° C for 1 h. Immediately afterwards, the cleavage mixture was treated with the Quick Blunting kit (New England Biolabs GmbH, Frankfurt am Main) according to the manufacturer's instructions and then purified using the QiaExll purification kit (Qiagen AG, Hilden, Germany) according to the manufacturer's instructions. The vector pretreated in this way was then cleaved with 10 units of Xbal in the enzyme-specific buffer system at 37 ° C for 1 h and then purified again using the QiaExll purification kit.
[00138] Preparation of the insert: the PCR fragment from lysE was cleaved with 10 units of each of the enzymes Avrll and EcoRV and then purified using the QiaExll purification kit according to the manufacturer's instructions.
[00139] Ligation: the vector and the insert were mixed at a molar ratio of 1: 5 and ligated using T4 DNA ligase at 16 ° C for 1 h. Chemically competent E. coli DH5alpha cells (Subcloning efficiency, Invitrogen GmbH, Karlsruhe, Germany) were transformed with 3 µl of the ligation mixture.
[00140] Transformants were identified based on their resistance to kanamycin on LB agar plates containing 50 pg / ml kanamycin sulfate. Plasmid DNA was isolated from 4 of said transformants, and plasmids were tested by restriction analysis for the presence of the 0.75 kb fragment as an insert. The recombinant plasmid produced in this way was called pVWExIlysE.
[00141] The nucleotide sequence of the 0.75 kb fragment in plasmid pVWEx1-lysE was determined by the dideoxy chain termination method, according to Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America (1977) 74: 5463-5467). For this, the complete pVWExI lysE plasmid insert was sequenced with the aid of the oligonucleotide primers pVW_1.p (5'-TGA GCG GAT AAC AAT TTC ACA C-3 ') and pVW_2.p (5'-CGA CGG CCA GTG AAT TCG AG-3 ') at Eurofins MWG Operon GmbH (Ebersberg, Germany).
[00142] The nucleotide sequence obtained was analyzed using the Clone Manager 9 program and is shown through SEQ ID No. 20. Example 2 Construction of the pK18mobsacB DarqFRGH vector for deletion of the arqFRGH region in Corynebacterium glutamicum
[00143] For this, first, the chromosomal DNA was isolated from C. glutamicum ATCC13032 by the method of Tauch et al. (1995, Plasmid 33: 168-179). The oligonucleotides mentioned below were selected based on the sequence of the C. glutamicum argFRGH genes in order to prepare the argFRGH deletion construct. Said deletion construct was generated with the aid of the polymerase chain reaction (PCR), more specifically, by the Gene SOEing method (Gene Splicing by Overlap Extension, Horton, Molecular Biotechnology 3: 93-98 (1995)). argFRGH_d1: 5-GGT GGT GCT AGO CCG GCG ATT TCT TTG CAC AT-3 '(see SEQ ID No. 24) argFRGH_d2: 5'-AAT GCT TAT CGA CGT ACC CCC CTG TGG TTG TGA AGT CAT A-3' (see SEQ ID No. 25) argFRGH_d3: 5'-GGG GTA CGT CGA TAA GCA TT-3 '(see SEQ ID No. 26) argFRGH_d4: 5'-GGT GGT ATG CAT GGT GAT GGT TCC GAA TGT TG-3' (see SEQ ID No. 27)
[00144] The oligonucleotide primers shown were purchased from Eurofins MWG Operon GmbH (Ebersberg, Germany). The PCR reaction was performed using the Phusion® DNA polymerase "Hot Start" (Biozym Scientific GmbH, D-31840 Hess, Oldendorf) in a thermocycler (Mastercycler, EppendorfAG, Hamburg).
[00145] The argFRGH_d2 initiator is composed of two regions. A part of the nucleotide sequence is complementary to the region of 1 bp upstream to 19 bp downstream of the codon of initiation of the argF gene. The other part of the nucleotide sequence is complementary to the region of nucleotide 1419 of the argH gene up to 5 nucleotides downstream of the argH gene.
[00146] With the aid of the polymerase chain reaction, the primers argFRGH_1 and argFRGH_2 allow a 543 bp DNA fragment to be amplified and the argFRGH_3 and arg- FRGH 4 primers allow a 513 bp DNA fragment to be amplified. The amplicons were produced by PCR, tested by electrophoresis on a 0.8% agarose gel, isolated from said agarose gel using the High Pure PCR Product Purification Kit (product no. 1732676, Roche Diagnostics GmbH, Mannheim , Germany), and used as a template for another PCR reaction using the primers argFRGH I and argFRGH_4. In this way, the 1036 bp DargFRGH deletion derivative was generated (see also SEQ ID No. 21). This includes 477 bp from the 3 'end of the argD gene, 19 bp from the 5' end of the argF gene, 15 bp from the 3 'end of the argH gene, and 420 bp from the 5' end of the cg1589 reading frame. The product amplified in this way was tested by electrophoresis on an agarose gel with a concentration of 0.8%.
The 1.04 kb DargFRGH PCR product (SEQ ID No. 21) was completely cleaved by the enzymes Ndel and Nsil. The fragment was subsequently purified using the PCR purification kit (Qiagen, Hilden, Germany). The DargFRGH deletion derivative pretreated in this way was used in conjunction with the mobilizable cloning vector pK18mobsacB (Schãfer et al. (1994), Gene 14: 69-73) for ligation. Said cloning vector had previously been completely cleaved by the restriction endonucleases Xbal and Pstl. This produced DNA ends compatible with the ends of the insert generated by the Ndel and Nsil divage. The vector prepared in this way was mixed with the fragment of DargFRGH at a molar ratio of 1: 5 and ligated using T4 DNA ligase (Amersham-Pharmacia, Freiburg, Germany) at 16 ° C for 1 hour. Chemically competent E. coli DH5alpha cells (Subcloning efficiency, Invitogen GmbH, Karlsruhe, Germany) were transformed with 3 pl of the ligation mixture. Transformants were identified based on their resistance to kanamycin on LB agar plates containing 50 pg / ml kanamycin sulfate. Plasmid DNA was isolated from 4 of said transformants (QIAprep Spin Miniprep Kit from Qiagen (Hilden)), and plasmids were tested by restriction analysis for the presence of the 1.04 kb fragment as an insert. The recombinant plasmid produced in this way was called pK18mobsacB_DargFRGH. The strain was called E.coli_DH5alpha / pK18mobsacB_DargFRGH.
[00148] The nucleotide sequence of the 1.04 kb fragment (SEQ ID No. 21) in plasmid pK18mobsacB_DargFRGH was determined by the dideoxy chain termination method, according to Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America (1977) 74: 5463-5467). For this, the complete insert of the plasmid pK18mobsacB_DargFRGH was sequenced and tested, as well for accuracy with the aid of the oligonucleotide primers M13 uni (-21) (5'-TGT AAA ACG ACG GCC AGT-3 ') and M13 rev (-49) (5-GAG CGG ATA ACA ATT TCA CAC AGG-3 ') at Eurofins MWG Operon (Ebersberg, Germany). Example 3 Preparation of the strain Corynebacterium glutamicum ATCC 13032 DarqFRGH
[00149] The vector mentioned in example 2, pK18mobsacB_DargFRGH, was transferred by conjugation according to a protocol by Schãfer et al. (Journal of Microbiology 172: 1663-1666 (1990)) in the strain of Corynebacterium glutamicum ATCC13032. For this purpose, the vector had previously been transformed into the E. coli S17-1 strain (Simon et al., Biotechnology 1: 784-791). The identity of the vector in S17-1 was tested in a similar way to detection in E. coli DHδalpha (see Example 2).
[00150] The pK18mobsacB and pK18mobsacB_DargFRGH vectors cannot self-replicate in C. glutamicum ATCC13032 and remain in the cell only if they are integrated into the chromosome after a recombination event. Clones with integrated pK18mobsacB_DargFRGH are selected by plating the LB agar conjugation mixture (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2aEd., Cold Spring Harbor, New York, 1989) supplemented with 15 mg / L of kanamycin and 50 mg / ml nalidixic acid. Established clones are plated by cooling on LB agar plates containing 25 mg / L kanamycin and incubated at 33 ° C for 16 hours. Mutants in which the plasmid was cut due to a second recombination event are selected by culturing the clones in liquid LB medium without selection for 20 hours and then cooling them on LB agar containing 10% sucrose, followed by incubation for 24 hours. hours.
[00151] The plasmid pK18mobsacB_DargFRGH, like the initial plasmid pK18mobsacB, contains, in addition to the kanamycin resistance gene, a copy of the sacB gene that encodes levansucrase from Bacillus subtilis. Sucrose-inducible expression leads to the formation of levansucrase that catalyzes the synthesis of the product levan, which is toxic to C. glutamicum. Consequently, only clones in which the integrated pK18mobsacB_DargFRGH was cut again established growth on LB agar containing sucrose. The cut may comprise plasmid cut along with the complete chromosomal copy of argFRGH or the incomplete copy that has the internal argFRGH deletion.
[00152] Approximately 40 to 50 colonies were tested for the phenotype "growth in the presence of sucrose" and "no growth in the presence of kanamycin". In order to prove that the deleted argFRGH allele had remained on the chromosome, approximately 20 colonies having the phenotype "growth in the presence of sucrose" and "no growth in the presence of kanamycin" were studied by the standard PCR method of Innis et al. (PCR Protocols. A Guide to Methods and Applications, 1990, Academic Press) with the help of the polymerase chain reaction. This involved amplifying, from the chromosomal DNA of the colonies, a fragment of DNA that carries the regions surrounding the deleted argFRGH region. The following primer oligonucleotides were selected for PCR. argFRGH dl (SEQ ID No. 24): 5'-GGT GGT GCT AGC CCG GCG ATT TCT TTG CAC AT- 3 'argFRGH_d4 (SEQ ID No. 27): 5'-GGT GGT ATG CATGGT GAT GGT TCC GAA TGT TG- 3 '
[00153] In control clones containing the full argFRGH locus, the primers allow an approximately 5.35 kb DNA fragment to be amplified. In clones having a deleted argFRGH locus, DNA fragments approximately 1.04 kb in size are amplified.
[00154] The amplified DNA fragments were identified by electrophoresis on an agarose gel with a concentration of 0.8%. Through this, it was shown that the strain carries an argFRGH allele deleted on the chromosome. The strain was called Corynebacterium glutamicum Delta argFRGH.
[00155] Example 4:
[00156] Expression of the lysE gene in Corynebacterium glutamicum ATCC 13032_Delta_argFRGH
[00157] Plasmid pVWExI LysE and empty plasmid pVWExI were introduced into the L-ornithine-forming strain - ATCC 13032_Delta_argFGH - through electroporation (Haynes et al., FEMS Microbiology Letters (1989) 61: 329-334). Transformants were identified based on their resistance to kanamycin on Case agar plates containing 25 pg / ml kanamycin. 5 isolated clones were subsequently tested for accuracy of the transformed plasmid. For this purpose, plasmid DNA was isolated (Plasmid Isolation Kit, Qiagen), and this DNA was tested by restriction analysis for the correct divination pattern. Thus, the strains of C. glutamicum ATCC 13032_Delta_argFRGH / pVWEx1_lysE and ATCC 13032_Delta_argFRGH / pVWEx1 were produced. Example 5: Preparation of L-ornithine using Corynebacterium glutamicum
[00158] In order to study its ability to produce L-ornithine, in each case, three clones of the ATCC 13032_Delta_argFRGH / pVWEx1_lysE strain and three clones of the ATCC 13032_Delta_argFRGH / pVWEx1 strain were pre-cultivated, in each case, in 10 ml of medium. test at 33 ° C for 16 h. For the production test, in each case, 10 ml of the test medium were inoculated with the pre-culture obtained so that the ODeoo (optical density at 600 nm) at the beginning was 0.1. Each clone was tested in three shaker bottles so that each strain was represented at the respective collection time by nine shaker bottles in total. The test medium was identical to the CgXII medium described in Keilhauer et al. (Journal of Bacteriology (1993) 175: 5593-5603), but it contained, in addition, 7.5 g / L of yeast extract (Difco), 25 pg / ml of kanamycin, 1 mM of IPTG (isopropyl be- D-thiogalactopyranoside) and 40 g / L of sucrose instead of glucose. For the sake of simplicity, the composition of the test medium is summarized in Table 2 below. Table 2

[00159] Cultivation was performed in 100 ml shaker flasks at 33 ° C and 200 rpm. The agitator deflection was 5 cm. Three cultures of a clone were collected after 24 and 48 hours. For this, samples were taken from the cultures and the optical density, sucrose content and L-ornithine content were determined. To determine the sucrose and L-ornithine levels, the cells were removed by rapid centrifugation (type 5415D bench centrifuge (Eppendorf) at 13000 rpm, 10 min, room temperature).
[00160] The optical density was determined at a wavelength of 660 nm, using a GENios microtiter plate photometer (Tecan, Reading, UK). The samples were diluted 1: 100 with demineralized water before measurement.
[00161] Sucrose was determined using a test system (Catalog No. 10 716 251 035) from R-Biopharm AG (Darmstadt, Germany). This involves the inversion of sucrose and the glucose formed is detected using a coupled enzyme test (hexokinase / glucose-6-phosphate dehydrogenase) through the formation of NADH.
[00162] Quantitative determination of the extracellular amino acid concentration from the culture supernatant was performed using reverse phase HPLC (Lindroth et al., Analytical Chemistry (1979) 51: 1167-1174), with the use of an HP1100 series HPLC (Hewlett-Packard, Waldbronn, Germany) with connected fluorescence detector (G1321A); system control and data evaluation were performed using HP ChemStation equipment (Hewlett-Packard). 1 pL of the amino acid solution to be analyzed was mixed in an automatic pre-column derivatization with 20 pl of ortho-phthaladehyde / 2-mercapto ethanol reagent (Pierce Europe BV, Oud-Beijerland, Netherlands). The resulting thio-substituted fluorescent isoindois (Jones et al., Journal of Chromatography (1983) 266: 471-482) were fractionated into a combination of pre-column (40'4 mm Hypersil ODS 5) and main column (Hypersil ODS 5 , both columns from CS-Chromatographie Service GmbH, Langerwehe, Germany) using a gradient program with a progressively non-polar phase (methanol). The eluent was sodium acetate (0.1 M; pH 7.2); the flow rate was 0.8 mL per minute. The fluorescence of the derivatized amino acids was detected at an excitation wavelength of 230 nm and an emission wavelength of 450 nm. The concentrations of L-ornithine and / or L-ornithine hydrochloride were calculated by comparison with an external standard and L-asparagine as an additional internal standard.
[00163] The molecular weight of L-ornithine hydrochloride is 168.6 g x mol'1 and that of L-ornithine is 132.1 g x mol1.
[00164] The yield was calculated by dividing the amount of L-ornithine formed (measured as L-ornithine hydrochloride) by the amount of sucrose consumed.
[00165] The results are shown in table 3.
[00166] Table 3: L-ornithine formation after 24 hours (Table 3A) and 48 hours (Table 3B) of incubation. Abbreviations: *: ATCC 13032_Delta_argFRGH; Orn-HCI: L-ornithine hydrochloride. Table 3A:
Table 3B:
Example 6: Sequencing and deposition of plasmid pEC7lysE
[00167] The plasmid pEC7lysE was made available in the form of an aqueous solution by Dr. Lothar Eggeling (Forschungszentrum Jülich GmbH, D-52425 Jülich), the corresponding author of the publication Bellmann et al. (Microbiology (2001) 147, 1765-1774 ).
[00168] An aliquot of the obtained DNA solution was used to transform competent Escherichia coli cells of the DH5alpha strain (subcloning efficiency, genotype: F-ψ80 / acZΔM15 Δ (ZacZYA-argF) U169 recM endM hsc / R17 (rK-, mk +) pho / X supE44 I-thi-1 gyrA96 relM) from Invitrogen GmbH (Paisley, UK) according to the manufacturer's instructions. The transformants were selected on Luria-Bertani agar supplemented with 50 pg / mL kanamycin.
[00169] A transformant called Escherichia coli DH5alpha / pEC7lysE (DM2204) was deposited in accordance with the Budapest Treaty with the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Brunswick, Germany) under deposition number DSM 23239 on 15 January 2010.
[00170] The plasmid pEC7lysE of the strain DSM 23239 was completely sequenced by customized DNA sequencing (Walking Service) at Eurofins MWG Operon GmbH (Martinsried, Germany). The sequence of pEC7lysE is shown as SEQ ID No. 29.

权利要求:
Claims (6)
[0001]
1. Process for the preparation of L-ornithine, characterized by the fact that the following steps are performed: a) fermentation of a bacterium that excretes L-ornithine selected from the group consisting of Corynebacterium, Bacillus, Streptomyces, Arthrobacter and Enterobacteriaceae that overexpresses a polynucleotide that encodes a polypeptide that has the activity of an L-ornithine exporter and whose amino acid sequence is identical to the amino acid sequence of SEQ ID No. 2, in a medium, b) accumulation of said L-ornithine in said medium, in which a fermentation broth is obtained, and c) in which the plasmid pEC7lysE, deposited in DSM23239, is excluded for overexpression.
[0002]
2. Process, according to claim 1, characterized by the fact that, in the case of Corynebacterium glutamicum, overexpression increases the level of export activity of L-ornithine by at least 10% compared to ATCC13032 or ATCC14067 or ATCC13869.
[0003]
3. Process according to claim 1 or 2, characterized by the fact that overexpression is obtained by one or more measures selected from the group comprising a) increasing the number of copies, and b) using a strong promoter.
[0004]
Process according to any one of claims 1 to 3, characterized in that the bacterium is Corynebacterium, preferably Corynebacterium glutamicum.
[0005]
5. Process according to any one of claims 1 to 4, characterized in that it is selected from the group comprising a batch process, fed batch process, repetitive fed batch process, and continuous process.
[0006]
Process according to any one of claims 1 to 5, characterized by the fact that L-ornithine or a liquid or solid product containing L-ornithine is recovered from the fermentation broth containing L-ornithine.

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引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US366072A|1887-07-05|Harry g- |

---

US2988489A|1957-09-19|1961-06-13|Kyowa Hakko Kogyo Kk|Method of producing l-ornithine by fermentation|

---

JPS427767Y1|1965-01-11|1967-04-18|

---

JPS4310996Y1|1965-12-28|1968-05-13|

---

GB1140827A|1966-10-06|1969-01-22|Kyowa Hakko Kogyo Kk|Fermentative preparation of l-ornithine|

---

JPS463194Y1|1967-06-29|1971-02-03|

---

US3668072A|1969-01-02|1972-06-06|Chugai Pharmaceutical Co Ltd|Fermentation process for the production of l-ornithine|

---

JPS5741912B2|1976-08-19|1982-09-06|

---

JPS5927691B2|1980-08-28|1984-07-07|Maruyama Mfg Co|

---

JPH0362396B2|1984-11-15|1991-09-25|Kyowa Hakko Kogyo Kk|

---

US4990487A|1988-03-11|1991-02-05|The University Of Tokyo|Superconductive optoelectronic devices|

---

US4889942A|1989-04-12|1989-12-26|Dow Corning Corporation|Process for synthesis of acylamino organosilicon compounds|

---

JP2817185B2|1989-04-20|1998-10-27|味の素株式会社|Method for producing L-ornithine by fermentation|

---

JPH03195494A|1989-12-26|1991-08-27|Toray Ind Inc|Production of l-proline by fermentation|

---

DE69106297T2|1990-09-28|1995-05-24|Kyowa Hakko Kogyo Kk|Process for the preparation of crystals of a salt of acidic amino acid and basic amino acid.|

---

US5693781A|1991-06-03|1997-12-02|Mitsubishi Chemical Corporation|Promoter DNA fragment from coryneform bacteria|

---

JP3195494B2|1994-06-10|2001-08-06|スター精密株式会社|Fluid pressurizer|

---

KR0161147B1|1995-05-12|1998-11-16|김은영|Process for the preparation of ornithine|

---

DE19548222A1|1995-12-22|1997-06-26|Forschungszentrum Juelich Gmbh|Process for the microbial production of amino acids through increased activity of export carriers|

---

US6861246B2|1999-07-07|2005-03-01|Degussa Ag|L-lysine-producing corynebacteria and process for the preparation of lysine|

---

DE60042552D1|1999-08-02|2009-08-27|Archer Daniels Midland Co|METABOLIC MANIPULATION OF THE PRODUCTION OF AMINO ACIDS|

---

JP4623825B2|1999-12-16|2011-02-02|協和発酵バイオ株式会社|Novel polynucleotide|

---

US6562601B2|2000-08-31|2003-05-13|Degussa Ag|Fermentation process for the preparation of L-threonine|

---

DE10047865A1|2000-09-27|2002-04-18|Degussa|New nucleotide sequences coding for the deaD gene|

---

EP1266966B1|2001-06-12|2009-04-29|Ajinomoto Co., Inc.|Method for producing L-lysine or L-arginine by using methanol assimilating bacterium|

---

US7252978B2|2001-07-25|2007-08-07|Ajinomoto Co., Inc.|Method for producing L-arginine|

---

BR0211723A|2001-08-06|2004-09-21|Degussa|Corineform bacteria that produce chemical compounds i|

---

US7160711B2|2001-08-06|2007-01-09|Degussa Ag|Coryneform bacteria which produce chemical compounds I|

---

CN101126075B|2001-08-06|2012-05-09|赢创德固赛有限公司|Coryneform bacteria which produce chemical compounds ii|

---

WO2005021772A1|2003-08-29|2005-03-10|Degussa Ag|Process for the preparation of l-lysine|

---

DE10359660A1|2003-12-18|2005-07-28|Basf Ag|Psod expression units|

---

EP1664318B1|2004-01-30|2009-09-23|Ajinomoto Co., Inc.|L-amino acid-producing microorganism and method for producing l-amino acid|

---

CN1594282A|2004-07-15|2005-03-16|武汉武大弘元股份有限公司|Method for producing L-ornithine hydrochloride|

---

KR100589121B1|2004-08-20|2006-06-14|주식회사 엠에이치투 바이오케미칼|Preparation Method of L-ornithine Using Enzymatic Reaction|

---

JP4881739B2|2004-09-28|2012-02-22|協和発酵バイオ株式会社|Process for producing L-arginine, L-ornithine or L-citrulline|

---

CA2583514C|2004-10-07|2013-07-02|Ajinomoto Co., Inc.|Method for producing basic substance|

---

DE102004061846A1|2004-12-22|2006-07-13|Basf Ag|Multiple promoters|

---

DE102005013676A1|2005-03-24|2006-09-28|Degussa Ag|Alleles of the zwf gene from coryneform bacteria|

---

WO2007105790A1|2006-03-15|2007-09-20|Kyowa Hakko Kogyo Co., Ltd.|Method for purification of amino acid|

---

EP2386650B1|2006-04-07|2013-07-03|Evonik Degussa GmbH|Methodfor producing L-amino acids using the gap promoter|

---

EP1881076A1|2006-07-17|2008-01-23|Evonik Degussa GmbH|Method for producing L-amino acids|

---

HUE025440T2|2006-10-24|2016-04-28|Basf Se|Method of reducing gene expression using modified codon usage|

---

KR100789274B1|2007-01-15|2008-01-02|씨제이 주식회사|Novel promoter nucleic acid molecule derived from Corynebacterium glutamicum recombinant vector comprising the promoter host cell comprising the recombinant vector and method of expressing a gene using the host cell|

---

CN101323866A|2007-06-14|2008-12-17|上海聚瑞生物技术有限公司|Method for producing L-ornithine by microorganism fermentation|

---

KR100898246B1|2007-08-17|2009-05-18|씨제이제일제당 |Novel Corynebacterium glutamicum promoter|

---

US20110117614A1|2008-04-30|2011-05-19|Evonik Degussa Gmbh|Production Process for Methionine Using Microorganisms with Reduced Isocitrate Dehydrogenase Activity|US8647642B2|2008-09-18|2014-02-11|Aviex Technologies, Llc|Live bacterial vaccines resistant to carbon dioxide , acidic PH and/or osmolarity for viral infection prophylaxis or treatment|

---

DE102011118019A1|2011-06-28|2013-01-03|Evonik Degussa Gmbh|Variants of the promoter of the glyceraldehyde-3-phosphate dehydrogenase-encoding gap gene|

---

KR101526047B1|2013-04-26|2015-06-04|상지대학교산학협력단|Microorganism having improved L-ornithin production by increasing the aminotransferase activity and process for preparing the L-ornithin employing the same|

---

EP2811028B1|2013-06-03|2017-02-01|Evonik Degussa GmbH|Process for producing L-valine employing recombinant Corynebacteria comprising the propionate-inducible ilvBN operon|

---

JP2017131111A|2014-06-03|2017-08-03|味の素株式会社|Method for producing L-amino acid|

---

CN104031934A|2014-06-05|2014-09-10|江南大学|Method for improving yield of corynebacterium crenatum arginine by excessively co-expressing phosphofructokinase and pyruvate kinase|

---

WO2016144247A1|2015-03-12|2016-09-15|Biopetrolia Ab|L-ornithine production in eukaryotic cells|

---

HUE055862T2|2016-04-20|2021-12-28|Centro De Investig Energeticas|Compositions and methods for enhanced gene expression of pklr|

---

CN106148440B|2016-08-10|2021-12-07|洛阳华荣生物技术有限公司|Production of agmatine by fermentation method|

---

US11180535B1|2016-12-07|2021-11-23|David Gordon Bermudes|Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria|

---

US11129906B1|2016-12-07|2021-09-28|David Gordon Bermudes|Chimeric protein toxins for expression by therapeutic bacteria|

---

CN107236769B|2017-06-26|2021-01-26|南京工业大学|Method for preparing L-ornithine and succinic acid by stages by utilizing membrane circulation bioreactor|

---

EP3456833A1|2017-09-18|2019-03-20|Evonik Degussa GmbH|Method for the fermentative production of l-amino acids|

---

EP3467099A1|2017-10-05|2019-04-10|Evonik Degussa GmbH|Method for the fermentative production of l-amino acids|

---

CN107739728A|2017-10-19|2018-02-27|江南大学|A kind of recombination bacillus coli of efficiently production Glucosamine and its application|

---

CN109161507B|2018-09-27|2020-07-14|南京工业大学|Corynebacterium glutamicum capable of producing L-ornithine at high yield and application thereof|

---

CN110079566B|2019-05-16|2020-08-04|黑龙江伊品生物科技有限公司|Method for producing L-lysine by fermentation of bacteria with modified ppc promoter|

---

CN110195087B|2019-05-16|2020-12-01|黑龙江伊品生物科技有限公司|Method for producing L-lysine by fermentation using bacteria with modified ppc gene|

---

CN111334535B|2020-03-26|2021-11-30|廊坊梅花生物技术开发有限公司|Recombinant strain for producing L-amino acid and application thereof|

法律状态:
2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-07-16| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-05-12| B25D| Requested change of name of applicant approved|Owner name: EVONIK OPERATIONS GMBH (DE) |

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2020-07-21| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-11-17| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 24/03/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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DE102010003419.3A|DE102010003419B4|2010-03-30|2010-03-30|Process for the fermentative production of L-ornithine|

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DE102010003419.3|2010-03-30|

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PCT/EP2011/054541|WO2011124477A2|2010-03-30|2011-03-24|METHOD FOR THE PRODUCTION OF L-ORNITHINE USING BACTERIA THAT OVEREXPRESS LysE|

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