阅读说明：本技术 使用改良的肠杆菌科菌株生产l-色氨酸的方法 (Method for producing L-tryptophan using improved strains of the enterobacteriaceae family ) 是由 M·里平 S·耶伦特鲁普 N·杜施 于 2019-01-30 设计创作，主要内容包括：本发明提供了一种生产L-色氨酸的方法,该方法包括在发酵培养基中培养产生L-色氨酸的微生物(属于肠杆菌科)；其中产生L-色氨酸的微生物已通过增强mdfA基因的表达水平或通过增强mdfA等位基因的表达水平而被修饰。(The present invention provides a method for producing L-tryptophan comprising culturing a microorganism (belonging to the family Enterobacteriaceae) that produces L-tryptophan in a fermentation medium, wherein the microorganism that produces L-tryptophan has been modified by increasing the expression level of the mdfA gene or by increasing the expression level of the mdfA allele.)

1. A method for producing L-tryptophan, comprising culturing in a fermentation medium a microorganism producing L-tryptophan, which microorganism belongs to the family Enterobacteriaceae (Enterobacteriaceae), characterized in that the L-tryptophan-producing microorganism has been modified by increasing the expression level of the mdfA gene or by increasing the expression level of the mdfA allele.

2. The method according to claim 1, characterized in that the mdfA allele is a polynucleotide selected from the group consisting of:

a) comprises the amino acid sequence of SEQ ID NO: 1;

b) under stringent conditions with SEQ ID NO: 1 to the complementary strand of the nucleic acid sequence;

c) a naturally occurring mutant or polymorphic form of a polynucleotide according to a) or b);

d) and SEQ ID NO: 1 has at least 80% sequence identity;

e) relative to SEQ ID NO: 1 comprises a substitution, deletion, insertion or addition of 1 to 60 nucleotides;

f) encodes a polypeptide comprising SEQ ID NO: 2;

g) a polynucleotide encoding a protein comprising an amino acid sequence set forth in SEQ ID NO: 2 comprises substitution, deletion, insertion or addition of 1 to 20 amino acids.

3. Method according to claim 1 or 2, characterized in that in said L-tryptophan-producing microorganism the expression level of the mdfA gene or mdfA allele is increased by increasing the copy number of the mdfA gene or mdfA allele by at least 1.

4. The method according to any of the preceding claims, characterized in that in said L-tryptophan-producing microorganism the copy number of the mdfA gene or mdfA allele is increased by at least 1 by integration of the gene into the chromosome of the microorganism.

5. The method according to any of the preceding claims, characterized in that in said L-tryptophan-producing microorganism the expression level of the mdfA gene or mdfA allele is enhanced by modifying the expression regulatory sequence of the gene.

6. The method according to any one of the preceding claims, characterized in that the expression level of the mdfA gene or mdfA allele is enhanced by using an inducible promoter.

7. The method according to any one of the preceding claims, characterised in that the mdfA gene or copy of the mdfA allele is integrated into the mtr locus whilst the mtr gene is deleted.

8. The method of claim 7, wherein the chromosomal environment of the mtr locus regulates expression of the mdfA gene or mdfA allele integrated into the mtr locus.

9. The method according to any of the preceding claims, characterized in that the L-tryptophan-producing microorganism is selected from the group consisting of the genera Escherichia (Escherichia), Erwinia (Erwina) and Providencia (Providencia).

10. The method according to any of the preceding claims, characterized in that the microorganism producing L-tryptophan is Escherichia coli (Escherichia coli).

11. A method for producing L-tryptophan-producing microorganisms by transformation, transduction, or conjugation, the method characterized in that the transformation, transduction, or conjugation is performed using a vector comprising:

a) comprises the amino acid sequence of SEQ ID NO: 1;

b) under stringent conditions with SEQ ID NO: 1 to the complementary strand of the nucleic acid sequence;

c) a naturally occurring mutant or polymorphic form of a polynucleotide according to a) or b);

d) and SEQ ID NO: 1 has at least 80% sequence identity;

e) relative to SEQ ID NO: 1 comprises a substitution, deletion, insertion or addition of 1 to 60 nucleotides;

f) encodes a polypeptide comprising SEQ ID NO: 2;

g) a polynucleotide encoding a protein comprising an amino acid sequence set forth in SEQ ID NO: 2 comprises substitution, deletion, insertion or addition of 1 to 20 amino acids;

and promoters which regulate the expression of polynucleotides a) -g).

12. An L-tryptophan-producing microorganism of the enterobacteriaceae family, characterized in that said microorganism has been modified by increasing the expression level of an mdfA gene or by increasing the expression level of an mdfA allele.

13. L-tryptophan-producing microorganism as claimed in claim 12, characterized in that the mdfA allele is a polynucleotide selected from the group consisting of:

a) comprises the amino acid sequence of SEQ ID NO: 1;

b) under stringent conditions with SEQ ID NO: 1 to the complementary strand of the nucleic acid sequence;

c) a naturally occurring mutant or polymorphic form of a polynucleotide according to a) or b);

d) and SEQ ID NO: 1 has at least 80% sequence identity;

e) relative to SEQ ID NO: 1 comprises a substitution, deletion, insertion or addition of 1 to 60 nucleotides;

f) encodes a polypeptide comprising SEQ ID NO: 2;

g) a polynucleotide encoding a protein comprising an amino acid sequence set forth in SEQ ID NO: 2 comprises substitution, deletion, insertion or addition of 1 to 20 amino acids.

14. The L-tryptophan-producing microorganism according to claim 12 or 13, wherein the copy of the mdfA gene or mdfA allele is integrated into the mtr locus while the mtr gene is deleted, and wherein optionally the chromosomal environment of the mtr locus regulates the expression of the mdfA gene or mdfA allele integrated into the mtr locus.

15. Use of the L-tryptophan-producing microorganism of any one of claims 12-14 in the production of L-tryptophan or in the production of a feed additive comprising L-tryptophan.

Technical Field

The present invention relates to a novel method for the fermentative production of L-tryptophan using a modified Enterobacteriaceae (Enterobacteriaceae) microorganism in which the expression level of mdfA gene is enhanced.

Background

L-Tryptophan is used in human medicine and in the pharmaceutical industry, in the food industry and in animal nutrition.

L-tryptophan can be produced by fermentation of strains of the Enterobacteriaceae family, in particular Escherichia coli (E.coli) and Serratia marcescens (Serratia marcocens), efforts are constantly being made to improve the production process because of its great significance.

Process improvements may relate to measures associated with fermentation technology, such as, for example, stirring and supply of oxygen, or the composition of the nutrient media, for example, the choice of the sugars used or the sugar concentration during the fermentation, or the work-up in product form, for example by ion exchange chromatography, or the intrinsic performance properties of the microorganism itself.

In wild-type strains, strict regulatory mechanisms prevent the production of metabolites, such as amino acids, in excess of those required by the strain and prevent release into the culture medium. Therefore, from the manufacturer's point of view, the construction of amino acid overproducing strains requires overcoming these metabolic regulations.

Methods of mutagenesis, selection and mutant selection are used to remove the control mechanisms and improve the performance characteristics of these microorganisms.

Due to the complexity of the biosynthetic pathway of aromatic L-amino acids, such as L-tryptophan, and due to their interconnection with many other metabolic pathways in the cell, it is not always possible to predict which genetic variations or modifications of a strain can achieve an improvement in L-tryptophan production.

Multidrug transporter MdfA is called proton-powered efflux pump (L ewinson et al, Proc. Natl. Acad. Sci. U.S.A.100(4) 1667-1672.) its additional function as a Na +/H + antiporter and its involvement in the production and accumulation of amino acids (e.g., L-lysine, L-arginine, L-threonine or L-histidine) has been reported in WO2007/069782A1, wherein bacteria of the Enterobacteriaceae family are disclosed that have increased expression levels of genes selected from nhaA, nhaB, nhaR, chaA, mdfA and combinations thereof.

However, WO2007/069782A1 does not mention the effect of enhanced mdfA expression levels on L-tryptophan productivity.

In view of the above, there is still a need to further optimize especially those strains of the Enterobacteriaceae family which are suitable for the production of L-tryptophan and to provide improved methods for the fermentative production of L-tryptophan using these microorganisms of the Enterobacteriaceae family.

Disclosure of Invention

The present invention provides a method for producing L-tryptophan comprising culturing a L-tryptophan-producing microorganism in a fermentation medium, said microorganism belonging to the family enterobacteriaceae, wherein said L-tryptophan-producing microorganism has been modified by increasing the expression level of the mdfA gene or by increasing the expression level of the mdfA allele.

Furthermore, the present invention relates to a method for the preparation of L-tryptophan-producing microorganisms by transformation, transduction or conjugation, wherein said transformation, transduction or conjugation is performed by a vector comprising:

a) comprises the amino acid sequence of SEQ ID NO: 1;

b) under stringent conditions with SEQ ID NO: 1 to the complementary strand of the nucleic acid sequence;

c) a naturally occurring mutant or polymorphic form of a polynucleotide according to a) or b);

d) and SEQ ID NO: 1 has at least 80% sequence identity;

e) relative to SEQ ID NO: 1 comprises a substitution, deletion, insertion or addition of 1 to 60 nucleotides;

f) encodes a polypeptide comprising SEQ ID NO: 2;

g) a polynucleotide encoding a protein comprising an amino acid sequence set forth in SEQ ID NO: 2 comprises substitution, deletion, insertion or addition of 1 to 20 amino acids;

and including promoters that regulate the expression of polynucleotides a) -g).

Furthermore, the present invention provides a microorganism of the enterobacteriaceae family which produces L-tryptophan, wherein said microorganism has been modified by increasing the expression level of the mdfA gene or by increasing the expression level of the mdfA allele.

Finally, the invention relates to the use of the above-mentioned microorganism for the production of L-tryptophan or the use of the above-mentioned microorganism for the production of a feed additive containing L-tryptophan.

Drawings

FIG. 1: map of replacement vector pKO3Darop containing Darop deletion fragment

FIG. 2: map of mdfA-containing replacement vector pKO3Darop

FIG. 3: map of replacement vector pKO3Dmtr containing Dmtr deletion fragment

FIG. 4: replacement vector pKO3Dmtr containing mdfA Gene map of mdfA

FIG. 5: map of plasmid pMU91

The specified lengths are to be understood as approximations. The abbreviations and names used have the following meanings:

CmR: chloramphenicol resistance gene

sacB: SacB gene from Bacillus subtilis

RepA: temperature-sensitive replication region of plasmid pSC101

pdhR': a portion of the coding region of the pdhR gene

mdfA: coding region of mdfA gene

Λ ampE: part of the coding region of the ampE Gene

Λ yhbW: part of coding region of yhbW gene

Λ deaD: part of the coding region of the deaD Gene

pSC 101: plasmid fragment pSC101

serA: the coding region of the serA gene which codes for D-3-phosphoglycerate dehydrogenase

TrpE476DCBA A part of the Tryptophan operon trp L EDCBA with the trpE476 allele

tetA: tetracycline resistance genes

Abbreviations for restriction enzymes have the following meanings

SalI: restriction endonucleases from Streptomyces albus G

HindIII: restriction endonucleases from Haemophilus influenzae Rd (Haemophilus influenzae Rd)

XhoI: restriction endonucleases from Xanthomonas (Xanthomonas holcicola)

Further details can be found in the examples.

Hereinafter, the present invention is illustrated by non-limiting examples and exemplary embodiments.

Detailed Description

In the work forming the basis of the present invention, it was surprisingly found that L-tryptophan productivity (productivity) can be significantly increased in strains of the enterobacteriaceae family which produce L-tryptophan by modulating the expression level of the mdfA gene or modulating the expression level of the mdfA allele, respectively, in an appropriate manner.

More specifically, the present invention provides a method for producing L-tryptophan comprising culturing in a fermentation medium a microorganism producing L-tryptophan, said microorganism belonging to the family Enterobacteriaceae, wherein said L-tryptophan-producing microorganism has been modified by increasing the expression level of the mdfA gene or by increasing the expression level of the mdfA allele.

For clarity and completeness, the coding region of the mdfA gene is set forth in SEQ ID NO: 1 is shown. The amino acid sequence of MdfA is described in SEQ ID NO: 2 in (c).

An allele is a variant of a given gene.

The mdfA allele according to the invention may be a polynucleotide selected from:

a) comprises the amino acid sequence of SEQ ID NO: 1;

b) under stringent conditions with SEQ ID NO: 1 to the complementary strand of the nucleic acid sequence;

c) a naturally occurring mutant or polymorphic form of a polynucleotide according to a) or b);

d) and SEQ ID NO: 1, having a sequence identity of at least 80%, at least 85% or at least 90%, preferably at least 95%;

e) relative to SEQ ID NO: 1 includes a polynucleotide of substitution, deletion, insertion or addition of 1 to 60 nucleotides;

f) encodes a polypeptide comprising SEQ ID NO: 2;

g) a polynucleotide encoding a protein comprising an amino acid sequence set forth in SEQ ID NO: 2 comprises substitution, deletion, insertion or addition of 1 to 20 amino acids.

The terms "enhance" or "enhanced" or "overexpressed" or "increased expression level", "enhanced expression level" or "overexpression" are used interchangeably herein and have similar meanings. In this case, these terms describe an increase in the intracellular activity of the enzyme activity encoded by the corresponding DNA, for example by increasing the copy number of the gene, by using a stronger promoter or by using an allele with increased activity and by combining these measures.

The expression level of the mdfA gene or mdfA allele can be enhanced by increasing the copy number of the gene or allele by at least 1.

In order to increase the expression level of the gene/allele, it is preferable to add a copy of the gene/allele on the chromosome. One or several additional copies may be present on the chromosomal genome, which may be introduced by recombinant methods known to those skilled in the art. Extrachromosomal genes can be carried by different types of plasmids or bacterial artificial chromosomes that differ in their origin of replication and therefore in their copy number in the cell. Corresponding to low, medium or high copy number plasmids with compact replication, they may be present in 1-5 copies, about 20 copies or up to 500 copies.

The copy number of the mdfA gene or mdfA allele can be increased by at least 1 by integrating the gene into the chromosome of the microorganism.

The expression level of the mdfA gene or mdfA allele can also be enhanced by modifying the expression regulatory sequence of the gene. As previously described, the mdfA gene or mdfA allele can be combined with a stronger promoter. These promoters may be inducible; they may be homologous or heterologous. The skilled person knows which promoter is most convenient.

When genes are organized in operons, their expression level can be enhanced by adding a supplementary copy of these genes under the control of a single promoter. Expression may also be enhanced by replacing the chromosomal wild-type promoter with an artificial promoter that is stronger than the wild-type promoter. Experts in the field know how to determine promoter strength.

The microorganisms according to the invention and the microorganisms used in the process according to the invention are each representatives of the family Enterobacteriaceae and are selected from the genera Escherichia (Escherichia), Erwinia (Erwinia) and Providencia (Providecia). The genus Escherichia is preferred. Particular mention must be made of the species Escherichia coli. Preferably, a microorganism with increased expression of the Trp operon is used.

The inventors have surprisingly found that particularly strong L-tryptophan productivity can be achieved in strains of the enterobacteriaceae family, in particular e.coli, that produce L-tryptophan in the case of integration of the mdfA gene or mdfA allele into a specific chromosomal or extra-chromosomal (extra-chromosomal) locus, more particularly, the inventors have found that the expression level of mdfA can be regulated by using specific chromosomal or extra-chromosomal loci for mdfA integration, thereby improving the fermentability of L-tryptophan when commercially relevant concentrations are accumulated in the fermentation broth.

As used in the context of the present invention, the term "modulation of expression levels" refers to setting expression levels that result in a balance of optimal yields of the amino acid (L-tryptophan) product.

The expression of stably integrated genes is strongly influenced by regulatory sequences in the chromosomal environment of the integration site, e.g., the combined effects of the start codon and flanking regions have been described (Stenstrom et al, Gene 273 (2): 259-65 (2001); Hui et al, EMBO Journal 3 (3): 623-9 (1984)).

In one embodiment, a copy of the mdfA gene or mdfA allele that has the native expression signal is integrated into the chromosomal mtr locus, thereby simultaneously deleting the mtr gene. Preferably, the mtr promoter remains intact as shown in SEQ ID NO: 25, i.e., the flanking regions may regulate the expression of the mdfA gene or mdfA allele.

Additionally or alternatively to the above, a copy of the mdfA gene or mdfA allele may be integrated into the chromosomal aroP locus, thereby simultaneously deleting the aroP gene. Preferably, the aroP promoter remains intact, i.e.the flanking regions may regulate the expression of the mdfA gene or the mdfA allele.

Thus, by using different integration loci, the mdfA expression level can be adapted to the specific needs of the abundance of mdfA protein in the cell membrane, since regulatory sequences of the flanking regions may have an influence.

The microorganism of the invention and the microorganism used in the method of the invention, respectively, can be prepared using the aforementioned methods for transformation, transduction, or conjugation. Preferably, the transformation, transduction, or conjugation is performed using a vector comprising:

a) comprises the amino acid sequence of SEQ ID NO: 1;

b) under stringent conditions with SEQ ID NO: 1 to the complementary strand of the nucleic acid sequence;

c) a naturally occurring mutant or polymorphic form of a polynucleotide according to a) or b);

d) and SEQ ID NO: 1, having a sequence identity of at least 80%, at least 85% or at least 90%, preferably at least 95%;

e) relative to SEQ ID NO: 1 comprises a substitution, deletion, insertion or addition of 1 to 60 nucleotides;

f) encodes a polypeptide comprising SEQ ID NO: 2;

g) a polynucleotide encoding a protein comprising an amino acid sequence set forth in SEQ ID NO: 2 comprises substitution, deletion, insertion or addition of 1 to 20 amino acids;

and promoters which regulate the expression of polynucleotides a) -g).

For the preparation of the strains of the Enterobacteriaceae family of the present invention, preference is given to using strains which already have the ability to enrich L-tryptophan in the cells and/or to secrete it into the nutrient medium surrounding the cells or to accumulate it in the fermentation broth (starting strains or parent strains).

More specifically, the strains used in the manner of the present invention have the ability to enrich or accumulate in cells and/or in nutrient media or fermentation broths for not more than (at least) 0.25g/l,. gtoreq.0.5 g/l,. gtoreq.1.0 g/l,. gtoreq.1.5 g/l,. gtoreq.2.0 g/l,. gtoreq.4 g/l,. gtoreq.10 g/l,. gtoreq.20 g/l,. gtoreq.30 g/l or. gtoreq.50 g/l L-tryptophan in not more than (at most) 120 hours,. ltoreq.96 hours,. ltoreq.48 hours,. ltoreq.36 hours,. ltoreq.24 hours or. ltoreq.12 hours.

Preferred recombinant microorganisms containing the nucleotide sequence of the present invention can produce L-tryptophan from glucose, sucrose, lactose, fructose, maltose, molasses, possibly starch, possibly cellulose or from glycerol and ethanol, and possibly from mixtures.

Examples of strains of the genus Escherichia, in particular of the species Escherichia coli, which are suitable for the parent strain and produce or secrete L-tryptophan are:

coli SV164(pGH5) (WO 94/08031)

Escherichia coli AGX17(pGX44) (NRR L B-12263) (U.S. Pat. No. 4,371,614)

Coli AGX6(pGX50) aroP (NRR L B-12264) (U.S. Pat. No. 4,371,614)

Escherichia coli AGX17/pGX50, pACKG4-pps (WO 97/08333)

Escherichia coli ATCC 31743 (CA 1182409)

Coli C534/pD2310, pDM136(ATCC 39795) (WO 87/01130)

Escherichia coli JB102/p 5L RPS2 (US 5,939,295).

The L-tryptophan-producing strain or L-tryptophan-secreting strain from the family enterobacteriaceae preferably has, among other things, one or more genetic or phenotypic characteristics selected from the group consisting of 5-methyl-D L-tryptophan resistance, 5-fluoro-tryptophan resistance, 4-methyl-D L-tryptophan resistance, 6-methyl-D L-tryptophan resistance, 4-fluoro-tryptophan resistance, 6-fluoro-tryptophan resistance, anthranilate resistance, indazole alanine (trpitazan) resistance, indole resistance, indoleacrylic acid resistance, phenylalanine requirement, tyrosine requirement, ability to potentially utilize sucrose, enhanced tryptophan operon, preferably anthranilate synthase, preferably in a feedback-resistant form, partially defective tryptophanyl-tRNA synthetase, attenuated tryptophan uptake, defective tryptophane enzyme, inactivated repressor protein, enhanced serine biosynthesis, enhanced phosphoenolpyruvate synthesis, enhanced D-erythrose-4-deoxyphosphate synthesis, enhanced 3-D-arabitol-7-AP-phosphate synthesis, enhanced branched-phosphate biosynthesis of DHAP 7 (DHAP).

Furthermore, for the production of L-tryptophan with strains of the Enterobacteriaceae family, it may be advantageous to additionally enhance one or more enzymes of the known biosynthetic pathway, or enzymes of anaplerotic metabolism (anaplerotic metabolism), or enzymes for the production of reduced nicotinamide adenine dinucleotide phosphate, or enzymes of glycolysis, or PTS enzymes, or enzymes of sulfur metabolism.

Furthermore, for the production of L-tryptophan, it may be advantageous to additionally shut down the undesirable secondary reactions (Nakayama: "Breeding of Amino Acid Producing Microorganisms", in: overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, L ondon, UK, 1982).

In accordance with the above, the present invention further provides an L-tryptophan-producing microorganism of the enterobacteriaceae family, wherein the microorganism has been modified by enhancing the expression level of an mdfA gene or by enhancing the expression level of an mdfA allele.

In one embodiment, the mdfA allele is a polynucleotide selected from the group consisting of:

a) comprises the amino acid sequence of SEQ ID NO: 1;

b) under stringent conditions with SEQ ID NO: 1 to the complementary strand of the nucleic acid sequence;

c) a naturally occurring mutant or polymorphic form of a polynucleotide according to a) or b);

d) and SEQ ID NO: 1, having a sequence identity of at least 80%, at least 85% or at least 90%, preferably at least 95%;

e) relative to SEQ ID NO: 1 comprises a substitution, deletion, insertion or addition of 1 to 60 nucleotides;

f) encodes a polypeptide comprising SEQ ID NO: 2;

g) a polynucleotide encoding a protein comprising an amino acid sequence set forth in SEQ ID NO: 2 comprises substitution, deletion, insertion or addition of 1 to 20 amino acids.

To enhance mdfA expression, the mdfA gene/allele is preferably integrated in the chromosomal mtr locus. In a specific embodiment, the mdfA gene or a copy of the mdfA allele is integrated in the mtr locus, while the mtr gene is deleted. Optionally, the chromosomal environment of the mtr locus remains intact and regulates expression of the mdfA gene or mdfA allele integrated in the mtr locus.

The L-tryptophan-producing microorganism can be used for producing L-tryptophan or used for producing L-tryptophan-containing feed additives.

The microorganisms according to the invention and the microorganisms used in the process according to the invention are cultivated in a batch process, a fed-batch process, a repeated fed-batch process or a continuous process, respectively (DE102004028859.3 or U.S. Pat. No. 5,763,230). A general description of these processes is given in the textbook Chmiel (Bioprozesstechnik 1.Einf ü hrung in dieBioverfahrentechnik [ Bioprocess technology 1.Introduction to Bioprocess chology ] (Gustav Fischer Verlag, Stuttgart,1991)) or the textbook Storhas (Bioreturnen und periphere Einrichtungen [ Bioreactors and peripheres ] (Vieweg Verlag, Braunschweid/Wiesbaden, 1994)).

In the case of a batch process, all starting materials, with the exception of some exceptions, for example, oxygen and pH correctors, are initially added in the form of a batch, and the microorganisms are cultured in the culture medium obtained.

In the case of a fed-batch process, the microorganisms are initially cultivated by a batch process (batch phase). The starting materials necessary for the preparation of the product, and if desired also a plurality of starting materials (feed stage), are then added to the culture in a continuous or discontinuous manner.

In the case of a repeated fed-batch process (a fed-batch process is concerned), wherein after completion of the fermentation, a portion of the fermentation broth obtained is used as inoculum to start a new repeated fed-batch process. This cycle may be repeated as many times as necessary. Repeated fed-batch processes are described, for example, in WO 02/18543 and WO 05/014843.

In the case of a continuous process, after a batch or fed-batch process, one or more, possibly all, of the starting materials are added continuously to the culture and the fermentation broth is removed at the same time. Continuous processes are described, for example, in patent documents US 5,763,230, WO 05/014840, WO 05/014841 and WO 05/014842. The culture medium must meet the requirements of the particular strain in a suitable manner. The culture media for different microorganisms are described in the Manual "Manual of Methods for general Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981). The terms medium, fermentation medium and nutrient medium or culture medium are interchangeable.

In general, the medium contains, inter alia, one or more carbon, nitrogen and phosphorus sources.

Sugars and carbohydrates, such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and possibly cellulose, may be used as carbon sources; oils and fats such as soybean oil, sunflower oil, peanut oil and coconut butter; fatty acids such as palmitic acid, stearic acid and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances may be used alone or as a mixture.

It is possible to use as nitrogen source organic nitrogen-containing compounds such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soybean flour and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or as a mixture.

It is possible to use phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts as phosphorus source.

In addition, the culture medium must contain metal salts, such as magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances such as amino acids and vitamins can be used in addition to the above-mentioned substances. In addition, suitable precursors may be added to the culture medium. The above starting materials may be added to the culture in the form of a single batch, or may be appropriately fed during the culture.

The fermentation is usually carried out at a pH of 5.5 to 9.0, more particularly 6.0 to 8.0. In order to control the pH of the culture, it is appropriate to use basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acidic compounds, such as phosphoric acid or sulfuric acid. To control the generation of foam, an antifoaming agent such as fatty acid polyglycol ester may be used. To maintain the stability of the plasmid, suitable selective substances, such as antibiotics, can be added to the medium. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures (e.g., air) are introduced into the culture.

The cultivation temperature is usually 25 ℃ to 45 ℃, preferably 30 ℃ to 40 ℃ the activity of the microorganism results in the enrichment or accumulation of L-tryptophan in the fermentation or culture broth, the cultivation is continued until the maximum amount of L-tryptophan is formed, the goal is usually reached within 10 hours to 160 hours, in a continuous process, there may be a longer cultivation time.

After completion of the fermentation, the resulting fermentation broth accordingly comprises a) biomass of the microorganism (cell mass) which is produced as a result of the propagation of the microbial cells, b) L-amino acid (L-tryptophan) which is formed during the fermentation, c) organic by-products which are formed during the fermentation, and d) constituents of the fermentation medium/culture medium used or of the starting material which are not consumed by the fermentation, for example vitamins such as thiamine or salts such as magnesium sulfate.

The resulting broth or fermentation broth can then be collected and L-tryptophan or a product containing L-tryptophan can be obtained or isolated.in one process variant, the fermentation broth is concentrated if desired, then L-tryptophan is purified or isolated in pure or nearly pure form.A typical method for purifying L-amino acids such as L-tryptophan is ion exchange chromatography, crystallization, extraction and treatment with activated carbon. the result is predominantly pure L-tryptophan containing ≥ 90%, > 95%, > 96%, > 97%, > 98% or ≥ 99% by weight.

In a further process variant, it is likewise possible to prepare the product from a fermentation liquor which is produced by removing the bacterial biomass present in the fermentation liquor to a complete extent (100%) or to an almost complete extent, i.e. more than or greater than (>) 90%, > 95%, > 97%, > 99%, and by leaving the remaining constituents of the fermentation liquor to a large extent in the product, i.e. to an extent of 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, or 90% to 100%, preferably more than or equal to (≧) 50%, ≧ 60%, ≧ 70%,. gtoreq.80%,. gtoreq.90%, or ≧ 95%, or to a complete extent (100%).

The biomass is removed or separated by using a separation method, such as centrifugation, filtration, decantation, flocculation, or a combination thereof. The resulting broth is then thickened or concentrated using known methods, for example by means of a rotary evaporator, by means of a thin-film evaporator, by means of a falling-film evaporator, by reverse osmosis, by nanofiltration or a combination thereof.

The concentrated broth is then processed by freeze drying, spray granulation or other methods to yield a preferably free-flowing fine powder. This free-flowing fine powder can then be converted into a coarse, highly free-flowing, storable and substantially dust-free product by a suitable compaction or granulation process. Here, water is completely removed to the extent of 90% or more, and thus the water content in the product is less than 10% by weight, less than 5% by weight, less than 4% by weight, or less than 3% by weight.

The L-tryptophan is isolated by ion exchange chromatography, preferably cation exchange chromatography, followed by post-column derivatization using ninhydrin, L-tryptophan can be analyzed to determine the concentration at one or more times during fermentation, see Spackman et al (Analytical Chemistry 30:1190-1206 (1958)). the post-column derivatization can also be performed using o-phthaldehyde instead of ninhydrin. overview on ion exchange chromatography can be found in Pickering (L C. GC (Magazine of Chromatographic science)7(6),484-487 (1989)).

Pre-column derivatization can likewise be carried out, for example using o-phthalaldehyde or phenyl isothiocyanate, and fractionating the resulting amino acid derivatives by reverse phase chromatography (RP), preferably in the form of high performance liquid chromatography (HP L C). this method is described, for example, in L index et al (Analytical Chemistry 51:1167-1174(1979)) and detection by photometric methods (absorption, fluorescence).

The process and the microorganism according to the invention are used for the fermentative preparation of L-tryptophan.