CN120683027A

CN120683027A - A recombinant microorganism for high L-tryptophan production, and its construction method and application

Info

Publication number: CN120683027A
Application number: CN202510558266.6A
Authority: CN
Inventors: 周本正; 杨梦雪
Original assignee: Shanghai Ruikang Biotechnology R & D Co ltd
Current assignee: Shanghai Ruikang Biotechnology R & D Co ltd
Priority date: 2025-04-29
Filing date: 2025-04-29
Publication date: 2025-09-23

Abstract

The invention belongs to the technical field of metabolic engineering, and particularly relates to a recombinant microorganism for high-yield L-tryptophan as well as a construction method and application thereof. The recombinant klebsiella oxytoca (Klebsiella oxytoca) for high-yield L-tryptophan is constructed by screening the knocked-out or added genes and through a genetic engineering technology. The recombinant klebsiella oxytoca disclosed by the invention takes glucose as a substrate, can be used for efficiently producing L-tryptophan under a fermentation condition, and has the yield of 60g/L and the yield of 0.23g/g. The invention provides a method for realizing efficient production of L-tryptophan by microorganisms by redirecting 2, 3-butanediol anabolic flux to L-tryptophan synthesis through pyruvic acid, which has the advantages of simple production process, low cost, high yield, important economic benefit and good prospect.

Description

Recombinant microorganism for high-yield L-tryptophan as well as construction method and application thereof

Technical Field

The invention belongs to the technical field of metabolic engineering, and particularly relates to a recombinant microorganism for high-yield L-tryptophan as well as a construction method and application thereof.

Background

Tryptophan (Tryptophan, L-Trp), D, L-alpha-amino-beta-indolyl propionic acid, belongs to an aromatic amino acid, and has a molecular formula of C ₁₁H₁₂O₂N₂ and a relative molecular mass of 204.21. Tryptophan has 3 isomers, namely L-type, D-type and raceme DL-type, wherein the left optical isomer, namely L-Trp, has biological activity and is one of essential amino acids of human body. L-Trp is white crystal or crystalline powder under normal temperature, has no smell, has a melting point of 289 ℃, has a solubility of 11.4g/L in water at normal temperature, is slightly soluble in ethanol, is insoluble in chloroform, and is relatively stable in alkaline solution.

Tryptophan is an important precursor for auxin biosynthesis in plants and has a structure similar to IAA (indole-3-acetic acid ) and is ubiquitous in higher plants. L-tryptophan, one of the 8 essential amino acids, cannot be synthesized in the mammalian body and must be taken up from the outside. Therefore, the L-tryptophan is widely applied to various fields of different industries such as food, medicine, feed and the like. In the food industry, L-tryptophan is used as a nutritional supplement, food enhancer, preservative, and the like. In addition, L-tryptophan can also be used as a precursor for producing the food pigment indigo pigment through fermentation. In the pharmaceutical manufacturing industry, L-tryptophan is commonly used in the fields of health products, biological medicines, medical raw materials and the like. In addition, L-tryptophan is used as a precursor of neurotransmitter 5-hydroxytryptamine and melatonin and is also used for synthesizing antidepressants and tranquilizer. In the feed industry, the L-tryptophan is used as a feed additive, and has positive effects on the aspects of growth and production, nutrition metabolism, immunity and the like of poultry and livestock. Up to now, the market demand for L-tryptophan has exceeded 28,000 t/year, and the market size of the product has been expanding. However, the relatively late production technology results in high production cost and low yield of L-tryptophan, thereby limiting the larger-scale application of the L-tryptophan.

In the conventional industrial production, fermentation methods of L-tryptophan are mainly classified into chemical synthesis methods and protein hydrolysis methods. However, the method for producing L-tryptophan by chemical synthesis has a plurality of defects such as high production cost, severe process conditions, environmental pollution and the like. Along with the development of gene recombination technology and the continuous analysis of model biological metabolic networks, the transformation of microorganisms to synthesize L-tryptophan by using genetic engineering means has received great attention.

Coli (ESCHERICHIA COLI) is the main strain for producing L-tryptophan by the current fermentation method. Along with the continuous development of metabolic engineering technology, the yield and the yield of the L-tryptophan produced by microbial fermentation are continuously improved. Guo et al obtained recombinant strain E.coli TRP12 by metabolic engineering means such as releasing negative feedback effect, enhancing tryptophan transport and enhancing precursor supply on the basis of mutagenesis strain E.coli TRP0, and after fermentation culture in a 5L tank, L-tryptophan concentration reached 52.1g/L and conversion rate reached 0.171g/g (Guo L et al, biotechnol bioeng.,2022,119 (3): 983-993). Xiong et al increased precursor supply by introducing a phosphoketolase pathway, a non-PTS system and an oxaloacetate carboxykinase pathway on the basis of a tryptophan-producing strain, and constructed recombinant strains with L-tryptophan conversion rate of 0.227g/g in a 5L fermenter (Xiong B et al, biotechnol bioeng.,2021,118 (3): 1393-1404). In addition, corynebacterium glutamicum (Corynebacterium glutamicum) has also been developed for l-tryptophan production. Dong et al metabolically engineered C.glutamicum by enhancing L-tryptophan biosynthetic pathway, central metabolic flux reprogramming, excavating metabolic bottlenecks, enhancing transport system and precursor supply, inhibiting competing pathways, and the like, the obtained recombinant strain can achieve L-tryptophan accumulation of 16.2g/L in shake flask fermentation with a conversion rate of 0.160g/g (Dong YF et al, bioRxiv.,2024,11.04.621991).

Although the existing microbial fermentation method has achieved remarkable results in terms of the yield and the yield of L-tryptophan, further expansion of the fermentation method and improvement of the yield or the yield of the L-tryptophan are still continuous pursuits in the field. Microorganisms such as klebsiella oxytoca (Klebsiella oxytoca) can metabolize carbohydrates such as glucose to synthesize 2, 3-butanediol or acetoin (Jantama K., metab Eng.,2015, 30:16-26) with high efficiency, and the precursor of the synthetic route of 2, 3-butanediol and acetoin is pyruvic acid, which means that K.oxytoca has strong glycolytic flux. Based on this, taking a metabolic flux redirection strategy is expected to redirect powerful 2, 3-butanediol and acetoin synthesis pathway metabolic fluxes to L-tryptophan synthesis.

Based on this, designing a strain construction strategy for high L-tryptophan production and applying the strategy to fermentation production of L-tryptophan remains a challenge in the art.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a recombinant microorganism for high-yield L-tryptophan and a construction method thereof, and aims to efficiently prepare L-tryptophan.

The invention provides a recombinant microorganism for producing L-tryptophan, wherein an original strain of the recombinant microorganism is selected from Klebsiella strains, and the recombinant microorganism overexpresses genes comprising phosphoenolpyruvate synthase gene ppsA and tryptophan operon partial gene cluster trpE ^fbr D.

Preferably, the nucleotide sequence of the tryptophan operon partial gene cluster trpE ^fbr D gene is shown in SEQ ID NO. 15.

Preferably, the starting strain is selected from at least one of klebsiella oxytoca, klebsiella plantarum or klebsiella pneumoniae.

The invention provides a recombinant microorganism for producing L-tryptophan, which is constructed by inactivating by-products and/or enzyme genes in a2, 3-butanediol synthesis pathway and genes related to enhancing L-tryptophan synthesis flux on the basis of an original strain, wherein the by-products are at least one of acetic acid, formic acid, succinic acid and lactic acid, and the genes related to L-tryptophan synthesis flux are at least one of L-tryptophan synthesis related enzyme genes, metabolic pathway related genes, efflux protein genes and endogenous strong promoter genes.

Preferably, the inactivation comprises a knockout or a knockout drop and the enhancement comprises overexpression.

Preferably, the byproduct and/or enzyme gene in the 2, 3-butanediol synthesis pathway is selected from at least one to sixteen of the pyruvate kinase encoding gene pykF, the tryptophan enzyme encoding gene tnaA, the transcription repressor trpR of the tryptophan operon, the attenuator trpL of the tryptophan operon, the pyruvate oxidase gene pox, the phosphotransacetylase gene pta, the acetate kinase ackA, the fumarate reductase subunit A gene frdA, the lactate dehydrogenase gene ldhD, the pyruvate formate lyase gene pflB, the alcohol dehydrogenase gene adhE, the alpha-acetolactate synthase gene budB, the alpha-acetolactate decarboxylase gene budA, the 2, 3-butanediol dehydrogenase gene budC, the glycerol dehydrogenase gene gldA, the 1, 3-propanediol dehydrogenase gene dhaT.

Preferably, the L-tryptophan synthesis flux-related gene is selected from at least one to thirteen of promoter P _budABC of 2, 3-butanediol synthesis gene cluster, 3-deoxy-d-arabinoheptulose 7-phosphate synthase mutein encoding gene aroG ^fbr, 3-phosphoglycerate dehydrogenase mutein encoding gene serA ^fbr, 3-dehydroquinic acid synthase encoding gene aroB, shikimate dehydrogenase encoding gene aroE, chorismate synthase encoding gene aroC, tryptophan operon part gene cluster trpDC, tryptophan operon part gene cluster trpBA, transketolase encoding gene tktA, aromatic amino acid efflux protein encoding gene ywkB, phosphoenolpyruvate carboxykinase gene pck, glutamine synthase encoding gene glnA, L-tryptophan efflux protein gene yddG.

The invention provides a construction method of a recombinant microorganism for producing L-tryptophan, which comprises the steps of inactivating by-products and/or enzyme genes in a2, 3-butanediol synthesis pathway on the basis of an original strain, and constructing genes related to L-tryptophan synthesis flux, wherein the by-products are at least one of acetic acid, formic acid, succinic acid and lactic acid, and the genes related to L-tryptophan synthesis flux are at least one of L-tryptophan synthesis related enzyme genes, metabolic pathway related genes, efflux protein genes and endogenous strong promoter genes.

The present invention provides the use of the recombinant microorganism for producing L-tryptophan as described in any one of the above for the production of L-tryptophan.

Preferably, the recombinant microorganism over-expressed with the phosphoenolpyruvate synthase gene ppsA and the tryptophan operon partial gene cluster trpE ^fbr D is cultured by using a fermentation medium containing glucose.

Preferably, the concentration of glucose in the fermentation medium is 80-100g/L;

And/or, the inoculation amount of the recombinant microorganism is 5-10% by volume when the recombinant microorganism is cultured;

and/or the stirring speed during the culture is 500-1000 rpm, and/or the pH during the culture is 6.8+ -0.1, and/or the temperature during the culture is 37+ -0.5 ℃, and/or the glucose concentration is maintained at 0-5g/L during the culture, and/or the time of the culture is 40-60 hours.

The invention provides a recombinant microorganism for high-yield L-tryptophan as well as a construction method and application thereof. The invention selects the knocked out or added genes, preferably a recombinant klebsiella oxytoca (Klebsiella oxytoca) with high L-tryptophan production. The recombinant klebsiella oxytoca can be used for efficiently producing L-tryptophan. The invention has the following characteristics and outstanding effects:

(1) The invention designs a construction strategy of a high-yield L-tryptophan engineering strain, which is characterized in that a system metabolism reconstruction is carried out on a 2, 3-butanediol or acetoin production strain, an exogenous L-tryptophan biosynthesis pathway is introduced on the basis of knocking out a byproduct synthesis related gene to inhibit synthesis of 2, 3-butanediol, acetic acid, formic acid, succinic acid and lactic acid, intracellular pyruvic acid anabolic flow is redirected from synthesis of 2, 3-butanediol to L-tryptophan production, the L-tryptophan production strain is constructed, and the engineering strain capable of efficiently producing L-tryptophan is further obtained by the strategies of enhancing the L-tryptophan synthesis flux, enhancing the L-tryptophan excretion and the like.

(2) The optimal Klebsiella oxytoca engineering strain takes glucose as a substrate, and produces L-tryptophan under the fermentation condition provided by the invention, the yield reaches 60g/L, and the yield is 0.23g/g.

(3) The recombinant strain constructed by the invention has the advantages of simple culture medium, low fermentation substrate and culture cost, high L-tryptophan yield of the engineering strain, single product component and easy separation.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 shows the L-tryptophan anabolic pathway of recombinant Klebsiella oxytoca of the invention.

Detailed Description

In the following examples and experimental examples, reagents and materials not specifically described are commercially available.

In the following examples, materials, reagents, plasmids, kits for exclusive use, strains, etc., were obtained commercially unless otherwise specified.

The nucleotide sequence information of the gene related to the invention is as follows:

the gene ppsA nucleotide sequence (SEQ ID NO. 1):

ATGTCCAACAATGGCTCGTCACCGCTGGTGCTTTGGTATAACCAACTCGGCATGAATGATGTAGACAGGGTTGGGGGCAAAAATGCCTCCCTGGGTGAAATGATTACTAATCTTTCCGGAATGGGTGTTTCCGTTCCGAATGGTTTCGCCACAACCGCCGACGCGTTTAACCAGTTTCTGGACCAAAGCGGCGTAAACCAGCGCATTTATGAACTGCTGGATAAAACGGATATTGACGATGTTACTCAGCTTGCGAAAGCGGGCGCGCAAATCCGCCAGTGGATTATCGACACTCCCTTCCAGCCTGAGCTGGAAAACGCCATCCGCGAAGCCTATGCACAGCTTTCCGCCGATGACGAAAACGCCTCTTTTGCGGTGCGCTCCTCCGCCACCGCAGAAGATATGCCGGACGCTTCTTTTGCCGGTCAGCAGGAAACCTTCCTCAACGTTCAGGGTTTTGACGCCGTTCTCGTGGCAGTGAAACATGTATTTGCTTCTCTGTTTAACGATCGCGCCATCTCTTATCGTGTGCACCAGGGTTACGATCACCGTGGTGTGGCGCTCTCCGCCGGTGTTCAACGGATGGTGCGCTCTGACCTCGCATCATCTGGCGTGATGTTCTCCATTGATACCGAATCCGGCTTTGACCAGGTGGTGTTTATCACTTCCGCATGGGGCCTTGGTGAGATGGTCGTGCAGGGTGCGGTTAACCCGGATGAGTTTTACGTGCATAAACCGACACTGGCGGCGAATCGCCCGGCTATCGTGCGCCGCACCATGGGGTCGAAAAAAATCCGCATGGTTTACGCGCCGACCCAGGAGCACGGCAAGCAGGTTAAAATCGAAGACGTACCGCAGGAACAGCGTGACATCTTCTCGCTGACCAACGAAGAAGTGCAGGAACTGGCAAAACAGGCCGTACAAATTGAGAAACACTACGGTCGCCCGATGGATATTGAGTGGGCGAAAGATGGCCACACCGGTAAACTGTTCATTGTGCAGGCGCGTCCGGAAACCGTGCGCTCACGCGGTCAGGTCATGGAGCGTTATACGCTGCATTCACAGGGTAAGATTATCGCCGAAGGCCGTGCTATCGGTCATCGCATCGGTGCGGGTCCGGTGAAAGTCATCCATGACATCAGCGAAATGAACCGCATCGAACCTGGCGACGTGCTGGTTACTGACATGACCGACCCGGACTGGGAACCGATCATGAAGAAAGCATCTGCCATCGTCACCAACCGTGGCGGTCGTACCTGTCACGCGGCGATCATCGCTCGTGAACTGGGCATTCCGGCGGTAGTGGGCTGTGGAGATGCAACAGAACGGATGAAAGACGGTGAGAACGTCACTGTTTCTTGTGCCGAAGGTGATACCGGTTACGTCTATGCGGAGTTGCTGGAATTTAGCGTGAAAAGCTCCAGCGTAGAAACGATGCCGGATCTGCCGTTGAAAGTGATGATGAACGTCGGTAACCCGGACCGTGCTTTCGACTTCGCCTGCCTACCGAACGAAGGCGTGGGCCTTGCGCGTCTGGAATTTATCATCAACCGTATGATTGGCGTCCACCCACGCGCACTGCTTGAGTTTGACGATCAGGAACCGCAGTTGCAAAACGAAATCCGCGAGATGATGAAAGGTTTTGATTCTCCGCGTGAATTTTACGTTGGTCGTCTGACTGAAGGGATCGCGACGCTGGGTGCCGCGTTTTATCCGAAGCGCGTCATTGTCCGTCTCTCTGATTTTAAATCGAACGAATATGCCAACCTGGTCGGTGGTGAGCGTTACGAGCCAGATGAAGAGAACCCGATGCTCGGCTTCCGTGGCGCGGGCCGCTATGTTTCCGACAGCTTCCGCGACTGTTTCGCGCTGGAGTGTGAAGCAGTGAAACGTGTGCGCAACGACATGGGACTGACCAACGTTGAGATCATGATCCCGTTCGTGCGTACCGTAGATCAGGCGAAAGCGGTGGTTGAAGAACTGGCGCGTCAGGGGCTGAAACGTGGCGAGAACGGGCTGAAAATCATCATGATGTGTGAAATCCCGTCCAACGCCTTGCTGGCCGAGCAGTTCCTCGAATATTTCGACGGCTTCTCAATTGGCTCAAACGATATGACGCAGCTGGCGCTCGGTCTGGACCGTGACTCCGGCGTGGTGTCTGAATTGTTCGATGAGCGCAACGATGCGGTGAAAGCACTGCTGTCGATGGCTATCCGTGCCGCGAAGAAACAGGGCAAATATGTCGGGATTTGCGGTCAGGGTCCGTCCGACCACGAAGACTTTGCCGCATGGTTGATGGAAGAGGGGATCGATAGCCTGTCTCTGAACCCGGACACCGTGGTGCAAACCTGGTTAAGCCTGGCTGAACTGAAGAAATAA

The gene budA nucleotide sequence (SEQ ID NO. 2):

ATGAACCATTCTGCTGAATGCTCTTGTGAAGAGAGCCTGTGTGAAACTCTACGAGGGTTTTCCGCGCAACATCCCGATAGCGTCATCTACCAGACCTCTCTGATGAGCGCGCTGTTGAGCGGCGTTTATGAAGGTAATACCACCATCGCTGATTTACTCACCCACGGCGATTTTGGCCTGGGAACCTTTAATGAACTGGACGGCGAGCTGATCGCGTTTAGCAGCGAGGTATACCAGCTGCGCGCCGACGGCAGCGCCCGTAAAGCCCGAATGGAACAGCGCACGCCGTTTGCGGTGATGACCTGGTTTCAGCCGCAGTATCGTAAAACGTTCGATAAACCGGTGAGCCGCCAGCAGTTGCACGACATTATCGACCAGCAAATACCCTCCGATAATCTCTTCTGCGCCTTGCGCATTAACGGTCATTTTCGTCATGCGCATACCCGCACCGTACCGCGCCAGACCCCGCCCTACCGGGCGATGACCGACGTGCTCGACGACCAGCCGGTATTCCGCTTCAACCAGCGCGAAGGGGTACTGGTCGGCTTCCGTACCCCGCAGCATATGCAGGGCATTAACGTTGCCGGCTACCACGAACATTTCATCACCGATGACCGTCAGGGCGGCGGTCATCTGCTCGATTATCAGCTCGACCACGGCGTGCTGACCTTTGGCGAAATCCACAAATTGATGATTGACCTTCCTGCCGATAGCGCCTTCCTGCAGGCGGATCTGCACCCGGACAATCTAGATGCCGCTATTCGCTCAGTCGAAAACTAA

mutein gene aroG ^fbr(D146N) nucleotide sequence (SEQ ID NO. 3):

ATGAATTATCAGAACGACGATTTACGCATCAAAGAAATCAAAGAGTTACTTCCTCCTGTCGCATTGCTGGAAAAATTCCCCGCTACTGAAAATGCCGCGAATACGGTTGCCCATGCCCGAAAAGCGATCCATAAGATCCTGAAAGGTAATGATGATCGCCTGTTGGTTGTGATTGGCCCATGCTCAATTCATGATCCTGTCGCGGCAAAAGAGTATGCCACTCGCTTGCTGGCGCTGCGTGAAGAGCTGAAAGATGAGCTGGAAATCGTAATGCGCGTCTATTTTGAAAAGCCGCGTACCACGGTGGGCTGGAAAGGGCTGATTAACGATCCGCATATGGATAATAGCTTCCAGATCAACGACGGTCTGCGTATAGCCCGTAAATTGCTGCTTGATATTAACGACAGCGGTCTGCCAGCGGCAGGTGAGTTTCTCAATATGATCACCCCACAATATCTCGCTGACCTGATGAGCTGGGGCGCAATTGGCGCACGTACCACCGAATCGCAGGTGCACCGCGAACTGGCATCAGGGCTTTCTTGTCCGGTCGGCTTCAAAAATGGCACCGACGGTACGATTAAAGTGGCTATCGATGCCATTAATGCCGCCGGTGCGCCGCACTGCTTCCTGTCCGTAACGAAATGGGGGCATTCGGCGATTGTGAATACCAGCGGTAACGGCGATTGCCATATCATTCTGCGCGGCGGTAAAGAGCCTAACTACAGCGCGAAGCACGTTGCTGAAGTGAAAGAAGGGCTGAACAAAGCAGGCCTGCCAGCACAGGTGATGATCGATTTCAGCCATGCTAACTCGTCCAAACAATTCAAAAAGCAGATGGATGTTTGTGCTGACGTTTGCCAGCAGATTGCCGGTGGCGAAAAGGCCATTATTGGCGTGATGGTGGAAAGCCATCTGGTGGAAGGCAATCAGAGCCTCGAGAGCGGGGAGCCGCTGGCCTACGGTAAGAGCATCACCGATGCCTGCATCGGCTGGGAAGATACCGATGCTCTGTTACGTCAACTGGCGAATGCAGTAAAAGCGCGTCGCGGGTAA

gene budB nucleotide sequence (SEQ ID No. 4):

GTGGATAATCAACATCAACCTCGCCAGTGGGCGCATGGCGCCGACCTGATCGTCAGCCAGCTGGAGGCCCAGGGCGTTCGCCAGGTCTTCGGCATTCCGGGGGCAAAAATCGATAAGGTATTTGATTCGCTACTGGACTCCTCAATTCGCATTATCCCGGTACGCCACGAGGCCAACGCCGCCTTTATGGCCGCTGCGGTTGGCCGCATCACAGGGAAAGCGGGCGTCGCGCTGGTCACTTCCGGCCCCGGCTGCTCCAATCTGATTACCGGTATGGCCACCGCTAACAGCGAAGGCGATCCGGTGGTGGCGCTGGGCGGTGCGGTAAAACGCGCCGACAAGGCCAAACAAGTGCACCAGAGTATGGATACGGTGGCGATGTTCAGCCCGGTAACCAAATACTCGGTGGAAGTCACTGCATCCGATGCGCTGGCGGAGGTCGTGTCTAACGCATTTCGCGCCGCCGAGCAGGGACGGCCGGGAAGCGCCTTCGTCAGTCTGCCGCAGGACGTCGTCGATGAGCCAGTCCACGCCAGGGTGCTGCCCGCCAGCGAAGCCCCGCAGACCGGCGCGGCCCCGGACGACGCCATTGAGCGAGTGGCGAAGATGATTGCCGGGGCAAAAAATCCGGTATTCCTGCTCGGTCTGATGGCCAGCCAGACGGAAAACAGCGCGGCGCTCCGCGAATTGCTGAAAAAAAGCCATATCCCGGTCACCAGTACCTATCAGGCCGCCGGCGCGGTAAATCAGGACCACTTTACGCGCTTTGCCGGACGGGTTGGGCTGTTCAATAACCAGGCCGGGGATCGGCTATTGCATCTCGCCGACCTGGTCATCTGCATTGGCTATAGCCCGGTGGAGTATGAACCGGCGATGTGGAATAACGGTAACGCCGCGCTGGTGCATATCGACGTACTGCCCGCTTATGAAGAGCGCAACTATACCCCGGATATCGAGCTGGTCGGCGATATCGCTGCCACGCTGCACAAACTCTCCGGGCGTATTGACCACCAGCTGGTTCTGTCGCCGCAGGCCGCGGAGATTCTTGTTGACCGCCAGCACCAGCGGGAACTGCTCGATCGCCGCGGCGCGCAGCTAAACCAGTTTGCGCTTCATCCGCTGCGCATCGTTCGCGCCATGCAGGATATCGTCAACAGCGACGTAACGCTGACCGTCGATATGGGGAGCTTTCATATCTGGATCGCCCGCTATCTCTACAGCTTCCGCGCTCGTCAGGTCATGATTTCCAACGGTCAACAGACCATGGGGGTGGCACTGCCGTGGGCGATTGGCGCCTGGCTGGTGAACCCGCAGCGCAAAGTGGTTTCGGTTTCCGGCGATGGCGGTTTCCTGCAATCCAGTATGGAGCTGGAGACCGCCGTAAGGCTAAAAGCTAACGTCCTGCATATCATCTGGGTCGATAACGGCTACAACATGGTGGCAATTCAGGAAGAGAAAAAATATCAGCGGCTATCCGGCGTTGAGTTCGGCCCGGTGGATTTTAAAGCCTATGCCGAAGCCTTCGGCGCCAGAGGGTTCGCGGTCGAGAGCGCCGCCGCCCTTGAGCCGACGCTGCGGGCGGCGATGGACGTCGATGGCCCCGCCGTGGTCGCCATCCCCGTCGATTACAGCGACAACCCGCTGCTGATGGGCCAGCTTCATCTCAGTCAACTACTTTGA

the gene tnaA nucleotide sequence (SEQ ID NO. 5):

ATGAAACGTATCCCTGAGCCGTTCCGCATTAAAATGGTCGAAAATATTCGTATGACCACCTTTGACGATCGCGTCAAAGCGCTGGAAGAGGCGGGCTATAACCCGTTTTTGCTCAATAGCCAGGACGTGTATATCGACCTGCTGACCGACTCCGGCACCGGCGCGATGAGCGACCATCAATGGGCCGGGCTGATGATGGGCGACGAAGCCTACGCCGGGTCGCGTAACTATCAGCATCTGTGTGAAAAGGTCAAAGAGATTATCGGCTATCCGTATACCATTCCCACCCACCAGGGGCGCGGCGCGGAGCAGATTCTGTTCCCAAGCCTGATTGCTCGCCGCAAGTCGGCGCATCCGGTGTTTATTTCCAACTTCCACTTCGATACTACCGCCGCTCACGTGGAGCTTAACGGCGCAAAAGCCATTAACGTGGTGACGCCGAAAGCCTTCGACACGACCTCGTGGTACGACTGGAAAGGCAACTTTGATATTGATTTACTGAAAGCGACCATCGCCGAGCACGGCGCGGAAAACGTGGCGGCGATTATTACTACCGTCACCTGTAACAGTTCCGGCGGCCAGCCGGTATCGCTGGCGAATATGCGCGAGGTGTATCAAATTGCGCGCCAGAACAATATTCCGGTGGTAATCGACTCGGCCCGCTTCTGCGAAAACGCCTGGTTTATTAAACAGCGCGAAGAGGGCTATGCCGATAAATCGGTGAAAGAGATTATTCTCGAGATGTACCAGTACGGCGATATGCTGACCATGTCCGCCAAGAAAGACCCGATGGTGAATATTGGCGGACTGTGCTGCTTCCGCGATGACGAAGAGCTCTTCAACGAAGTGCGCATTCGCTGCGTGCCGATGGAAGGCTTTGTCACCTACGGCGGCCTGGCGGGGCGCGATATGGAGGCGCTGGCTATCGGTCTGGAAGAAGGGACCAACGAGGATTACCTCGCTTATCGCATCAATCAGGTGGAGTACCTCGGTGAACGCCTGCGCGAAGGCGGCATTCCGATTCAGTATCCGACCGGCGGTCATGCGGTGTTCGTCGATGCTAAGCTGCTGCTGCCGCATATTCCGCCGGAGCAGTTCCCGGCGCACGCGCTAAACAACGAGCTGTATCTGGAAGCGGGCATTCGCAGCGTGGAAATCGGCTCCCTGCTGCTGGGACGCGACCCGGAAACCGGTAAGCAGAAGGCGTCGCCGATGGAGCTGCTGCGCCTGACCATTCCGCGTCGGGTTTATACCAACGATCATATGGACTATATCGCTGATGCGCTGATCGCCGTTAAGGCGCGCGCCGCCACGATTAAGGGCCTGACCTTTACCTATGAACCGCCGGTACTGCGCCACTTCGTCGCCAGACTGAAACCGGTGAAATAA

Promoter P _budABC nucleotide sequence (SEQ ID No. 6):

ATCGAAAACGTCTCAAACAATCATAGATTCTATATTGGAACTGTGAGCTGAATCG CGTCAACATTTATTTAACCTTTCTGATATTCGTTGAACGAGGAAGCGGGTA

gene trpL nucleotide sequence (SEQ ID No. 7):

GATGACTCCTGCTGGACATGAACCTGTACCCGTCATACTTTAAGTCACAAGCAGATTGACTGATGTTCTGCAGCTTAAGGGATTAAGGATAGTGATAAGCGCATCATCATACCGACTGATAGAGAAAATCTGCAAACGGGGGTTGACTTTAATCTCTCGGACTAGTTAACTAGTACGCAAGTTCACATGAAGGGGTATCATCAATGAAAACGCAACTCATCACTCTGCACGGCTGGTGGCGCACCTCCTGTTATCGGGCGGCGTGATCGCGTTTTGCACTCAGCATACAGATACCCGGCCCGCCAATGAGCGGGCTTTTTATTGGACAAATTATTACAGCGAACAGGCGACAACAAATA

the gene trpR nucleotide sequence (SEQ ID NO. 8):

ATGACCCAACAATCCCCCTATTCAGCAGCGGTAGCCGAACAGCGTCATCAGGAGTGGCTGCGTTTTGTCGCGCTCTTACAGCAGGCGTACGCCGACGATCTTCATCTGCCGCTTTTACAGCTTATGCTGACCCCCGACGAGCGCGAGGCGCTGGGTACGCGGGTACGTATTATTGAAGAGCTGCTGCGCGGTGAGATGAGCCAGCGCGAGCTAAAAAATGAACTCGGCGCCGGCATCGCGACCATCACCCGCGGTTCGAACAGCCTCAAATCAGCGCCGCCGGAGCTGCGTTTATGGCTGGAGCAGTCGTTGTTTAACGCTGGCGATAAATAG

gene aroB nucleotide sequence (SEQ ID No. 9):

ATGGAGAGGATTGTCGTTACTCTCGGGGAACGTAGTTACCCAATTACCATCGCATCTGGTTTGTTTAATGAACCAGCTTCATTCTTACCGCTGAAATCGGGCGAGCAGGTCATGTTGGTCACCAACGAAACCCTGGCTCCTCTGTATCTCGATAAGGTCCGCGGCGTACTTGAACAGGCGGGTGTTAACGTCGATAGCGTTATCCTCCCTGACGGCGAGCAGTATAAAAGCCTGGCTGTACTCGATACCGTCTTTACGGCGTTGTTACAAAAACCGCATGGTCGCGATACTACGCTGGTGGCGCTTGGCGGCGGCGTAGTGGGCGATCTGACCGGCTTCGCGGCGGCGAGTTATCAGCGCGGTGTCCGTTTCATTCAAGTCCCGACGACGTTACTGTCGCAGGTCGATTCCTCCGTTGGCGGCAAAACTGCGGTCAACCATCCCCTCGGTAAAAACATGATTGGCGCGTTCTACCAACCTGCTTCAGTGGTGGTGGATCTCGACTGTCTGAAAACGCTTCCCCCGCGTGAGTTAGCGTCGGGGCTGGCAGAAGTCATCAAATACGGCATTATTCTTGACGGTGCGTTTTTTAACTGGCTGGAAGAGAATCTGGATGCGTTGTTGCGTCTGGACGGTCCGGCAATGGCGTACTGTATTCGCCGTTGTTGTGAACTGAAGGCAGAAGTTGTCGCCGCCGACGAGCGCGAAACCGGGTTACGTGCTTTACTGAATCTGGGACACACCTTTGGTCATGCCATTGAAGCTGAAATGGGGTATGGCAATTGGTTACATGGTGAAGCGGTCGCTGCGGGTATGGTGATGGCGGCGCGGACGTCGGAACGTCTCGGGCAGTTTAGTTCTGCCGAAACGCAGCGTATTATAACCCTGCTCAAGCGGGCTGGGTTACCGGTCAATGGGCCGCGCGAAATGTCCGCGCAGGCGTATTTACCGCATATGCTGCGTGACAAGAAAGTCCTTGCGGGAGAGATGCGCTTAATTCTTCCGTTGGCAATTGGTAAGAGTGAAGTTCGCAGCGGCGTTTCGCACGAGCTTGTTCTTAACGCCATTGCCGATTGTCAATCAGCGTAA

Gene ldhD nucleotide sequence (SEQ ID No. 10):

ATGAAAATCGCTGTGTACAGTACGAAACAGTACGACAAGAAGTATCTGCAGCATGTCAATGATGCATACGGCTTTGAACTGGAATTTTTTGACTTCCTGCTCACCGAAAAGACCGCCAAAACCGCCAACGGCTGTGAAGCGGTATGCATTTTCGTTAACGATGACGGTAGCCGCCCGGTACTTGAAGAACTGAAAGCCCACGGCGTGAAGTACATCGCGCTGCGCTGCGCCGGGTTCAACAACGTTGACCTCGACGCCGCGAAAGAGCTGGGCCTGCGGGTAGTACGCGTCCCGGCCTACTCGCCGGAAGCGGTCGCTGAGCACGCAATCGGCATGATGATGTCGCTGAACCGCCGCATTCATCGCGCCTATCAGCGCACTCGCGATGCTAACTTCTCCCTTGAGGGGCTGACCGGCTTCACTATGCACGGTAAAACCGCTGGCGTTATCGGCACCGGTAAGATTGGCGTTGCCGCGCTGCGCATCCTTAAAGGTTTCGGTATGCGCCTGCTGGCGTTTGATCCCTATCCAAGCGCCGCCGCGCTGGATATGGGCGTGGAGTATGTCGATCTGGAAACGCTATACCGGGAGTCCGATGTTATCTCCCTGCACTGCCCGCTGACCGATGAGAACTATCATTTGCTGAACCATGCCGCGTTCGATCGGATGAAAGATGGGGTGATGATCATCAACACCAGTCGCGGCGCGCTTATCGATTCGCAGGCAGCGATCGATGCCCTGAAGCACCAGAAAATTGGCGCGCTGGGGATGGACGTGTATGAGAACGAACGCGATCTGTTCTTTGAAGATAAGTCTAACGACGTTATTCAGGACGATGTCTTCCGCCGTCTTTCCGCCTGCCACAACGTTCTGTTTACCGGTCACCAGGCGTTTTTGACCGCAGAGGCGTTGATCAGTATCTCGCAGACCACCCTCGACAACCTGCGTCAGGTGGATGCTGACGAAACCTGCCCTAACGCACTGGTCTGA

gene aroE nucleotide sequence (SEQ ID No. 11):

ATGGAAACCTATGCTGTTTTTGGTAATCCGATAGCCCACAGCAAATCGCCATTCATTCATCAGCAATTTGCTCAGCAACTGAATATTGAACATCCCTATGGGCGCGTGTTGGCACCCATCAATGATTTCATCAACACACTGAACGCTTTCTTTAGTGCTGGTGGTAAAGGTGCGAATGTGACGGTGCCTTTTAAAGAAGAGGCTTTTGCCAGAGCGGATGAGCTTACTGAACGGGCAGCGTTGGCTGGTGCTGTTAATACCCTCATGCGGTTAGAAGATGGACGCCTGCTGGGTGACAATACCGATGGTGTAGGCTTGTTAAGCGATCTGGAACGTCTGTCTTTTATCCGCCCTGGTTTACGTATTCTGCTTATCGGCGCTGGTGGAGCATCTCGCGGCGTACTACTGCCACTCCTTTCCCTGGACTGTGCGGTGACAATAACTAATCGGACGGTATCCCGCGCGGAAGAGTTGGCTAAATTGTTTGCGCACACTGGCAGTATTCAGGCGTTGAGTATGGACGAACTGGAAGGTCATGAGTTTGATCTCATTATTAATGCAACATCCAGTGGCATCAGTGGTGATATTCCGGCGATCCCGTCATCGCTCATTCATCCAGGCATTTATTGCTATGACATGTTCTATCAGAAAGGAAAAACTCCTTTTCTGGCATGGTGTGAGCAGCGAGGCTCAAAGCGTAATGCTGATGGTTTAGGAATGCTGGTGGCACAGGCGGCTCATGCCTTTCTTCTCTGGCACGGTGTTCTGCCTGACGTAGAACCAGTTATAAAGCAATTGCAGGAGGAATTGTCCGCGTGA

the gene adhE nucleotide sequence (SEQ ID NO. 12):

ATGGCTGTTACTAATGTCGCTGAACTTAACGCACTCGTAGAGCGCGTAAAAAAAGCCCAGCGTGAATATGCCAGTTTCACTCAAGAACAAGTAGACAAAATCTTCCGCGCCGCCGCTCTGGCCGCTGCAGATGCTCGAATCCCTCTCGCTAAGATGGCCGTTGCCGAATCCGGCATGGGCATCGTCGAAGATAAAGTGATCAAAAACCACTTTGCTTCCGAATACATCTATAACGCCTATAAAGATGAAAAGACCTGCGGCGTCCTGTCTGAAGATGACACCTTTGGTACTATTACAATCGCTGAACCGATTGGTATCATCTGCGGTATCGTCCCGACCACTAACCCGACTTCAACCGCTATCTTTAAATCTCTGATTAGCCTGAAGACTCGTAACGCCATCATCTTCTCCCCGCATCCGCGTGCTAAAGATGCAACCAATAAAGCAGCTGATATCGTACTGCAGGCGGCTATCGCTGCCGGCGCGCCGAAAGATCTGATTGGTTGGATTGACCAGCCTTCCGTAGAACTGTCCAATGCGCTGATGCATCACCCGGATATCAACCTGATCCTCGCCACCGGCGGTCCGGGCATGGTTAAAGCAGCGTACAGCTCTGGTAAACCGGCAATCGGCGTAGGCGCGGGTAACACTCCCGTTGTTATCGACGAAACGGCTGATATCAAACGCGCCGTCGCATCCGTACTGATGTCTAAAACCTTCGATAACGGCGTTATTTGTGCTTCCGAGCAGTCCGTAGTCGTTGTAGATTCCGTATACGACGCCGTTCGCGAACGTTTCTCCAGCCACGGCGGCTACCTGCTGCAGGGCCAGGAGCTGAAAGCGGTTCAGAATATCATTCTGAAAAATGGCGCGCTGAACGCCGCTATCGTTGGTCAACCTGCGTATAAAATCGCTGAGCTGGCAGGCTTCACCGTTCCTGTGTCCACCAAGATTCTGATTGGTGAAGTCACCGACGTTGATGAAAGTGAGCCGTTTGCCCACGAAAAACTGTCCCCGACGCTGGCGATGTATCGTGCGAAGAACTTCGAAGACGCGGTTGATAAAGCGGAAAAACTGGTTGCTATGGGCGGTATCGGCCACACTTCCTGCCTGTATACCGACCAGGATAACCAGCCAGAACGCGTTGCTTACTTCGGTCAGTTGATGAAAACCGCACGTATCCTGATCAACACCCCGGCTTCTCAGGGTGGTATCGGTGACCTGTATAACTTCAAACTCGCACCTTCCCTGACTCTGGGTTGTGGTTCATGGGGTGGTAACTCCATCTCGGAAAACGTTGGTCCTAAGCACCTGATCAACAAGAAAACCGTTGCTAAGCGAGCTGAAAACATGTTGTGGCACAAACTTCCGAAATCTATCTACTTCCGTCGTGGCTCACTGCCAATCGCGCTGGATGAAGTGATTACTGATGGTCACAAACGCGCGCTGATCGTCACCGACCGTTTCCTGTTTAACAACGGCTACGCAGACCAGATCACTTCCGTACTGAAAGCGGCCGGCGTTGAAACAGAAGTTTTCTTTGAAGTGGAAGCTGACCCGACTCTGACCATCGTCCGTAAAGGCGCTGAGCTGGCGAACTCTTTCAAACCGGACGTGATTATCGCCCTGGGCGGCGGTTCCCCGATGGACGCGGCGAAAATCATGTGGGTCATGTACGAACATCCGGAAACCCACTTCGAAGAGCTGGCGCTGCGCTTTATGGATATCCGTAAGCGTATCTACAAGTTCCCGAAAATGGGCGTTAAAGCCAAAATGGTGGCGATCACCACCACGTCTGGTACCGGCTCTGAAGTCACGCCGTTCGCCGTTGTTACCGATGATGCAACCGGGCAGAAATACCCGCTGGCTGACTATGCCCTGACCCCGGATATGGCGATTGTTGATGCCAACCTGGTCATGGAAATGCCGAAATCCCTGTGTGCTTTCGGTGGTCTGGATGCGGTGACCCATGCGCTGGAAGCTTACGTTTCCGTACTGGCCTCCGAGTTCTCCGACGGTCAGGCTCTGCAGGCGCTGAAGCTGCTGAAAGAAAACCTGCCGGCGTCTTACCATGAAGGTTCTAAGAACCCGGTAGCGCGTGAGCGCGTACACAGTGCTGCAACGATCGCCGGTATTGCGTTTGCTAACGCCTTCCTGGGTGTATGTCACTCCATGGCGCACAAGCTTGGTTCTCAGTTCCACATTCCTCACGGTCTGGCGAACGCCCTGTTGATCAGTAACGTTATCCGCTATAACGCCAACGACAACCCGACCAAACAGACGGCATTCAGCCAGTACGACCGTCCGCAGGCCCGTCGTCGCTATGCGGAAATCGCTGACCACCTGGGTCTGACTGCACCGGGTGACCGCACCGCAGCGAAAATCGAAAAACTGCTTGGCTGGCTGGAAGAAATTAAAGCTGAACTGGGTATTCCTAAGTCTATCCGCGAAGCTGGCGTGCAGGAAGCTGACTTCCTGGCCCACGTTGATAAGCTGTCTGAAGACGCATTCGATGACCAGTGCACCGGCGCCAACCCGCGCTATCCGCTGATTGCCGAGCTGAAACAGATTCTGCTGGATACCTACTACGGTCGCCATTACGTCGAAGGCGGCGTGGAAGAGAAGAAAGAAGCCGCGCCGGGTAAAGCTGAGAAAAAAGCGAAGAAATCCGCTTAA

The aroC nucleotide sequence (SEQ ID NO. 13):

ATGGCTGGAAACACAATTGGACAACTCTTTCGCGTAACCACCTTCGGCGAATCGCACGGGCTGGCGCTCGGCTGCATCGTCGATGGTGTTCCGCCAGGCATTCCGCTGACGGAAGCGGACCTGCAACATGACCTCGACCGTCGTCGCCCTGGGACATCGCGCTATACCACCCAGCGCCGCGAGCCGGATCAGGTCAAAATTCTCTCCGGTGTTTTTGAAGGCGTTACTACCGGCACCAGCATTGGCTTGTTGATCGAAAACACTGACCAGCGCTCTCAGGATTACAGTGCGATTAAGGACGTTTTCCGTCCAGGCCATGCCGATTACACCTACGAACAAAAATACGGTCTGCGCGATTATCGCGGCGGTGGACGTTCTTCCGCCCGCGAAACCGCCATGCGCGTGGCGGCAGGAGCTATTGCCAAAAAATATCTCGCCGAGAAATTTGGTATTGAAATCCGTGGCTGCCTGACCCAGATGGGCGACATTCCGCTGGATATCAAAGACTGGTCGCAGGTCGAGCAAAATCCGTTTTTTTGCCCGGACCCCGACAAAATCGACGCGTTAGACGAGTTGATGCGTGCGCTGAAAAAAGAGGGCGACTCCATCGGCGCTAAAGTCACCGTTGTTGCCAGTGGCGTTCCTGCCGGACTTGGCGAGCCGGTCTTTGACCGCCTGGATGCTGACATCGCCCATGCGCTGATGAGCATCAACGCGGTGAAAGGCGTGGAAATTGGCGACGGCTTTGACGTGGTGGCGCTGCGCGGCAGCCAGAACCGCGATGAAATCACCAAAGACGGTTTCCAGAGCAACCATGCGGGCGGCATTCTCGGCGGTATCAGCAGCGGGCAGCAAATCATTGCCCATATGGCGCTGAAACCGACCTCCAGCATTACCGTGCCGGGTCGTACCATTAACCGCTTTGGCGAAGAAGTTGAGATGATCACCAAAGGCCGTCACGATCCCTGTGTCGGGATCCGCGCAGTGCCGATCGCAGAAGCGATGCTGGCGATCGTTTTAATGGATCACCTGTTACGGCAACGGGCGCAAAATGCCGATGTGAAGACTGATATTCCACGCTGGTAA

the frdA nucleotide sequence of the gene (SEQ ID NO. 14):

GTGCAAACTTTTCAAGCCGATCTTGCCGTTATTGGCGCAGGCGGAGCAGGTCTTCGTGCTGCAATTGCCGCCGCGCAAGCTAATCCAAATGCTAAAATCGCTCTAATTTCAAAAGTCTATCCTATGCGCAGCCATACCGTTGCTGCTGAAGGGGGTTCCGCTGCCGTCGCGCAGGATCATGATAGCTTTGAGTACCATTTCCACGATACTGTTGCAGGCGGCGACTGGCTATGCGAACAGGATGTCGTCGACTACTTCGTTCATCACTGCCCGACGGAAATGACCCAGCTTGAGCAGTGGGGTTGTCCGTGGAGCCGCCGCCCTGACGGCAGCGTCAACGTCCGGCGCTTCGGCGGTATGAAGATCGAGCGAACCTGGTTCGCAGCCGATAAAACCGGCTTTCATATGCTGCACACCCTGTTCCAGACCTCGCTTCAGTTCCCCCAAATCCAACGCTTCGACGAACACTTTGTCCTCGATATCCTGGTCGACGAAGGTCAGGCGCGCGGTCTGGTCGCGATGAATATGATGGAGGGTACGCTGGTACAGATCCGCGCCAACGCGGTAGTTCTGGCGACCGGCGGCGCAGGACGCGTATATCGCTACAATACCAACGGCGGGATCGTCACCGGCGATGGTATGGGTATGGCGCTCAGCCACGGCGTACCGCTGCGCGATATGGAGTTCGTGCAATATCACCCAACCGGCCTGCCGGGTTCCGGTATTCTGATGACGGAAGGCTGCCGCGGTGAAGGCGGTATTCTGGTCAACAAGAATGGCTACCGCTATCTGCAGGATTACGGCATGGGCCCGGAAACTCCGCTGGGCGAGCCAAAAAACAAATATATGGAGCTGGGTCCGCGCGACAAAGTCTCCCAGGCTTTCTGGCACGAATGGCGTAAAGGCAACACGATCCCAACGCCGCGAGGCGACGTGGTCTACCTCGATCTGCGTCATTTGGGCGAGAAAAAGCTGCTTGAACGTCTGCCGTTTATCTGTGAACTCTCAAAAGCCTACGTGGGCGTCGATCCGGTGAAAGATCCCATTCCGGTACGTCCAACCGCGCACTACACCATGGGCGGTATCGAAACCGACCAGCAGTGTGAAACCCGTATTAAAGGGCTGTTTGCCGTTGGCGAATGCTCTTCTGTCGGTTTGCACGGCGCTAACCGTCTCGGCTCCAACTCGCTGGCGGAGCTGGTGGTGTTTGGTCGTTTGGCCGGTGAACAGGCCATGCAGCGCGCCACCGAAGCGGGCGAAGCCAATAGCACCGCGCTGGACGCGCAGGTCGTCGATATCGAGAAGCGCCTGAAGGACCTGGTGAATCAGGAAGGTAACGAAAACTGGGCGAAGATCCGTGATGAAATGGGTCTGTCGATGGAAGAAGGCTGCGGCATCTACCGTACGCCGGAGCTCATGCAGAAAACGGTTGATAAGCTGGCCGAGCTGCAGGAGCGCTTCAAGCGCGTGCGTATCACCGACACCTCCAGCGTGTTCAATACCGACCTGCTGTATACCATCGAACTGGGTCATGGTCTGAACGTCGCCGAATGTATGGCGCATTCCGCCATCGCGCGTAAAGAGTCCCGCGGCGCGCATCAGCGTCTGGATGAAGGCTGCACCGAGCGTGACGACGTCAACTTCCTCAAGCATACCCTCGCCTTCCGCGACGCCAACGGTAATACCACTCTGGAGTACAGTGATGTGAAAATCACCACTCTGCCGCCGGCAAAACGCGTGTACGGGGCGGAAGCGGAAGCAGCCGAGAAGAAGGAGACGACGAATGGCTGA

The mutein gene trpE ^fbr(S40F) D nucleotide sequence (SEQ ID NO. 15):

ATGCAAACACAAAAACCGACTCTCGAACTGCTAACCTGCGAAGGCGCTTATCGCGACAATCCCACCGCGCTTTTTCACCAGTTGTGTGGGGATCGTCCGGCAACGCTGCTGCTGGAATTCGCAGATATCGACAGCAAAGATGATTTAAAAAGCCTGCTGCTGGTAGACAGTGCGCTGCGCATTACAGCTTTAGGTGACACTGTCACAATCCAGGCACTTTCCGGCAACGGCGAAGCCCTCCTGGCACTACTGGATAACGCCCTGCCTGCGGGTGTGGAAAGTGAACAATCACCAAACTGCCGTGTGCTGCGCTTCCCCCCTGTCAGTCCACTGCTGGATGAAGACGCCCGCTTATGCTCCCTTTCGGTTTTTGACGCTTTCCGTTTATTGCAGAATCTGTTGAATGTACCGAAGGAAGAACGAGAAGCCATGTTCTTCGGCGGCCTGTTCTCTTATGACCTTGTGGCGGGATTTGAAGATTTACCGCAACTGTCAGCGGAAAATAACTGCCCTGATTTCTGTTTTTATCTCGCTGAAACGCTGATGGTGATTGACCATCAGAAAAAAAGCACCCGTATTCAGGCCAGCCTGTTTGCTCCGAATGAAGAAGAAAAACAACGTCTCACTGCTCGCCTGAACGAACTACGTCAGCAACTGACCGAAGCCGCGCCGCCGCTGCCAGTGGTTTCCGTGCCGCATATGCGTTGTGAATGTAATCAGAGCGATGAAGAGTTCGGTGGCGTAGTGCGTTTGTTGCAAAAAGCGATTCGCGCTGGAGAAATTTTCCAGGTGGTGCCATCTCGCCGTTTCTCTCTGCCCTGCCCGTCACCGCTGGCGGCCTATTACGTGCTGAAAAAGAGTAATCCCAGCCCGTACATGTTTTTTATGCAGGATAATGATTTCACCCTATTTGGCGCGTCGCCGGAAAGCTCGCTCAAGTATGATGCCACCAGCCGCCAGATTGAGATCTACCCGATTGCCGGAACACGCCCACGCGGTCGTCGCGCCGATGGTTCACTGGACAGAGATCTCGACAGCCGTATTGAACTGGAAATGCGTACCGATCATAAAGAGCTGTCTGAACATCTGATGCTGGTTGATCTCGCCCGTAATGATCTGGCACGCATTTGCACCCCCGGCAGCCGCTACGTCGCCGATCTCACCAAAGTTGACCGTTATTCCTATGTGATGCACCTCGTCTCTCGCGTAGTCGGCGAACTGCGTCACGATCTTGACGCCCTGCACGCTTATCGCGCCTGTATGAATATGGGGACGTTAAGCGGTGCGCCGAAAGTACGCGCTATGCAGTTAATTGCCGAGGCGGAAGGTCGTCGCCGCGGCAGCTACGGCGGCGCGGTAGGTTATTTCACCGCGCATGGCGATCTCGACACCTGCATTGTGATCCGCTCGGCGCTGGTGGAAAACGGTATCGCCACCGTGCAAGCGGGTGCTGGTGTAGTCCTTGATTCTGTTCCGCAGTCGGAAGCCGACGAAACCCGTAACAAAGCCCGCGCTGTACTGCGCGCTATTGCCACCGCGCATCATGCACAGGAGACTTTCTGATGGCTGACATTCTGCTGCTCGATAATATCGACTCTTTTACGTACAACCTGGCAGATCAGTTGCGCAGCAATGGGCATAACGTGGTGATTTACCGCAACCATATTCCGGCGCAAACCTTAATTGAACGCCTGGCGACCATGAGCAATCCGGTGCTGATGCTTTCTCCTGGCCCCGGTGTGCCGAGCGAAGCCGGTTGTATGCCGGAACTCCTCACCCGCTTGCGTGGCAAGCTGCCCATTATTGGCATTTGCCTCGGACATCAGGCGATTGTCGAAGCTTACGGGGGCTATGTCGGTCAGGCGGGCGAAATTCTCCACGGTAAAGCCTCCAGCATTGAACATGACGGTCAGGCGATGTTTGCCGGATTAACAAACCCGCTGCCGGTGGCGCGTTATCACTCGCTGGTTGGCAGTAACATTCCGGCCGGTTTAACCATCAACGCCCATTTTAATGGCATGGTGATGGCAGTACGTCACGATGCGGATCGCGTTTGTGGATTCCAGTTCCATCCGGAATCCATTCTCACCACCCAGGGCGCTCGCCTGCTGGAACAAACGCTGGCCTGGGCGCAGCAGAAACTAGAGCCAGCCAACACGCTGCAACCGATTCTGGAAAAACTGTATCAGGCGCAGACGCTTAGCCAACAAGAAAGCCACCAGCTGTTTTCAGCGGTGGTGCGTGGCGAGCTGAAGCCGGAACAACTGGCGGCGGCGCTGGTGAGCATGAAAATTCGCGGTGAGCACCCGAACGAGATCGCCGGGGCAGCAACCGCGCTACTGGAAAACGCAGCGCCGTTCCCGCGCCCGGATTATCTGTTTGCTGATATCGTCGGTACTGGCGGTGACGGCAGCAACAGTATCAATATTTCTACCGCCAGTGCGTTTGTCGCCGCGGCCTGTGGGCTGAAAGTGGCGAAACACGGCAACCGTAGCGTCTCCAGTAAATCTGGTTCGTCCGATCTGCTGGCGGCGTTCGGTATTAATCTTGATATGAACGCCGATAAATCGCGCCAGGCGCTGGATGAGTTAGGTGTATGTTTCCTCTTTGCGCCGAAGTATCACACCGGATTCCGCCACGCGATGCCGGTTCGCCAGCAACTGAAAACCCGCACCCTGTTCAATGTGCTGGGGCCATTGATTAACCCGGCGCATCCGCCGCTGGCGTTAATTGGTGTTTATAGTCCGGAACTGGTGCTGCCGATTGCCGAAACCTTGCGCGTGCTGGGGTATCAACGCGCGGCGGTGGTGCACAGCGGCGGGATGGATGAAGTTTCATTACACGCGCCGACAATCGTTGCCGAACTGCATGACGGCGAAATTAAAAGCTATCAGCTCACCGCAGAAGACTTTGGCCTGACACCCTACCACCAGGAGCAACTGGCAGGCGGAACACCGGAAGAAAACCGTGACATTTTAACACGTTTGTTACAAGGTAAAGGCGACGCCGCCCATGAAGCAGCCGTCGCTGCGAACGTCGCCATGTTAATGCGCCTGCATGGCCATGAAGATCTGCAAGCCAATGCGCAAACCGTTCTTGAGGTACTGCGCAGTGGTTCCGCTTACGACAGAGTCACCGCACTGGCGGCACGAGGGTAA

Gene pflB nucleotide sequence (SEQ ID No. 16):

ATGTCCGAGCTTAATGAAAAGTTAGCCACAGCCTGGGAAGGTTTTGCGAAAGGTGACTGGCAGAACGAAGTCAACGTCCGCGACTTCATCCAGAAAAACTATACCCCGTACGAAGGTGACGAGTCCTTCCTGGCTGGCGCAACTGAAGCGACCACCAAGCTGTGGGACACCGTAATGGAAGGCGTTAAACAGGAAAACCGCACTCATGCGCCTGTTGATTTTGATACTTCCCTTGCGTCCACCATCACCTCTCATGACGCTGGCTACATCGAGAAAGGTCTCGAGAAAATCGTTGGTCTGCAGACTGAAGCGCCGCTGAAACGTGCGATTATCCCGTTCGGTGGTATCAAAATGGTAGAAGGCTCCTGCAAAGCGTACAATCGCGAGCTGGACCCAATGCTGAAGAAAATCTTCACTGAATACCGTAAAACCCACAACCAGGGCGTGTTTGACGTTTACACCAAAGACATCCTGAACTGCCGTAAATCTGGCGTTCTGACCGGTCTGCCGGATGCCTATGGTCGTGGTCGTATCATCGGTGACTACCGTCGCGTTGCGCTGTACGGTATCGACTTCCTGATGAAAGACAAATACGCTCAGTTCGTCTCTCTGCAAGACAAACTGGAAAGCGGTGAAGATCTGGAAGCAACCATCCGTCTGCGTGAAGAAATCTCTGAGCAGCACCGTGCGCTGGGTCAGATCAAAGAAATGGCGGCTAAATATGGCTGCGATATCTCTGGTCCTGCTACCACCGCTCAGGAAGCTATCCAGTGGACCTACTTTGGTTACCTGGCTGCCGTTAAATCTCAGAACGGCGCGGCAATGTCCTTCGGTCGTACCTCCAGCTTCCTGGACATCTTCATCGAACGTGACCTGAAAGCCGGTAAAATCACCGAGCAAGACGCTCAGGAAATGATTGACCACCTGGTCATGAAACTGCGTATGGTTCGTTTCCTGCGTACTCCAGAATATGATGAACTGTTCTCCGGTGACCCGATTTGGGCAACTGAATCCATCGGCGGTATGGGCGTTGACGGCCGTACTCTGGTAACCAAAAACAGCTTCCGTTTCCTGAACACCCTGTACACCATGGGGCCGTCTCCGGAGCCGAACATCACCATTCTGTGGTCTGAAAAACTGCCGCTGAGCTTCAAAAAATTCGCTGCAAAAGTGTCCATCGATACCTCTTCTCTGCAGTATGAGAACGATGACCTGATGCGCCCAGACTTCAACAACGATGACTACGCTATCGCTTGCTGCGTAAGCCCGATGGTTGTTGGTAAGCAAATGCAGTTCTTCGGTGCTCGCGCTAACCTCGCGAAAACCATGCTGTACGCAATCAACGGCGGCGTTGATGAAAAACTGAAAATGCAGGTTGGTCCTAAATCTGAACCGATCAAAGGCGACGTTCTGAACTTCGATGAAGTGATGGACCGCATGGATCACTTCATGGACTGGCTGGCTAAACAGTACGTCACTGCGCTGAACATCATCCACTACATGCACGACAAGTACAGCTACGAAGCCTCCCTGATGGCGCTGCACGACCGTGATGTTATCCGCACTATGGCATGTGGTATCGCAGGTCTGTCCGTTGCTGCTGACTCCCTGTCTGCAATCAAATATGCGAAAGTTAAACCGATTCGTGATGAAAACGGTCTGGCTGTCGACTTCGAAATCGAAGGCGAATACCCGCAGTTTGGTAACAACGACTCTCGCGTCGATGATATGGCCGTTGACCTGGTTGAACGTTTCATGAAGAAAATTCAGAAACTGCACACCTACCGTAACGCTATCCCGACTCAGTCCGTTCTGACCATCACCTCTAACGTTGTGTATGGTAAGAAAACTGGTAACACCCCAGACGGTCGTCGCGCTGGCGCTCCGTTCGGACCAGGTGCTAACCCGATGCACGGCCGTGACCAGAAAGGTGCAGTTGCCTCTCTGACCTCCGTTGCTAAACTGCCGTTTGCTTACGCGAAAGATGGTATCTCTTATACCTTCTCTATCGTTCCGAACGCACTGGGTAAAGACGACGAAGTTCGTAAAACTAACCTCGCAGGCCTGATGGATGGTTACTTCCACCACGAAGCGTCCATCGAAGGCGGTCAGCACCTGAACGTCAACGTCATGAACCGCGAAATGCTGCTCGACGCGATGGAAAACCCGGAAAAATATCCGCAGCTGACCATCCGCGTATCCGGTTATGCAGTACGTTTTAACTCCCTGACTAAAGAACAGCAGCAGGACGTTATTACTCGTACCTTCACTCAGACCATGTAA

gene trpDC nucleotide sequence (SEQ ID No. 17):

ATGGCTGACATTCTGCTGCTCGATAATATCGACTCTTTTACGTACAACCTGGCAGATCAGTTGCGCAGCAATGGGCATAACGTGGTGATTTACCGCAACCATATTCCGGCGCAAACCTTAATTGAACGCCTGGCGACCATGAGCAATCCGGTGCTGATGCTTTCTCCTGGCCCCGGTGTGCCGAGCGAAGCCGGTTGTATGCCGGAACTCCTCACCCGCTTGCGTGGCAAGCTGCCCATTATTGGCATTTGCCTCGGACATCAGGCGATTGTCGAAGCTTACGGGGGCTATGTCGGTCAGGCGGGCGAAATTCTCCACGGTAAAGCCTCCAGCATTGAACATGACGGTCAGGCGATGTTTGCCGGATTAACAAACCCGCTGCCGGTGGCGCGTTATCACTCGCTGGTTGGCAGTAACATTCCGGCCGGTTTAACCATCAACGCCCATTTTAATGGCATGGTGATGGCAGTACGTCACGATGCGGATCGCGTTTGTGGATTCCAGTTCCATCCGGAATCCATTCTCACCACCCAGGGCGCTCGCCTGCTGGAACAAACGCTGGCCTGGGCGCAGCAGAAACTAGAGCCAGCCAACACGCTGCAACCGATTCTGGAAAAACTGTATCAGGCGCAGACGCTTAGCCAACAAGAAAGCCACCAGCTGTTTTCAGCGGTGGTGCGTGGCGAGCTGAAGCCGGAACAACTGGCGGCGGCGCTGGTGAGCATGAAAATTCGCGGTGAGCACCCGAACGAGATCGCCGGGGCAGCAACCGCGCTACTGGAAAACGCAGCGCCGTTCCCGCGCCCGGATTATCTGTTTGCTGATATCGTCGGTACTGGCGGTGACGGCAGCAACAGTATCAATATTTCTACCGCCAGTGCGTTTGTCGCCGCGGCCTGTGGGCTGAAAGTGGCGAAACACGGCAACCGTAGCGTCTCCAGTAAATCTGGTTCGTCCGATCTGCTGGCGGCGTTCGGTATTAATCTTGATATGAACGCCGATAAATCGCGCCAGGCGCTGGATGAGTTAGGTGTATGTTTCCTCTTTGCGCCGAAGTATCACACCGGATTCCGCCACGCGATGCCGGTTCGCCAGCAACTGAAAACCCGCACCCTGTTCAATGTGCTGGGGCCATTGATTAACCCGGCGCATCCGCCGCTGGCGTTAATTGGTGTTTATAGTCCGGAACTGGTGCTGCCGATTGCCGAAACCTTGCGCGTGCTGGGGTATCAACGCGCGGCGGTGGTGCACAGCGGCGGGATGGATGAAGTTTCATTACACGCGCCGACAATCGTTGCCGAACTGCATGACGGCGAAATTAAAAGCTATCAGCTCACCGCAGAAGACTTTGGCCTGACACCCTACCACCAGGAGCAACTGGCAGGCGGAACACCGGAAGAAAACCGTGACATTTTAACACGTTTGTTACAAGGTAAAGGCGACGCCGCCCATGAAGCAGCCGTCGCTGCGAACGTCGCCATGTTAATGCGCCTGCATGGCCATGAAGATCTGCAAGCCAATGCGCAAACCGTTCTTGAGGTACTGCGCAGTGGTTCCGCTTACGACAGAGTCACCGCACTGGCGGCACGAGGGTAAATGATGCAAACCGTTTTAGCGAAAATCGTCGCAGACAAGGCGATTTGGGTAGAAGCCCGCAAACAGCAGCAACCGCTGGCCAGTTTTCAGAATGAGGTTCAGCCGAGCACGCGACATTTTTATGATGCGCTACAGGGTGCGCGCACGGCGTTTATTCTGGAGTGCAAGAAAGCGTCGCCGTCAAAAGGCGTGATCCGTGATGATTTCGATCCAGCACGCATTGCCGCCATTTATAAACATTACGCTTCGGCAATTTCGGTGCTGACTGATGAGAAATATTTTCAGGGGAGCTTTAATTTCCTCCCCATCGTCAGCCAAATCGCCCCGCAGCCGATTTTATGTAAAGACTTCATTATCGACCCTTACCAGATCTATCTGGCGCGCTATTACCAGGCCGATGCCTGCTTATTAATGCTTTCAGTACTGGATGACGACCAATATCGCCAGCTTGCCGCCGTCGCTCACAGTCTGGAGATGGGGGTGCTGACCGAAGTCAGTAATGAAGAGGAACAGGAGCGCGCCATTGCATTGGGAGCAAAGGTCGTTGGCATCAACAACCGCGATCTGCGTGATTTGTCGATTGATCTCAACCGTACCCGCGAGCTTGCGCCGAAACTGGGGCACAACGTGACGGTAATCAGCGAATCCGGCATCAATACTTACGCTCAGGTGCGCGAGTTAAGCCACTTCGCTAACGGTTTTCTGATTGGTTCGGCGTTGATGGCCCATGACGATTTGCACGCCGCCGTGCGCCGGGTGTTGCTGGGTGAGAATAAAGTATGTGGCCTGACGCGTGGGCAAGATGCTAAAGCAGCTTATGACGCGGGCGCGATTTACGGTGGGTTGATTTTTGTTGCGACATCACCGCGTTGCGTCAACGTTGAACAGGCGCAGGAAGTGATGGCTGCGGCACCGTTGCAGTATGTTGGCGTGTTCCGCAATCACGATATTGCCGATGTGGTGGACAAAGCTAAGGTGTTATCGCTGGCGGCAGTGCAACTGCATGGTAATGAAGAACAGCTGTATATCGATACGCTGCGTGAAGCTCTGCCAGCACATGTTGCCATCTGGAAAGCATTAAGCGTCGGTGAAACCCTGCCCGCCCGCGAGTTTCAGCACGTTGATAAATATGTTTTAGACAACGGCCAGGGTGGAAGCGGGCAACGTTTTGACTGGTCACTATTAAATGGTCAATCGCTTGGCAACGTTCTGCTGGCGGGGGGCTTAGGCGCAGATAACTGCGTGGAAGCGGCACAAACCGGCTGCGCCGGACTTGATTTTAATTCTGCTGTAGAGTCGCAACCGGGCATCAAAGACGCACGTCTTTTGGCCTCGGTTTTCCAGACGCTGCGCGCATATTAA

Gene poxnucleotide sequence (SEQ ID No. 18):

ATGAAACAGACCGTGGCGGCATACATTGCCAAAACTCTAGAACAGGCTGGCGTTAAACGTATTTGGGGCGTGACCGGGGATTCTCTTAATGGCTTAAGCGACAGCCTGAACCGCATGGGGACCATCGACTGGATGCCCACGCGCCATGAAGAAGTTGCCGCCTTCGCCGCCGGAGCGGAAGCGCAGCTTACCGGCGAACTGGCGGTGTGCGCCGGTTCCTGCGGGCCGGGCAACCTGCATCTGATTAACGGTCTTTTCGACTGCCACCGCAACCATGTTCCGGTGCTGGCCATCGCCGCCCATATTCCTTCCAGCGAAATCGGCAGCGGTTACTTTCAGGAAACGCATCCGCAGGAGCTGTTTCGCGAATGCAGCCACTACTGCGAACTGGTCTCCTCGCCGGAGCAGATCCCGCAGGTGCTGGCCATCGCTATGCGCAAAGCGGTGATTAACCGCGGCGTCTCGGTAGTGGTGCTCCCCGGCGACGTTGCGCTAAAACCCGCGCCGGAAAGCGCCAGCAGCCACTGGTACCACGCCCCGCTGCCGCAGGTGACGCCGGCAGAAGAAGAACTGAAAAAACTGGCCCAGCTGCTGCGCTATTCGAGCAATATCGCGCTGATGTGCGGCAGCGGCTGCGCCGGAGCGCATAAAGAGCTGGTTGAGTTCGCCGCGAAGCTGAAAGCGCCTATCGTTCACGCGCTGCGCGGCAAAGAGCACGTTGAATACGATAACCCTTATGATGTTGGGATGACCGGGCTGATAGGCTTCTCCTCCGGTTTCCATACCATGATGAACGCTGACACCCTGGTGCTGCTCGGCACCCAGTTCCCCTATCGCGCCTTCTACCCGACGGACGCAAAAATTATTCAGATCGATATCAACCCCGGCAGTATCGGCGCGCACAGTAAAGTGGACATGGCGCTGATCGGCGATATCAAATCCACCCTCAGCGCGCTGCTGCCGCATCTGGAAGAGAAAACCGACCGCAAGTTCCTCGACAAAGCCCTGGAACACTATCGCGACGCGCGCAAGGGGCTCGATGACCTGGCGAAGCCAAGCGATAAAACCCTTCATCCGCAGTATCTGGCGCAGCGGATCAGCCACTTTGCCGACGACGACGCCATTTTTACCTGTGACGTCGGAACGCCAACCGTGTGGGCTGCGCGCTATTTACAGATGAACGGCAAGCGCCGCCTGATCGGATCGTTCAACCACGGTTCGATGGCTAACGCCATGCCGCAGGCCATCGGCGCGAAGGCCACCGCCCCCGACCGGCAGGTCGTGGCAATGTGCGGCGACGGCGGCTTTAGCATGCTGATGGGGGATTTTCTCTCGCTGGCGCAAATGAAGCTGCCGGTGAAGATCGTTATCTTCAATAACAGCGTCCTGGGCTTTGTCGCGATGGAGATGAAGGCCGGCGGCTATCTGACAGACGGCACCGAGCTGCATGCGACCAACTTTGCCCGCATCGCCGAGGCCTGCGGCATTAAGGGCATCCGTGTCGAAAAAGCGGCTGACGTAGACCAGGCGTTGCAAACCGCCTTCAGTACCGACGGCCCGGTCCTGGTGGATGTGGTGGTCGCCAGTGAAGAACTGGCGATGCCGCCGCAGATCAAACTGGAGCAGGCCAAAGGTTTCAGCCTTTACATGCTGCGCGCGATTATTAGCGGCCGCGGCGATGAGGTTATCGAACTGGCGAAAACTAACTGGCTAAGGTAA

the gene trpBA nucleotide sequence (SEQ ID NO. 19):

ATGACAACATTACTTAACCCCTATTTTGGTGAGTTTGGCGGCATGTACGTGCCACAAATCCTGATGCCTGCTCTGCGCCAGCTGGAAGAAGCTTTTGTCAGTGCGCAAAAAGATCCTGAATTTCAGGCTCAGTTCAACGACCTGCTGAAAAACTATGCCGGGCGTCCAACCGCGCTGACCAAATGCCAGAACATTACAGCCGGGACGAACACCACGCTGTATCTCAAGCGTGAAGATTTGCTGCACGGCGGCGCGCATAAAACTAACCAGGTGCTGGGGCAGGCGTTGCTGGCGAAGCGGATGGGTAAAACCGAAATCATCGCCGAAACCGGTGCCGGTCAGCATGGCGTGGCGTCGGCCCTTGCCAGCGCCCTGCTCGGCCTGAAATGCCGTATTTATATGGGTGCCAAAGACGTTGAACGCCAGTCGCCTAACGTTTTTCGTATGCGCTTAATGGGTGCGGAAGTGATCCCGGTGCATAGCGGTTCCGCGACGCTGAAAGATGCCTGTAACGAGGCGCTGCGCGACTGGTCCGGTAGTTACGAAACCGCGCACTATATGCTGGGCACCGCAGCTGGCCCGCATCCTTATCCGACCATTGTGCGTGAGTTTCAGCGGATGATTGGCGAAGAAACCAAAGCGCAGATTCTGGAAAGAGAAGGTCGCCTGCCGGATGCCGTTATCGCCTGTGTTGGCGGCGGTTCGAATGCCATCGGCATGTTTGCTGATTTCATCAATGAAACCAACGTCGGCCTGATTGGTGTGGAGCCAGGTGGTCACGGTATCGAAACTGGCGAGCACGGCGCACCGCTAAAACATGGTCGCGTGGGTATCTATTTCGGTATGAAAGCGCCGATGATGCAAACCGAAGACGGGCAGATTGAAGAATCTTACTCCATCTCCGCCGGACTGGATTTCCCGTCTGTCGGCCCACAACACGCGTATCTTAACAGCACTGGACGCGCTGATTACGTGTCTATTACCGATGATGAAGCCCTTGAAGCCTTCAAAACGCTGTGCCTGCACGAAGGGATCATCCCGGCGCTGGAATCCTCCCACGCCCTGGCCCATGCGTTGAAAATGATGCGCGAAAACCCGGATAAAGAGCAGCTACTGGTGGTTAACCTTTCCGGTCGCGGCGATAAAGACATCTTCACCGTTCACGATATTTTGAAAGCACGAGGGGAAATCTGATGGAACGCTACGAATCTCTGTTTGCCCAGTTGAAGGAGCGCAAAGAAGGCGCATTCGTTCCTTTCGTCACGCTCGGTGATCCGGGCATTGAGCAGTCATTGAAAATTATCGATACGCTAATTGAAGCCGGTGCTGACGCGCTGGAGTTAGGTATCCCCTTCTCCGACCCACTGGCGGATGGCCCGACGATTCAAAACGCCACTCTGCGCGCCTTTGCGGCAGGTGTGACTCCGGCACAATGTTTTGAAATGCTGGCACTGATTCGCCAGAAACACCCGACCATTCCCATTGGCCTGTTGATGTATGCCAATCTGGTGTTTAACAAAGGCATTGATGAGTTTTATGCCCAGTGCGAAAAAGTCGGCGTCGATTCGGTGCTGGTTGCCGATGTGCCAGTTGAAGAGTCCGCGCCCTTCCGCCAGGCCGCGTTGCGTCATAATGTCGCACCTATCTTCATCTGCCCGCCAAATGCCGATGACGACCTGCTGCGCCAGATAGCCTCTTACGGTCGTGGTTACACCTATTTGCTGTCACGAGCAGGCGTGACCGGCGCAGAAAACCGCGCCGCGTTACCCCTCAATCATCTGGTTGCGAAGCTGAAAGAGTACAACGCTGCACCTCCATTGCAGGGATTTGGTATTTCCGCCCCGGATCAGGTAAAAGCAGCGATTGATGCAGGAGCTGCGGGCGCGATTTCTGGTTCGGCCATTGTTAAAATCATCGAGCAACATATTAATGAGCCAGAGAAAATGCTGGCGGCACTGAAAGTTTTTGTACAACCGATGAAAGCGGCGACGCGCAGTTAA

Gene pta nucleotide sequence (SEQ ID NO. 20):

GTGGTGCAGTTTTGTATCCCGCCCGATACTGGCGGTAACGAAAGAGGATATATCGTGTCCCGTACAATTATGCTGATCCCTACCGGAACCAGCGTAGGCCTGACCAGCGTCAGCCTCGGTGTTGTCCGTGCTATGGAACGTAAAGGCGTGCGCCTGAGCGTCTTTAAACCAATCGCTCAACCACGCGCCGGTGGCGATGCGCCAGACCAGACGACGACCATCATTCGCGCGAACTCCGACCTTCCGGCCGCAGAACCGTTGAAGATGAGCCACGTTGAGTCGCTGCTCTCCAGCAACCAGAAAGACGTGCTGATGGAAGAGATCATCGCCAACTACCACGCCAACGCTCAGGATGCTGAAGTGGTGCTGGTTGAAGGTCTGGTCCCGACCCGCAAACATCAGTTTGCCCAGTCGCTGAACTACGAAATCGCCAAAACGCTGAACGCTGAAATCGTCTTCGTCATGTCCCAGGGTACCGATACTCCGGAACAGCTGAAAGAACGCGTTGAGCTGACTCGCAGCAGCTTCGGCGGCGCGAAAAACACCAGCATTACCGGCGTAATCGTCAACAAGCTGAATGCGCCAGTTGACGAACAGGGCCGTACTCGCCCTGACCTGTCAGAAATCTTCGACGACTCTTCCAAAGCGAAAGTCGTGAAGATCGATCCGGCTCAGCTGCAGAACGGTAGCCCATTACCGGTTCTGGGCGCGGTGCCGTGGAGCTTCGATCTGATCGCCACCCGCGCGATCGATATGGCGCGTCACCTGAACGCCACCATCATCAACGAAGGCGACATTAATACCCGCCGCGTGAAGTCCGTGACCTTCTGCGCACGCAGCATTCCACACATGCTGGAGCACTTCCGCGCAGGATCGCTGCTGGTCACTTCCGCAGACCGTCCCGACGTTCTGGTCGCCGCCTGCCTGGCCGCGATGAACGGCGTGGAAATCGGCGCCATCCTGCTGACCGGCGGCTACGAAATGGACGAGCGCATCAGCAAGCTGTGCGAACGCGCATTCGCGACCGGCCTGCCGGTATTTATGGTCAACACCAATACCTGGCAGACCTCCCTGAGCCTGCAGAGCTTCAACCTGGAAGTTCCGGTTGACGATCACGAGCGCATCGAGAAAGTTCAGGAATACGTGGCTAACTACATTAACGCTGACTGGATCGAGTCGCTCACCGCGACTTCCGAGCGTAGCCGTCGCCTCTCTCCGCCAGCCTTCCGCTATCAGCTCACCGAGCTGGCGCGTAAAGCAGGCAAGCGCGTTGTTCTGCCAGAAGGTAACGAACCGCGTACCGTTAAAGCCGCCGCTATCTGCGCCGAGCGCGGTATCGCCACCTGCGTCCTGCTGGGCAACCCCGAAGAAATCACCCGCGTTGCCGCGTCTCAGGGCGTAGAGCTGGGCGCCGGGATTGAAATCGTCGATCCGGAAGTGGTTCGCGAAAGCTACGTAGCTCGCCTGGTCGAGCTGCGTAAGAGCAAGGGTATGACCGAAGCGGTTGCTCGCGAGCAGCTGGAAGACAACGTGGTTCTTGGCACCCTGATGCTGGAGCAAGACGAAGTTGACGGTCTGGTCTCCGGCGCGGTTCACACCACCGCCAACACCATCCGTCCGCCACTGCAGCTGATCAAAACGGCCCCTGGCAGCTCTCTGGTCTCCTCCGTATTCTTCATGCTGCTGCCGGAACAGGTTTACGTTTACGGCGACTGCGCGATCAACCCGGATCCGACCGCTGAGCAGCTGGCTGAAATCGCCATTCAGTCTGCTGAATCCGCAATTGCCTTTGGCATCGAGCCGCGCGTGGCCATGCTCTCCTACTCCACCGGCACTTCAGGCGCAGGCAGCGATGTAGAGAAAGTTCGCGAAGCAACTCGTCTGGCGCAGGAAAAACGTCCTGACCTGATGATCGACGGCCCGCTGCAGTATGATGCTGCGGTAATGGCTGACGTTGCGAAATCCAAAGCGCCGAACTCCCCGGTTGCCGGACGCGCTACCGTGTTCATCTTCCCGGATCTGAACACCGGTAACACCACTTACAAAGCCGTACAGCGTTCCGCCGACCTGATCTCCATCGGGCCGATGCTCCAAGGCATGCGTAAGCCGGTGAACGACCTGTCCCGCGGCGCGCTGGTTGACGATATCGTCTATACCATCGCCCTGACCGCGATTCAGTCGTCCCAGCAGCAAGCATAA

The gene tktA nucleotide sequence (SEQ ID No. 21):

ATGTCCTCACGTAAAGAGCTTGCCAATGCTATTCGTGCGCTGAGCATGGACGCAGTACAGAAAGCCAAATCCGGTCACCCGGGTGCCCCTATGGGTATGGCTGACATTGCCGAAGTCCTGTGGCGTGATTTCCTGAAACACAACCCGCAGAATCCGTCCTGGGCTGACCGTGACCGCTTCGTGCTGTCCAACGGCCACGGCTCCATGCTGATCTACAGCCTGCTGCACCTCACCGGTTACGATCTGCCGATGGAAGAACTGAAAAACTTCCGTCAGCTGCACTCTAAAACTCCGGGTCACCCGGAAGTGGGTTACACCGCTGGTGTGGAAACCACCACCGGTCCGCTGGGTCAGGGTATTGCCAACGCAGTCGGTATGGCGATTGCAGAAAAAACGCTGGCGGCGCAGTTTAACCGTCCGGGCCACGACATTGTCGACCACTACACCTACGCCTTCATGGGCGACGGCTGCATGATGGAAGGCATCTCCCACGAAGTTTGCTCTCTGGCGGGTACGCTGAAGCTGGGTAAACTGATTGCATTCTACGATGACAACGGTATTTCTATCGATGGTCACGTTGAAGGCTGGTTCACCGACGACACCGCAATGCGTTTCGAAGCTTACGGCTGGCACGTTATTCGCGACATCGACGGTCATGACGCGGCATCTATCAAACGCGCAGTAGAAGAAGCGCGCGCAGTGACTGACAAACCTTCCCTGCTGATGTGCAAAACCATCATCGGTTTCGGTTCCCCGAACAAAGCCGGTACCCACGACTCCCACGGTGCGCCGCTGGGCGACGCTGAAATTGCCCTGACCCGCGAACAACTGGGCTGGAAATATGCGCCGTTCGAAATCCCGTCTGAAATCTATGCTCAGTGGGATGCGAAAGAAGCAGGCCAGGCGAAAGAATCCGCATGGAACGAGAAATTCGCTGCTTACGCGAAAGCTTATCCGCAGGAAGCCGCTGAATTTACCCGCCGTATGAAAGGCGAAATGCCGTCTGACTTCGACGCTAAAGCGAAAGAGTTCATCGCTAAACTGCAGGCTAATCCGGCGAAAATCGCCAGCCGTAAAGCGTCTCAGAATGCTATCGAAGCGTTCGGTCCGCTGTTGCCGGAATTCCTCGGCGGTTCTGCTGACCTGGCGCCGTCTAACCTGACCCTGTGGTCTGGTTCTAAAGCAATCAACGAAGATGCTGCGGGTAACTACATCCACTACGGTGTTCGCGAGTTCGGTATGACCGCGATTGCTAACGGTATCTCCCTGCACGGTGGCTTCCTGCCGTACACCTCCACCTTCCTGATGTTCGTGGAATACGCACGTAACGCCGTACGTATGGCTGCGCTGATGAAACAGCGTCAGGTGATGGTTTACACCCACGACTCCATCGGTCTGGGCGAAGACGGCCCGACTCACCAGCCGGTTGAGCAGGTCGCTTCTCTGCGCGTAACCCCGAACATGTCTACATGGCGTCCGTGTGACCAGGTTGAATCCGCGGTCGCGTGGAAATACGGTGTTGAGCGTCAGGACGGCCCGACCGCACTGATCCTCTCCCGTCAGAACCTGGCGCAGCAGGAACGAACTGAAGAGCAACTGGCAAACATCGCGCGCGGTGGTTATGTGCTGAAAGACTGCGCCGGTCAGCCGGAACTGATTTTCATCGCTACCGGTTCAGAAGTTGAACTGGCTGTTGCTGCCTACGAAAAACTGACTGCCGAAGGCGTGAAAGCGCGCGTGGTGTCCATGCCGTCTACCGACGCATTTGACAAGCAGGATGCTGCTTACCGTGAATCCGTACTGCCGAAAGCGGTTACTGCACGCGTTGCTGTAGAAGCGGGTATTGCTGACTACTGGTACAAGTATGTTGGCCTGAACGGTGCTATCGTCGGTATGACCACCTTCGGTGAATCTGCTCCGGCAGAGCTGCTGTTTGAAGAGTTCGGCTTCACTGTTGATAACGTTGTTGCGAAAGCAAAAGAACTGCTGTAA

The gene pykF nucleotide sequence (SEQ ID No. 22):

ATGAAAAAGACCAAAATTGTTTGCACCATCGGTCCGAAAACCGAATCCGAAGAGATGTTGACCAAAATGCTGGACGCCGGTATGAACGTTATGCGTCTGAACTTCTCTCACGGTGACTATGCGGAACACGGCCAGCGCATCCAGAACCTGCGCAATGTGATGAGCAAAACGGGCAAGAAAGCGGCAATCCTTCTGGATACCAAAGGTCCGGAAATCCGTACTATTAAGCTGGAAGGCGGTAACGACGTCTCCCTGAAAGCGGGCCAGACTTTCACCTTCACCACCGACAAATCGGTGGTAGGTAATAACGAAATCGTTGCGGTGACCTATGAAGGCTTCACCAGCGACCTGAGCGTCGGCAACACCGTACTGGTCGACGATGGTCTGATCGGTATGGAAGTCACCGCGATCGAAGGTAACAAAGTTGTTTGTAAAGTGCTGAACAACGGCGACCTCGGCGAGAACAAAGGCGTTAACCTGCCGGGCGTATCTATCGCTCTGCCGGCGCTGGCTGAGAAAGACAAACAGGACCTGATCTTCGGTTGCGAGCAGGGCGTGGACTTTGTTGCGGCCTCCTTTATCCGTAAGCGTTCCGACGTCGTGGAAATCCGCGAGCACCTGAAAGCTCACGGCGGCGAGAAGATCCAGATCATCTCTAAAATTGAAAACCAGGAAGGCCTGAACAACTTCGATGAAATCCTCGAAGCTTCTGACGGCATCATGGTTGCGCGTGGCGACCTGGGCGTTGAAATCCCGGTTGAAGAAGTTATCTTCGCGCAGAAAATGATGATCGAAAAATGTATCCGCGCGCGTAAAGTCGTTATTACCGCGACCCAAATGCTGGATTCCATGATCAAAAACCCGCGTCCGACCCGTGCGGAAGCGGGCGACGTGGCTAACGCCATCCTCGACGGCACCGATGCTGTCATGCTCTCAGGCGAATCCGCAAAAGGTAAATATCCGCTGGAAGCGGTCACCATCATGGCGACCATCTGCGAACGTACCGACCGCGTGATGACCAGCCGTCTTGAGTACAACAACGATAACCGTAAGCTGCGCATCACCGAAGCGGTCTGCCGCGGTGCGGTTGAAACGGCAGAAAAACTGGAAGCGCCGCTGATCGTCGTCGCGACCCAGGGCGGTAAATCCGCGCGCGCCGTACGTAAATACTTCCCGGATGCAACCATCCTGGCGCTGACCACCAACGAAACCACTGCGCGTCAGCTGGTGCTGAGCAAAGGCGTGGTAGCGCAGCTGGTTGAAGAGATCGCCTCTACCGACGCTTTCTATCACCTCGGTAAAGAGCTGGCGCTGCAGAGCGGCCTGGCGCGCAAAGGCGACGTGGTTGTTATGGTTTCCGGCGCATTAGTCCCGAGCGGAACCACCAATACCGCTTCCGTGCACGTGCTGTAA

mutein gene serA ^{fbr(H344A,N346A,N364A)} nucleotide sequence (SEQ ID NO. 23):

ATGGCAAAGGTATCGCTGGAGAAAGACAAGATTAAGTTTCTGCTGGTAGAAGGCGTGCACCAAAAGGCGCTGGAAAGCCTTCGTGCAGCTGGTTACACCAACATCGAATTTCACAAAGGCGCGCTGGATGATGAACAATTAAAAGAATCCATCCGCGATGCCCACTTCATCGGCCTGCGATCCCGTACCCATCTGACTGAAGACGTGATCAACGCCGCAGAAAAACTGGTCGCTATTGGCTGTTTCTGTATCGGAACAAACCAGGTTGATCTGGATGCGGCGGCAAAGCGCGGGATCCCGGTATTTAACGCACCGTTCTCAAATACGCGCTCTGTTGCGGAGCTGGTGATTGGCGAACTGCTGCTGCTATTGCGCGGCGTGCCGGAAGCCAATGCTAAAGCGCACCGTGGCGTGTGGAACAAACTGGCGGCGGGTTCTTTTGAAGCGCGCGGCAAAAAGCTGGGTATCATCGGCTACGGTCATATTGGTACGCAATTGGGCATTCTGGCTGAATCGCTGGGAATGTATGTTTACTTTTATGATATTGAAAATAAACTGCCGCTGGGCAACGCCACTCAGGTACAGCATCTTTCTGACCTGCTGAATATGAGCGATGTGGTGAGTCTGCATGTACCAGAGAATCCGTCCACCAAAAATATGATGGGCGCGAAAGAAATTTCACTAATGAAGCCCGGCTCGCTGCTGATTAATGCTTCGCGCGGTACTGTGGTGGATATTCCGGCGCTGTGTGATGCGCTGGCGAGCAAACATCTGGCGGGGGCGGCAATCGACGTATTCCCGACGGAACCGGCGACCAATAGCGATCCATTTACCTCTCCGCTGTGTGAATTCGACAACGTCCTTCTGACGCCACACATTGGCGGTTCGACTCAGGAAGCGCAGGAGAATATCGGCCTGGAAGTTGCGGGTAAATTGATCAAGTATTCTGACAATGGCTCAACGCTCTCTGCGGTGAACTTCCCGGAAGTCTCGCTGCCACTGCACGGTGGGCGTCGTCTGATGCACATCGCCGAAGCCCGTCCGGGCGTGCTAACTGCGCTGAACAAAATCTTCGCCGAGCAGGGCGTCGCCATCGCCGCGCAATATCTGCAAACTTCCGCCCAGATGGGTTATGTGGTTATTGATATTGAAGCCGACGAAGACGTTGCCGAAAAAGCGCTGCAGGCAATGAAAGCTATTCCGGGTACCATTCGCGCCCGTCTGCTGTACTAA

gene budC nucleotide sequence (SEQ ID No. 24):

ATGAAAAAAGTCGCACTCGTGACCGGCGCAGGCCAGGGTATCGGTAAAGCTATCGCCCTTCGCCTGGTTCAAGATGGCTTTGCCGTGGCCATCGCCGATTATAACGATGCCACCGCACAGGCGGTTGCTGACGAAATTAACCAGCACGGCGGCCAGGCGCTGGCGGTGAAGGTCGATGTCTCGAAACGCGATCAGGTTTTTGCCGCCGTTGAGCAGGCGCGTAAGGGCCTTGGCGGTTTTGACGTGATCGTTAACAACGCCGGGGTCGCGCCCTCTACGCCTATCGAAGAGATTCGCGAGGACGTCATCGATAAAGTCTACAATATCAACGTTAAGGGCGTTATCTGGGGCATTCAGGCCGCGGTAGATGCGTTTAAAAAAGAGGGCCACGGCGGCAAGATCATCAACGCCTGCTCCCAGGCGGGCCACGTGGGTAACCCGGAACTGGCGGTCTACAGTTCAAGCAAGTTTGCCGTGCGCGGCCTGACGCAAACCGCCGCCCGCGATCTGGCGCATCTGGGAATTACCGTTAACGGCTACTGCCCGGGGATCGTCAAAACCCCCATGTGGGCGGAAATTGACCGTCAGGTTTCCGAAGCGGCGGGCAAACCGCTGGGCTACGGAACCCAGGAATTTGCGAAACGCATTACCCTCGGTCGTCTTTCCGAACCGGAAGACGTCGCGGCCTGCGTCTCTTATCTGGCCGGTCCGGACTCCAACTACATGACCGGTCAGTCGCTGCTGATCGATGGCGGTATGGTATTCAGTTAA

the gene glnA nucleotide sequence (SEQ ID NO. 25):

GTGGCGTTTGAAACCCCGGAAGAAATTGTCAAGTTCATCAAGGATGAAAACGTCGAGTTCGTTGACGTTCGATTCACCGACCTTCCCGGCACCGAGCAGCACTTCAGCATCCCAGCTGCCAGCTTCGATGCAGATACAATCGAAGAAGGTCTCGCATTCGACGGATCCTCGATCCGTGGCTTCACCACGATCGACGAATCTGACATGAATCTCCTGCCAGACCTCGGAACGGCCACCCTTGATCCATTCCGCAAGGCAAAGACCCTGAACGTTAAGTTCTTCGTTCACGATCCTTTCACCCGCGAGGCATTCTCCCGCGACCCACGCAACGTGGCACGCAAGGCAGAGCAGTACCTGGCATCCACCGGCATTGCAGACACCTGCAACTTCGGCGCCGAGGCTGAGTTCTACCTCTTCGACTCCGTTCGCTACTCCACCGAGATGAACTCCGGCTTCTACGAAGTAGATACCGAAGAAGGCTGGTGGAACCGTGGCAAGGAAACCAACCTCGACGGCACCCCAAACCTGGGCGCAAAGAACCGCGTCAAGGGTGGCTACTTCCCAGTAGCACCATACGACCAAACCGTTGACGTGCGCGATGACATGGTTCGCAACCTCGCAGCTTCCGGCTTCGCTCTTGAGCGTTTCCACCACGAAGTCGGTGGCGGACAGCAGGAAATCAACTACCGCTTCAACACCATGCTCCACGCGGCAGATGATATCCAGACCTTCAAGTACATCATCAAGAACACCGCTCGCCTCCACGGCAAGGCTGCAACCTTCATGCCTAAGCCACTGGCTGGCGACAACGGTTCCGGCATGCACGCTCACCAGTCCCTCTGGAAGGACGGCAAGCCACTCTTCCACGATGAGTCCGGCTACGCAGGCCTGTCCGACATCGCCCGCTACTACATCGGCGGCATCCTGCACCACGCAGGCGCTGTTCTGGCGTTCACCAACGCAACCCTGAACTCCTACCACCGTCTGGTTCCAGGCTTCGAGGCTCCAATCAACCTGGTGTACTCACAGCGCAACCGTTCCGCTGCTGTCCGTATCCCAATCACCGGATCCAACCCGAAGGCAAAGCGCATCGAATTCCGCGCTCCAGACCCATCAGGCAACCCATACCTGGGCTTTGCAGCGATGATGATGGCCGGCCTCGACGGCATCAAGAACCGCATCGAGCCACACGCTCCAGTGGACAAGGACCTCTACGAACTACCACCAGAGGAAGCTGCATCCATTCCACAGGCACCAACCTCCCTGGAAGCATCCCTGAAGGCACTGCAGGAAGACACCGACTTCCTCACCGAGTCTGACGTCTTCACCGAGGATCTCATCGAGGCGTACATCCAGTACAAGTACGACAACGAGATCTCCCCAGTTCGCCTGCGCCCAACCCCGCAGGAATTCGAATTGTACTTCGACTGCTAA

The nucleotide sequence of the gene dhaT (SEQ ID NO. 26):

ATGAGCTATCGTATATTTGATTATCTGGTGCCAAACGTGAACTTTTTTGGTCCCAATGCTGTTTCTGTCGTCGGCGAACGCTGTAAGCTGCTGGGCGGTAAAAAAGCGCTGCTGGTCACCGATAAAGGTCTGCGGGCCATCAAAGACGGCGCGGTGGATAAAACTCTCGGCTATCTGCAGGAAGCCGGTATTGAGGTGGTGGTTTTTGATGGCGTCGAGCCAAATCCAAAAGACACCAACGTGCGCGATGGCCTGGCGGTCTTTCGTCAAGAACAGTGCGATATTATCGTCACCGTCGGCGGCGGTAGCCCTCATGACTGCGGTAAAGGGATTGGTATCGCGGCTACCCACGAAGGGGATCTTTACCGCTATGCCGGCATAGAAACCCTGGCTAACCCACTGCCGCCGATCGTCGCGGTGAATACCACCGCCGGTACCGCCAGCGAAGTGACCCGCCACTGCGTACTGACCAACACCCAAACAAAAGTGAAGTTCGTTATCGTCAGCTGGCGTAACCTGCCCTCGGTCTCCATCAATGACCCGCTGCTCATGCTTGGCAAACCGGCCCCGCTGACCGCCGCAACCGGCATGGATGCGTTAACCCACGCCGTGGAGGCCTATATTTCCAAAGATGCCAACCCGGTCACCGATGCCGCCGCGATCCAGGCCATCCGCCTGATCGCCCGGAACTTGCGCCAGGCCGTCGCGCTGGGCAGCAACCTGCAAGCCCGCGAAAACATGGCCTACGCTTCCCTGCTGGCGGGAATGGCTTTCAATAACGCCAACCTCGGTTACGTTCACGCCATGGCCCATCAGCTTGGCGGCCTGTACGATATGCCGCACGGCGTGGCGAACGCGGTTCTGTTGCCGCACGTTGCGCGCTACAACCTGATCGCCAATCCGGAAAAGTTTGCCGACATCGCCGAATTTATGGGAGAAAACATCCACGGACTTTCAACCATGGACGCCGCCGAGTCGGCCATTTGCGCCATCGCCCGCCTCTCTTCCGATATCGGCATTCCGCAGCATCTGAGCGAGCTGGGAGTTAAAGAGGCTGACTTCCCCTATATGGCGGAGATGGCCTTGAAAGACGGCAACGCCTTCTCAAATCCGCGCAAGGGTAACGAGAAAGAGATTGTCGCTATCTTCCGCCAGGCTTTCTGA

Gene ywkB nucleotide sequence (SEQ ID No. 27):

TTGAGCATCTTAGATATCTTAATCCTCCTGGCGCCGATCTTCTTTGTTATCGTGCTGGGTTGGTTTGCAGGACATTTTGGAAGTTATGATGCCAAGTCGGCAAAAGGGGTAAGTACGTTAGTAACGAAATACGCACTTCCAGCTCACTTTATCGCTGGTATTTTGACAACTTCCAGAAGTGAATTTTTATCACAAGTACCTTTAATGATTTCTTTAATTATTGGGATTGTTGGTTTCTATATCATCATTCTTTTGGTTTGCAGATTTATATTCAAGTATGATTTAACGAACTCATCTGTATTTTCTTTGAACTCTGCACAGCCGACATTCGCATTTATGGGTATCCCGGTATTGGGAAGCTTATTCGGAGCGAATGAAGTTGCGATTCCGATCGCGGTCACAGGTATCGTGGTTAACGCGATTCTTGATCCGCTCGCGATCATTATCGCTACTGTTGGTGAGTCTTCTAAGAAAAACGAAGAGAGTGGCGACAGCTTCTGGAAGATGACAGGAAAATCAATCCTGCATGGTCTTTGTGAGCCGCTTGCAGCTGCTCCGTTAATCAGTATGATCTTGGTGCTGGTTTTCAATTTCACTCTTCCTGAGCTGGGTGTTAAAATGCTTGATCAGCTTGGAAGCACAACATCTGGTGTTGCTCTCTTCGCTGTTGGTGTTACCGTTGGTATTCGTAAAATTAAACTCAGTATGCCGGCTATCGGTATTGCGTTACTAAAAGTTGCGGTTCAGCCTGCGTTAATGTTCCTGATTGCTCTTGCTATCGGACTTCCAGCTGACCAAACAACAAAAGCAATCCTTCTTGTTGCATTCCCTGGTTCTGCCGTTGCAGCCATGATTGCGACTCGTTTCGAGAAACAAGAAGAAGAAACTGCAACTGCGTTTGTGGTCAGTGCGATTCTGTCATTGATTTCACTTCCAATCATTATCGCGCTTACTGCGTAA

the gene gldA nucleotide sequence (SEQ ID NO. 28):

ATGGATCGCATTATTCAATCACCCGGTAAGTACATTCAGGGCGCGGACGCAATTTCTCGCCTTGGCGGATACCTCAAACCGCTGGCTGAGCGCTGGCTCATCGTTGGCGATAAATTTGTACTCGGTTTTGCCGAAGAAAAACTGCGTAAAAGCCTGGCAGATGCCGATCTGGTTGGCGAAATCGCGCCGTTTGGCGGCGAATGCTCACACAATGAAATCAACCGCTTGCGTGATATTGCCGGTTCCGCCCGCTGCACAGCCGTGCTGGGTATCGGCGGCGGCAAGACGCTGGATACCGCAAAAGCATTAGCACACTTTATGAATCTGCCGGTGGTGATTGCCCCGACGATTGCCTCCACCGATGCCCCCTGCAGCGCGCTCTCTGTCATCTATACCGATGACGGCGAGTTTGATAGCTACCTGATGCTGCCGCGCAACCCGAATATGGTCATTGTCGATACGCAAATCGTTGCCGGAGCCCCGGCGCGCCTGCTGGCGGCCGGTATCGGCGACGCGCTGGCGACCTGGTTTGAAGCGCGTGCCTGCTCCCGCAGCGGCGCCACCACCATGGCGGGCGGCAAATGTACTCAGGCAGCGCTGGCGCTGGCTGAACTGTGCTACAACACGCTGATTGAAGAAGGTGAAAAAGCGATGCTTGCGGCAGAGCAGCACGTTGTGACGCCAGCGCTGGAGCGGGTGATTGAAGCCAACACCTACCTGAGCGGCGTCGGCTTTGAAAGCGGCGGCCTGGCGGCTGCGCACGCTATCCACAACGGCCTGACCGCGATCCCGGATGCTCACCACTACTATCATGGTGAAAAAGTAGCTTTCGGTACGCTCACGCAGCTGGTACTGGAAAACGCGCCGGTAGAAGAGATCGAAACCGTAGCGGCGCTGTGCCATAGCGTCGGACTGCCTATCACCCTGGCGCAGCTGGATATTAAAGCCGATATCCCAGGCAAAATGCGTACCGTAGCGGAAGCCGCCTGCGCAGAAGGCGAAACCATCCATAATATGCCCGGCGGCGCAACGCCGGATCAGGTTTATGCCGCGCTTCTGGTTGCCGACCAGTACGGTCAACGCTTCCTGCAAGAGTGGGAATAA

the nucleotide sequence of gene pck (SEQ ID NO. 29):

ATGAACTCAGTTGATTTGACCGCTGATTTACAAGCCTTATTAACATGTCCAAATGTGCGTCATAATTTATCAGCAGCACAGCTAACAGAAAAAGTCCTCTCCCGAAACGAAGGCATTTTAACATCCACAGGTGCTGTTCGCGCGACAACAGGCGCTTACACAGGACGCTCACCTAAAGATAAATTCATCGTGGAGGAAGAAAGCACGAAAAATAAGATCGATTGGGGCCCGGTGAATCAGCCGATTTCAGAAGAAGCGTTTGAGCGGCTGTACACGAAAGTTGTCAGCTATTTAAAGGAGCGAGATGAACTGTTTGTTTTCGAAGGATTTGCCGGAGCAGACGAGAAATACAGGCTGCCGATCACTGTCGTAAATGAGTTCGCATGGCACAATTTATTTGCGCGGCAGCTGTTTATCCGTCCGGAAGGAAATGATAAGAAAACAGTTGAGCAGCCGTTCACCATTCTTTCTGCTCCGCATTTCAAAGCGGATCCAAAAACAGACGGCACTCATTCCGAAACGTTTATTATTGTCTCTTTCGAAAAGCGGACAATTTTAATCGGCGGAACTGAGTATGCCGGTGAAATGAAGAAGTCCATTTTCTCCATTATGAATTTCCTGCTGCCTGAAAGAGATATTTTATCTATGCACTGCTCCGCCAATGTCGGTGAAAAAGGCGATGTCGCCCTTTTCTTCGGACTGTCAGGAACAGGAAAGACCACCCTGTCGGCAGATGCTGACCGCAAGCTGATCGGTGACGATGAACATGGCTGGTCTGATACAGGCGTCTTTAATATTGAAGGCGGATGCTACGCTAAGTGTATTCATTTAAGCGAGGAAAAGGAGCCGCAAATCTTTAACGCGATCCGCTTCGGGTCTGTTCTCGAAAATGTCGTTGTGGATGAAGATACACGCGAAGCCAATTATGATGATTCCTTCTATACTGAAAACACGCGGGCAGCTTACCCGATTCATATGATTAATAACATCGTGACTCCAAGCATGGCCGGCCATCCGTCAGCCATTGTATTTTTGACGGCTGATGCCTTCGGAGTCCTGCCGCCGATCAGCAAACTAACGAAGGAGCAGGCGATGTACCATTTTTTGAGCGGTTACACGAGTAAGCTTGCCGGAACCGAACGTGGTGTCACGTCTCCTGAAACGACGTTTTCTACATGCTTCGGCTCACCGTTCCTGCCGCTTCCTGCTCACGTCTATGCTGAAATGCTCGGCAAAAAGATCGATGAACACGGCGCAGACGTTTTCTTAGTCAATACCGGATGGACCGGGGGCGGCTACGGCACAGGCGAACGAATGAAGCTTTCTTACACTAGAGCAATGGTCAAAGCAGCGATTGAAGGCAAATTAGAGGATGCTGAAATGATAACTGACGATATTTTCGGCCTGCACATTCCGGCCCATGTTCCTGGCGTTCCTGATCATATCCTTCAGCCTGAAAACACGTGGACCAACAAGGAAGAATACAAAGAAAAAGCAGTCTACCTTGCAAATGAATTCAAAGAGAACTTTAAAAAGTTCGCACATACCGATGCCATCGCCCAGGCAGGCGGCCCTCTCGTATAA

Gene ackA nucleotide sequence (SEQ ID No. 30):

ATGTCGAGTAAGTTAGTACTGGTTCTGAACTGCGGTAGCTCCTCTCTGAAATTCGCTATCCTTGATGCCGTCAACGGTGACGAATACCTGTCCGGTTTAGCAGAATGTTTCCATCTTCCAGAAGCCCGTATCAAATGGAAAATGGATGGCAGCAAACAAGAAGCCGAGCTAGGCGCTGGCGCTGCTCACAGTGAAGCGCTGAACTTTATCGTTAACACTATTCTGGCACAAAAACCAGAACTGTCTGCTCAGCTGACCGCAATCGGCCATCGTATCGTTCACGGCGGTGAGAAATACACCAGCTCCGTTGTTATCGATGACTCCGTCATTCAGGGCATTAAAGATTCCGCATCTTTCGCTCCGCTGCACAACCCTGCGCACCTGATCGGTATCGCTGAAGCGCTGAAATCCTTCCCGCATCTGAAAGATAAGAACGTTGCCGTGTTCGACACCGCGTTCCACCAGACCATGCCGGAAGAATCTTACCTCTATGCCCTGCCGTACAGCCTGTACAAAGAACACGGCGTACGTCGCTACGGCGCGCACGGTACCAGCCACTTCTATGTGACTCAGGAAGCTGCGAAAGTTCTGAACAAACCGGTTGAAGAGCTGAACATCATCACCTGCCATCTGGGCAACGGTGGTTCCGTTTCCGCTATCCGTAACGGTAAATGCGTTGACACCTCTATGGGTCTGACTCCGCTGGAAGGCCTGGTTATGGGTACGCGTTCTGGCGATATCGACCCGGCAATCATTTTCCATCTGCACGATGCCCTGGGCATGAGCGTAGACGCCATCAACAAAATGCTGACCAAAGAGTCCGGTCTGCTCGGCCTGACCGAAGTGACCAGCGACTGCCGCTATGTTGAAGACAACTACCAGGACAAAGCGGATGCTAAACGCGCTATGGACGTTTACTGCCACCGCCTGGCAAAATACATCGGTTCCTACACCGCGCTGATGGATGGCCGTCTGGACGCGGTTATTTTCACCGGCGGTATCGGTGAGAACGCGGCGATGGTTCGCGAACTGTCTCTGGGCAAACTGGGCGTACTGGGCTTTGAAGTTGATCACGAGCGCAACCTGGCTGCCCGTTTCGGCAAGTCTGGCTTCATCAACAAAGAAGGCACTCGCCCGGCTATCGTCATTCCGACCAACGAAGAGCTGGTCATCGCGCAAGACGCCAACCGTCTGACCGCCTGA

EXAMPLE 1 construction of L-tryptophan-producing strains from Klebsiella oxytoca K.oxytoca CICC21518

The original strain Klebsiella oxytoca K.oxytoca CICC21518 is purchased from China center for type culture Collection of industrial microorganisms, and the deposit number is CICC21518. Oxytoca CICC21518 is gram-negative, grown aerobically or facultatively anaerobically, the optimum culture temperature being 37 ℃. VP (Voges-Proskauer) of the oxoma CICC21518 reacted positively and was able to metabolize citrate for growth. Klebsiella oxytoca has a broad substrate spectrum, delays fermentation of lactose, fermentation of mannitol, inositol, sorbitol, melibiose, adonitol and raffinose.

1.1 Substitution of the alpha-acetolactate decarboxylase Gene budA with the phosphoenolpyruvate synthase Gene ppsA from ESCHERICHIA COLI W3110

The length of the phosphoenolpyruvate synthase gene ppsA is 2379 bases, and the nucleotide sequence is shown as SEQ ID NO. 1. The length of the alpha-acetolactate decarboxylase gene budA is 780 bases, and the nucleotide sequence of the alpha-acetolactate decarboxylase gene budA is shown as SEQ ID NO. 2.

(1) Construction of Gene replacement vector genomic DNA of K.oxyloca CICC21518 was prepared by conventional methods, and the process was carried out by referring to the method for small preparation of bacterial genome in the "Fine programming molecular biology guide" published by scientific Press, and genomic DNA of Klebsiella oxytoca K.oxyloca CICC21518 was extracted. The upstream and downstream homology arms of the budA gene were amplified by PCR using genomic DNA of K.oxyloca CICC21518 as template for subsequent gene replacement. The middle replacement gene ppsA was amplified using the E.coli W3110 genome as template. And (3) carrying out recombinant PCR by taking the obtained upstream homology arm and the gene ppsA as templates, carrying out recombinant PCR on the obtained recombinant fragment and the upstream homology arm and the downstream homology arm, and then obtaining the delta budA, wherein the gene substitution fragment of ppsA comprises enzyme cutting sites of EcoRI and BamHI at two ends.

The suicide plasmid pR6KmobsacB is subjected to double digestion by using restriction enzymes EcoRI and BamHI, and the digested product is recovered by using nucleic acid gel and then is connected with a gene replacement fragment by using T5 exonuclease, so as to obtain a gene replacement plasmid pR6 KmobsacB-delta budA:: ppsA.

(2) And (3) gene knockout step, inoculating Escherichia coli S17-1 lambda pir carrying the plasmid obtained in the step (1), culturing Klebsiella oxytoca K.oxytoca CICC21518 at 37 ℃ overnight, and transferring the strain to be cultured at 37 ℃ until OD _600nm is about 0.6-0.8. 5mL of E.coli bacterial liquid and 1mL of Klebsiella oxytoca bacterial liquid were collected, and the bacterial bodies were collected by centrifugation at 6500rpm for 3 minutes, washed twice with 0.85% physiological saline, mixed with 100. Mu.L of physiological saline, and then dropped onto LB plates, and were placed in a 37℃incubator overnight for cultivation. After the bacterial membrane is washed by 0.85% normal saline to collect cells, the cells are collected by centrifugation at 6500rpm for 3 minutes, the cells are washed twice by 0.85% normal saline, diluted by 4-10 times and coated on an M9 solid plate containing 20% citrate and added with kanamycin, the cells are cultured for 36-48 hours at 37 ℃, the grown single colony is picked up and cultured in LB culture medium containing kanamycin at 37 ℃ and the bacterial solution PCR verification is carried out by using upstream and downstream primers, so that the correct single exchange target bacteria capable of simultaneously producing long fragments and short fragments by PCR are obtained.

Transferring the correct single-exchange target strain into a non-resistant LB (LB) medium, culturing at 37 ℃ overnight, transferring into a LB medium containing 15% sucrose, culturing at 37 ℃ for 10-12 hours, transferring for two generations, gradient diluting, coating on a LB solid medium containing 15% sucrose, culturing at 37 ℃ overnight, picking up grown single colonies, performing bacterial solution PCR (polymerase chain reaction) verification by using an upstream primer and a downstream primer, extracting a single colony genome with the correct PCR verification band size, and performing genome temperature gradient PCR verification by using the primers, wherein the correct band size is the double-exchange target strain.

1.2 Substitution of the alpha-acetolactate synthase Gene budB with the 3-deoxy-D-arabinoheptulose 7-phosphate synthase mutein-encoding Gene aroG ^fbr(D146N) from ESCHERICHIA COLI W3110

The length of the aroG ^fbr(D146N) sequence of the 3-deoxy-D-arabinoheptulose 7-phosphate synthase mutant protein gene is 1053 bases, and the nucleotide sequence is shown as SEQ ID NO. 3. The length of the alpha-acetolactate synthase gene budB is 1680 bases, and the nucleotide sequence is shown in SEQ ID NO. 4.

The α -acetolactate synthase encoding gene budB was replaced with the 3-deoxy-D-arabinoheptulose 7-phosphate synthase mutein encoding gene aroG ^fbr(D146N) from ESCHERICHIA COLI W3110, and the construction and procedure of the vector were referred to the procedure of the present example, in step 1.1, for replacement of the budA gene with the ppsA gene. The klebsiella acidogens used in this step were performed on a 1.1 basis.

1.3A Tryptophan enzyme encoding Gene tnaA knockout

The tryptophan enzyme encoding gene tnaA has a length of 1416 bases and a nucleotide sequence shown in SEQ ID NO. 5.

Knock-out vector construction the upstream and downstream homology arms of the tnaA gene were amplified by PCR. And (3) carrying out recombination by taking the obtained upstream and downstream homology arms as templates, and amplifying the recombined fragments by PCR to obtain a truncated fragment of the tnaA, wherein both ends of the truncated fragment comprise enzyme cutting sites of EcoRI and BamHI.

The truncated recombinant fragment of tnaA and suicide plasmid pR6KmobsacB are respectively subjected to double digestion by restriction enzymes EcoRI and BamHI, and the digested products are recovered by nucleic acid gel and then are connected with a gene replacement fragment by using T5 exonuclease, so that gene replacement plasmid pR6 KmobsacB-delta tnaA is obtained. Gene manipulation procedure the ppsA gene was replaced with the budA gene in step 1.1 of this example. The klebsiella acidogens used in this step were performed on a 1.2 basis.

1.4 Replacement of the attenuator trpL of the tryptophan operon with the promoter P _budABC of the endogenous 2, 3-butanediol synthesis pathway gene cluster

The length of the promoter P _budABC sequence of the endogenous 2, 3-butanediol synthesis pathway gene cluster is 106 bases, and the nucleotide sequence is shown as SEQ ID NO. 6. The attenuator trpL of the tryptophan operon has a sequence length of 45 bases and the nucleotide sequence of the attenuator is shown as SEQ ID NO. 7.

Construction and procedure of the attenuator trpL vector in which the tryptophan operon was replaced with the promoter P _budABC of the endogenous 2, 3-butanediol synthesis pathway gene cluster were described with reference to the procedure of the present example in which the budA gene was replaced with the ppsA gene in step 1.1. The klebsiella acidogens used in this step were performed on a 1.3 basis.

1.5 Transcription repressor trpR knockout of tryptophan operon

The length of the transcription inhibitor trpR sequence of the tryptophan operon is 327 bases, and the nucleotide sequence of the transcription inhibitor trpR sequence is shown as SEQ ID NO. 8.

Construction and procedure of the transcription repressor trpR knockout vector of tryptophan operon refer to the procedure of the tnaA gene knockout in step 1.3 of this example. The klebsiella acidogens used in this step were performed on a 1.4 basis.

1.6 Substitution of lactate dehydrogenase Gene ldhD with 3-dehydroquinic acid synthase Gene aroB from E.coli W3110

The length of the aroB sequence of the 3-dehydroquinic acid synthase gene from E.coli W3110 is 1089 bases, and the nucleotide sequence is shown in SEQ ID NO. 9. The lactic dehydrogenase gene ldhD has a sequence length of 990 bases and a nucleotide sequence shown in SEQ ID NO. 10.

Construction and procedure of vector for substitution of lactate dehydrogenase gene ldhD with 3-dehydroquinic acid synthase gene aroB from e.coli W3110 reference was made to the procedure of substitution of budA gene with ppsA gene in step 1.1 of this example. The klebsiella acidogens used in this step were performed on a 1.6 basis.

1.7 Substitution of the ethanol dehydrogenase Gene adhE with the shikimate dehydrogenase encoding Gene aroE from E.coli W3110

The sequence length of the shikimate dehydrogenase encoding gene aroE from E.coli W3110 is 819 bases, and the nucleotide sequence is shown in SEQ ID NO. 11. The length of the adhE sequence of the alcohol dehydrogenase gene is 780 bases, and the nucleotide sequence of the adhE is shown as SEQ ID NO. 12.

Construction and procedure of substitution of the ethanol dehydrogenase gene adhE vector with shikimate dehydrogenase encoding gene aroE from E.coli W3110 reference is made to the procedure of substitution of the budA gene with ppsA gene in step 1.1 of this example. The klebsiella acidogens used in this step were performed on a 1.7 basis.

1.8 Substitution of the fumaric reductase subunit A Gene frdA with the chorismate synthase encoding Gene aroC from E.coli W3110

The length of the aroC sequence of the coding gene of the branched acid synthase from E.coli W3110 is 1086 bases, and the nucleotide sequence is shown as SEQ ID NO. 13. The length of the fumaric acid reductase subunit A gene sequence is 1791 bases, and the nucleotide sequence is shown as SEQ ID NO. 14.

Construction and procedure of the vector for substituting the branched acid synthase encoding gene aroC from E.coli W3110 for the fumaric acid reductase subunit A gene frdA reference is made to the procedure for substituting the ppsA gene for the budA gene in step 1.1 of this example. The klebsiella acidogens used in this step were performed on a 1.8 basis.

1.9 Substitution of the pyruvate formate lyase Gene pflB with the Tryptophan operon partial Gene Cluster trpE ^fbr(S40F) D from E.coli W3110

The tryptophan operon partial gene cluster trpE ^fbr(S40F) D from E.coli W3110 has a length of 3158 bases and a nucleotide sequence shown in SEQ ID NO. 15. The pyruvic acid lyase gene pflB has a sequence length of 2283 bases and a nucleotide sequence shown in SEQ ID NO. 16.

Construction and procedure of the vector for replacing the pyruvate formate lyase gene pflB with the tryptophan operon partial gene cluster trpE ^fbr(S40F) D from E.coli W3110 refer to the procedure for replacing the budA gene with the ppsA gene in step 1.1 of this example. The klebsiella acidogens used in this step were performed on a 1.8 basis.

1.10 Substitution of the pyruvate oxidase Gene pox with the Tryptophan operon partial Gene Cluster trpDC from E.coli W3110

The tryptophan operon partial gene cluster trpDC from E.coli W3110 has a sequence length of 2958 bases and a nucleotide sequence shown in SEQ ID NO. 17. The pyruvate oxidase gene pox gene sequence is 1719 bases in length, and the nucleotide sequence is shown as SEQ ID NO. 18.

Construction and procedure of substitution of the pyruvate oxidase gene pox vector with the tryptophan operon partial gene cluster trpDC from E.coli W3110 reference is made to the procedure of substitution of the budA gene with the ppsA gene in step 1.1 of this example. The klebsiella acidogens used in this step were performed on a 1.9 basis.

1.11 Substitution of the phosphotransacetylase Gene pta with the Tryptophan operon partial Gene Cluster trpBA from E.coli W3110

The tryptophan operon partial gene cluster trpBA from E.coli W3110 has a length of 2000 bases and a nucleotide sequence shown in SEQ ID NO. 19. The length of the phosphotransacetylase pta gene sequence is 2199 bases, and the nucleotide sequence is shown as SEQ ID NO. 20.

Construction and procedure of substitution of the phosphotransacetylase gene pta vector with the tryptophan operon partial gene cluster trpBA from E.coli W3110 reference is made to the procedure of substitution of the budA gene with the ppsA gene in step 1.1 of this example. The klebsiella acidogens used in this step were performed on a 1.10 basis.

1.12 Substitution of the pyruvate kinase Gene pykF with the transketolase encoding Gene tktA from E.coli W3110

The sequence length of the transketolase coding gene tktA from E.coli W3110 is 1992 bases, and the nucleotide sequence is shown as SEQ ID NO. 21. The pyruvic acid kinase gene pykF has 1413 bases in length and the nucleotide sequence shown in SEQ ID No. 22.

Construction and procedure of the pyruvate kinase gene pykF vector replaced by the transketolase encoding gene tktA from e.coli W3110 reference is made to the procedure of the ppsA gene replacement budA gene in step 1.1 of this example. The klebsiella acidogens used in this step were performed on a 1.11 basis.

1.13 Substitution of 2, 3-butanediol dehydrogenase Gene budC with the 3-phosphoglycerate dehydrogenase mutein-encoding Gene serA ^fbr(H344A ^{,N346A,N364A)} from E.coli W3110

The sequence length of the 3-phosphoglycerate dehydrogenase mutant protein coding gene serA ^fbr(H344A ^{,N346A,N364A)} from E.coli W3110 is 1233 bases, and the nucleotide sequence is shown as SEQ ID NO. 23. The length of the sequence of the 2, 3-butanediol dehydrogenase gene budC is 771 base, and the nucleotide sequence of the gene is shown as SEQ ID NO. 24.

Construction and procedure of the vector for substituting 2, 3-butanediol dehydrogenase gene budC with serA ^fbr(H344A ^{,N346A,N364A)}, a 3-phosphoglycerate dehydrogenase mutein-encoding gene from E.coli W3110, reference is made to the procedure of the present example for substituting the budA gene with ppsA gene in step 1.1. The klebsiella acidogens used in this step were performed on a 1.12 basis.

1.14 Substitution of the 1, 3-propanediol dehydrogenase Gene dhaT with the glutamine synthase-encoding gene glnA from Corynebacterium glutamicum C.glutamicum ATCC13032

The glutamine synthase encoding gene glnA from Corynebacterium glutamicum C.glutamicum ATCC13032 has a length of 1434 bases and a nucleotide sequence shown in SEQ ID NO. 25. The length of the dhaT sequence of the 1, 3-propanediol dehydrogenase gene is 1164 bases, and the nucleotide sequence is shown as SEQ ID NO. 26.

The procedure for the construction and manipulation of the dhaT vector in which the gene glnA encoding the glutamine synthase from Corynebacterium glutamicum C.glutamicum ATCC13032 was used in place of the 1, 3-propanediol dehydrogenase gene was referred to as the procedure for the replacement of the budA gene by the ppsA gene in step 1.1 of this example. The klebsiella acidogens used in this step were performed on a 1.13 basis.

1.15 Substitution of the Glycerol dehydrogenase Gene gldA with the aromatic amino acid efflux protein Gene ywkB from Bacillus subtilis (Bacillus subtilis) 168

The sequence length of the aromatic amino acid efflux protein gene ywkB from B.subtilis 168 is 960 bases, and the nucleotide sequence is shown as SEQ ID NO. 27. The length of the gldA sequence of the glycerol dehydrogenase gene is 1104 bases, and the nucleotide sequence of the gldA sequence is shown as SEQ ID NO. 28.

Construction and procedure of the gldA vector in which the glycerol dehydrogenase gene was replaced with the aromatic amino acid efflux protein gene ywkB derived from Bacillus subtilis (Bacillus subtilis) 168 were referred to the procedure in which the budA gene was replaced with the ppsA gene in step 1.1 of this example. The klebsiella acidogens used in this step were performed on a 1.14 basis.

1.16 Substitution of the acetate kinase Gene ackA with the phosphoenolpyruvate carboxykinase Gene pck from Bacillus subtilis (Bacillus subtilis) 168

The phosphoenolpyruvate carboxykinase gene pck from B.subtilis 168 has a length of 1584 bases and the nucleotide sequence is shown in SEQ ID NO. 29. The length of the acetate kinase gene ackA sequence is 1203 bases, and the nucleotide sequence is shown as SEQ ID NO. 30.

Construction and procedure of substituting phosphoenolpyruvate carboxykinase gene pck from Bacillus subtilis (Bacillus subtilis) 168 for acetate kinase gene ackA vector the procedure of substituting the ppsA gene for budA gene in step 1.1 of this example was referred to. The klebsiella acidogens used in this step were performed on a 1.15 basis.

The resulting recombinant klebsiella oxytoca was designated Klebsiella oxytoca TRP-27, and the genotype thereof was K.oxytoca CICC21518ΔbudA::ppsAΔbudB::aroG^fbr(D146N)ΔtnaAΔtrpL::P_budABCΔtrpRΔldhD::aroBΔadhE::aroEΔfrdA::aroCΔpflB::trpE^fbr(S40F)DΔpox::trpDCΔpta::trpBAΔpykF::tktAΔbudC::serA^{fbr(H344A,N346A,N364A)}ΔdhaT::glnAΔgldA::ywkBΔackA::pck., and the L-tryptophan anabolic pathway of the recombinant bacterium of this example is shown in FIG. 1.

EXAMPLE 2 recombinant Klebsiella oxytoca K.oxytoca TRP-27 production of L-tryptophan by shake flask fermentation with glucose as substrate

(1) Plating, namely streaking the recombinant strain K.oxyoca TRP-27 on LB medium containing agar with the mass-volume ratio of 1.8%, and culturing at 37 ℃ for 10 hours;

(2) Seed culture, namely picking a single colony on the flat plate in the step (1) by using a sterile gun tip under the sterile condition, inoculating the single colony into 5mL of LB liquid medium, carrying out shaking culture for 10 hours at 37 ℃, and then inoculating the single colony into 100mL of LB liquid medium for shaking culture for 10 hours at 37 ℃ according to the inoculum size of 1% (v/v);

(3) Shake flask fermentation culture, in which the bacterial liquid obtained in the step (2) is inoculated into a fermentation medium containing 80g/L glucose according to an inoculum size of 5% (v/v) under aseptic conditions. The fermentation conditions are that the culture temperature is 37 ℃, the culture mode is shaking culture, the rotating speed is 180 turns per minute, the pH value is regulated to be 6.8 by ammonia water, and meanwhile, the concentration of the l-tryptophan in the fermentation liquid is measured by high performance liquid chromatography analysis of the fermentation sample. Stopping fermentation when glucose is not consumed any more, and obtaining L-tryptophan from the fermentation broth;

The result shows that the recombinant strain K.oxyoca TRP-27 is cultured for 60 hours, 80g/L of glucose is consumed, the concentration of L-tryptophan reaches 14.6g/L, and the yield of L-tryptophan reaches 0.183g/g.

Wherein the formula of the LB culture medium in the steps (1) - (2) comprises 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, pH 7.0 and sterilization at 121 ℃ for 20 minutes.

The formula of the fermentation medium in the step (3) is 80g/L of glucose and 1mL of yeast powder 5g/L,K₂HPO₄ 10g/L,NaH₂PO₄ 2g/L,NH₄SO₄ 10g/L,MgSO₄·7H₂O 0.7g/L,1000× microelement solution, wherein the formula of the 1000X microelement solution is ：CaCl₂·2H₂O 3g/L,ZnCl₂ 3g/L,FeCl₃·2H₂O 20g/L,MnCl₂·2H₂O 11g/L,CuCl₂·2H₂O 1g/L,CoCl₂·2H₂O 2g/L,H₃BO₃ 0.35g/L,NaMoO₄·2H₂O 0.024g/L., and in other embodiments, the content of glucose can be adjusted within the range of 80-90 g/L.

The detection method of the fermentation product L-tryptophan comprises the following steps:

The fermentation broth sample was centrifuged at 12,000rpm for 1min, 100. Mu.L of the sample supernatant was taken, 400. Mu.L of 0.5mol/L sodium bicarbonate (pH=9.0) and 100. Mu.L of 1%2, 4-dinitrofluorobenzene (2, 4-Dinitrofluorobenzene, DNFB) were added, and after shaking and mixing, light was blocked at 60℃for 1h. After cooling to room temperature, 0.01mol/L KH ₂PO₄ (pH 7.0) 1mL was added, the mixture was shaken and centrifuged, and the supernatant was filtered through a 0.22 μm filter, and then L-tryptophan was detected by high performance liquid chromatography. Specific liquid phase detection conditions are as follows:

The liquid chromatograph is Agilent 1260, the chromatographic column is Yuehu Welchrom C ₁₈ (4.6 mm. Times.250 mm,5 μm), the detector is a VWD detector, the detection wavelength is 254nm, the mobile phase A is 0.1% trifluoroacetic acid (TFA) aqueous solution, the mobile phase B is acetonitrile, and gradient elution is carried out by using the mobile phases A and B with different proportions, specifically, the liquid chromatograph comprises 0-10min,5% B, 10-13min,80% B, 13.1-18min,5% B, the flow rate is 1mL/min, the column temperature is 30 ℃, the sample injection amount is 5 mu L, and the analysis time is 18min.

EXAMPLE 3 recombinant Klebsiella oxytoca TRP-27 production of L-tryptophan by fed-batch fermentation with glucose as substrate

(3) 7.5L fermentation tank culture, namely inoculating the bacterial liquid obtained in the step (2) into a fermentation medium containing 60g/L glucose according to an inoculum size of 10% (v/v) under aseptic conditions. The fermentation condition is that the liquid loading amount is 5L, the culture temperature is 37 ℃, the culture mode is stirring culture, the stirring rotation speed is 500 revolutions per minute, the cascade rotation speed of dissolved oxygen level and ventilation are maintained at 15% -20%, the pH is regulated to 6.8+ -0.1, and 500g/L glucose mother liquor is added according to the glucose concentration, so that the glucose concentration is maintained at 0g/L. And stopping fermentation after 40 hours, and obtaining the L-tryptophan from the fermentation broth.

The result shows that the recombinant strain K.oxytoca TRP-27 is cultured for 60 hours, glucose is consumed for 272.7g/L, the concentration of L-tryptophan reaches 60.0g/L, the yield of L-tryptophan reaches 0.23g/g, the highest yield and conversion rate are achieved so far, and the L-tryptophan high-yield strain is obtained by taking Klebsiella oxytoca (K.oxytoca) as a chassis strain for the first time.

The method for detecting L-tryptophan as a product in the above steps, and the LB medium formulation and the fermentation medium formulation were the same as those in example 2, except that the concentration of glucose in the fermentation medium formulation of this example was 60g/L.

The technical scheme of the invention is further described through experiments.

Experimental example 1 blocking 2, 3-butanediol synthesis pathway and enhancing L-tryptophan synthesis pathway

The experimental example selects Klebsiella oxytoca K.oxytoca CICC21518 of Klebsiella genus as a production host to enhance accumulation of precursor pyruvic acid by blocking 2, 3-butanediol synthesis, and simultaneously enhance expression of phosphoenolpyruvate synthase and feedback inhibition insensitive 3-deoxy-D-arabinoheptulose 7-phosphate synthase to achieve redirection of 2, 3-butanediol anabolic stream to L-tryptophan synthesis by pyruvic acid.

Wherein, the gene knockout and the gene integration adopted by the invention both adopt two-step homologous recombination technology, and the specific operation process refers to the embodiment. Meanwhile, in the experimental example, phosphoenolpyruvate synthase ppsA (with the nucleotide sequence of SEQ ID NO. 1) and feedback inhibition insensitive 3-deoxy-7-phosphoheptanoate synthase gene aroG ^fbr(D146N) (with the nucleotide sequence of SEQ ID NO. 3) from escherichia coli are selected, and the ppsA gene is integrated at the site of 2, 3-butanediol synthesis pathway key gene alpha-acetolactate decarboxylase gene budA (with the nucleotide sequence of SEQ ID NO. 2) in the K.oxoma genome, and recombinant strain K.oxoma TRP-2 is obtained, and ppsA and aroG ^fbr(D146N) genes are respectively integrated at the site of 2, 3-butanediol synthesis pathway key gene alpha-acetolactate decarboxylase gene budA (with the nucleotide sequence of SEQ ID NO. 2) and alpha-acetolactate synthase gene budB (with the nucleotide sequence of SEQ ID NO. 4) in the K.oxoma genome by referring to the above-mentioned gene integration technology.

The starting strain K.oxyloca TRP-0, K.oxyloca TRP-2 and the recombinant strain K.oxyloca TRP-4 were shake-flask cultured for 60 hours in the same manner as in example 2, and the fermentation broth was subjected to metabolite analysis by High Performance Liquid Chromatography (HPLC).

TABLE 1 influence of blocking the 2, 3-butanediol synthesis pathway and enhancing the L-tryptophan synthesis pathway

The fermentation results are shown in Table 1, and the results show that the primary fermentation product of the original strain K.oxytoca CICC21518 is 2, 3-butanediol, budA and budB are knocked out, ppsA and aroG ^fbr(D146N) are added, 2, 3-butanediol is hardly accumulated after the recombinant strain K.oxytoca TRP-4 is fermented and cultured, and 0.8g/L of L-tryptophan is accumulated.

Experimental example 2 blocking the L-tryptophan degradation pathway

This experimental example knocks out the L-tryptophan enzyme encoding gene tnaA (SEQ ID NO. 5) of K.oxyoca CICC21518 to enhance L-tryptophan accumulation.

Wherein the gene knockout adopted in the invention adopts a two-step homologous recombination technology, and the specific operation process refers to the example 1. Recombinant strain K.oxyloca TRP-5 was obtained by knocking out the tryptophan enzyme tnaA from recombinant strain K.oxyloca TRP-4.

Recombinant strains K.oxyloca TRP-4 and K.oxyloca TRP-5 were shake-cultured for 60h in the same manner as in example 2, and the fermentation broth was subjected to metabolite analysis by High Performance Liquid Chromatography (HPLC).

TABLE 2 blocking the effect of the L-tryptophan degradation pathway on L-tryptophan production

The fermentation results are shown in Table 2, and the results show that the L-tryptophan accumulation amount of the recombinant strain K.oxyloca TRP-5 is increased to 1.3g/L after the tryptophan enzyme tnaA is knocked out.

Experimental example 3 introduction of endogenous strong promoter to enhance expression of tryptophan operon Gene

This experimental example attempted to introduce an endogenous strong promoter P _budABC (SEQ ID NO. 6) in place of thrL (SEQ ID NO. 7) to enhance transcription of the L-tryptophan synthesis gene cluster.

Wherein the gene integration adopted in the invention adopts a two-step homologous recombination technology, and the specific operation process refers to the example 1. On the basis of recombinant strain K.oxyloca TRP-5, introducing a strong promoter P _budABC to the attenuator thrL site to obtain recombinant strain K.oxyloca TRP-6.

Recombinant strains K.oxyloca TRP-5 and K.oxyloca TRP-6 were shake-cultured for 60h in the same manner as in example 2, and the fermentation broth was subjected to metabolite analysis by High Performance Liquid Chromatography (HPLC).

TABLE 3 introduction of endogenous strong promoters to enhance Tryptophan operon Gene expression

The fermentation results are shown in Table 3, and the results show that the accumulation amount of L-tryptophan of the recombinant strain K.oxyoca TRP-6 is obviously improved to 5.2g/L after the thrL is replaced by the strong promoter P _budABC.

Experimental example 4 screening different Rate-limiting genes to increase L-tryptophan production

In order to further enhance the L-tryptophan production, the present experimental example screened the rate-limiting gene of the L-tryptophan synthesis pathway to ensure a strong expression level of the gene involved in L-tryptophan synthesis. Specifically, first, the transcription repressor TRP (SEQ ID NO. 8) of the L-tryptophan operon was inactivated on the basis of the recombinant strain K.oxyloca TRP-6 to obtain the recombinant strain K.oxyloca TRP-7. Then, on the basis of the recombinant strain K.oxyoca TRP-7, the expression of shikimic acid synthesis key gene and L-tryptophan synthesis key gene is enhanced to increase the L-tryptophan accumulation level. Specifically, the expression of the 3-dehydroquinic acid synthase encoding gene aroB (SEQ ID No. 9), shikimate dehydrogenase encoding gene aroE (SEQ ID No. 11), chorismate synthase encoding gene aroC (SEQ ID No. 13), tryptophan operon part gene cluster trpE ^fbr D (SEQ ID No. 15), tryptophan operon part gene cluster trpDC (SEQ ID No. 17), tryptophan operon part gene cluster trpBA (SEQ ID No. 19) and transketolase encoding gene tktA (SEQ ID No. 21) from e.coli W3110 was enhanced.

Wherein the gene integration and gene knockout adopted in the experimental example adopts a two-step homologous recombination technology, and the specific operation process refers to the above-mentioned example 1. The above genes were introduced into the recombinant strain K.oxyloca TRP-7 at the site of D-lactate dehydrogenase gene ldhD (SEQ ID NO. 10), and in addition, the present experimental example further integrates all of the selected rate-limiting genes into different gene sites of K.oxyloca TRP-7, the gene sites being respectively adhE(SEQ ID NO.12)、frdA(SEQ ID NO.14)、pflB(SEQ ID NO.16)、pox(SEQ ID NO.18)、pta(SEQ ID NO.20)、pykF(SEQ ID NO.22).

The obtained series of recombinant K.oxyroca were shake-flask cultured for 60 hours in the same manner as in example 2, and the fermentation broth was subjected to metabolite analysis by High Performance Liquid Chromatography (HPLC).

TABLE 4 Effect of expression of different rate-limiting genes on improving L-tryptophan production

As a result, as shown in Table 4, the recombinant strains obtained by enhancing different rate-limiting genes showed different levels of improvement in L-tryptophan production, and the recombinant strain (TRP-11) having enhanced trpE ^fbr(S40F) D rate-limiting gene had the best improvement effect. Further, all the screened speed-limiting genes are integrated on different gene loci of the K.oxytoca TRP-7, so that the strain K.oxytoca TRP-22 can accumulate 10.1g/L of L-tryptophan.

Experimental example 5 enhancement of L-serine Synthesis to increase L-tryptophan production

In the L-tryptophan synthesis pathway, L-serine participates in a tryptophan synthase catalyzed reaction, which is the last step in L-tryptophan synthesis. The supply of L-serine is thus advantageous for L-tryptophan production. However, L-serine synthesis is mainly limited by the strict feedback inhibition of 3-phosphoglycerate dehydrogenase by L-serine.

Therefore, the invention adopts a two-step homologous recombination technology to insert a 3-phosphoglycerate dehydrogenase mutant protein coding gene serA ^{fbr(H344A,N346A,N364A)} (SEQ ID NO. 23) from E.coli W3110 into a site of a 2, 3-butanediol dehydrogenase gene budC (SEQ ID NO. 24) on the basis of a recombinant strain K.oxytoca TRP-22, so as to obtain the recombinant strain K.oxytoca TRP-23.

The recombinant K.oxyloca obtained was shake-flask cultured for 60h according to the culture method of example 2, and the fermentation broth was subjected to metabolite analysis by High Performance Liquid Chromatography (HPLC).

TABLE 5 influence of overexpression of feedback-insensitive 3-phosphoglycerate dehydrogenase on the increase of L-tryptophan production

As a result, as shown in Table 5, the L-tryptophan accumulation level was increased by overexpressing the feedback-insensitive 3-phosphoglycerate dehydrogenase, and the obtained strain K.oxylocaTRP-23 was able to accumulate 12.2g/L of L-tryptophan.

Experimental example 6 enhancement of glutamine supply to enhance L-tryptophan production

In the L-tryptophan synthesis pathway, chorismate is a key intermediate metabolite thereof, as well as a precursor for L-phenylalanine and L-tyrosine. However, chorismate enters the L-tryptophan synthesis mainly by aminobenzoic acid synthase, which requires glutamine for its participation. Thus the supply of glutamine facilitates L-tryptophan production. The invention adopts a two-step homologous recombination technology, and based on recombinant strain K.oxytoca TRP-23, a glutamine synthase coding gene glnA (SEQ ID NO. 25) from corynebacterium glutamicum C.glutamicum ATCC13032 is inserted into a locus of 1, 3-propanediol dehydrogenase gene dhaT (SEQ ID NO. 26) to obtain recombinant strain K.oxytoca TRP-24.

TABLE 6 influence of enhanced glutamine supply on enhanced L-tryptophan production

As shown in Table 6, the L-tryptophan accumulation level was increased by introducing the heterologous glutamine synthase encoding gene glnA, and the obtained strain K.oxylocaTRP-24 was able to accumulate 13.0g/L of L-tryptophan.

Experimental example 7 enhancement of L-tryptophan efflux pathway and enhancement of L-tryptophan production

The activity and expression of key enzymes in the L-tryptophan synthesis pathway are subject to severe feedback inhibition by L-tryptophan. In order to alleviate this negative feedback effect, in addition to the known introduction of negative feedback insensitive mutation sites in key enzymes, reducing intracellular L-tryptophan concentration is also an important means. In addition, the intracellular L-tryptophan with too high concentration can inhibit the growth of the strain, and the intracellular L-tryptophan concentration can be effectively reduced by improving the excretion of the L-tryptophan. Thus, the present invention employs a two-step homologous recombination technique, based on recombinant strain K.oxytoca TRP-24, an aromatic amino acid efflux protein gene ywkB (SEQ ID NO. 27) from Bacillus subtilis (Bacillus subtilis) 168 and an L-tryptophan efflux protein gene yddG from Escherichia coli W3110 are inserted into the sites of glycerol dehydrogenase gene gldA (SEQ ID NO. 28), respectively, to obtain recombinant strains K.oxytoca TRP-25 and TRP-26.

TABLE 7 enhancement of the effect of L-tryptophan efflux on L-tryptophan production

As a result, as shown in Table 7, the L-tryptophan accumulation level of the recombinant strain was increased by introducing the heterologous L-tryptophan efflux protein. In particular, the strain K.oxylocaTRP-25 obtained by introducing the aromatic amino acid efflux protein gene ywkB can accumulate more L-tryptophan, and the shake flask fermentation yield reaches 14.0g/L.

Experimental example 8 enhancement of expression of phosphoenolpyruvate carboxykinase to increase L-tryptophan production

Phosphoenolpyruvate carboxykinase can catalyze the conversion of oxaloacetate to phosphoenolpyruvate, the latter a precursor for L-tryptophan synthesis. Thus, enhancing the expression of phosphoenolpyruvate carboxykinase may be advantageous for L-tryptophan production. The invention adopts a two-step homologous recombination technology, and based on a recombinant strain K.oxytoca TRP-26, a phosphoenolpyruvate carboxykinase gene pck (SEQ ID NO. 29) from bacillus subtilis (Bacillus subtilis) 168 is inserted into a site of an acetate kinase gene ackA (SEQ ID NO. 30) to obtain the recombinant strain K.oxytoca TRP-27.

TABLE 8 Effect of enhancing expression of phosphoenolpyruvate carboxykinase on enhanced L-tryptophan production

As a result, as shown in Table 8, the strain K.oxylocaTRP-27 obtained by enhancing the expression of phosphoenolpyruvate carboxykinase was able to accumulate 14.6g/L of L-tryptophan by shake flask fermentation.

The experimental examples show that the metabolic flux can be changed from 2, 3-butanediol to L-tryptophan by introducing different sources of L-tryptophan synthesis key genes in combination with the metabolic pathway of Klebsiella oxytoca. Further combines the optimization of the L-tryptophan synthesis pathway, the blocking of the L-tryptophan degradation pathway, the enhancement of the precursor substance supply and the screening of the transport protein, and greatly promotes the stabilization of the L-tryptophan metabolic flow and the accumulation of products. Thus, a genetically engineered strain for efficiently producing L-tryptophan is obtained.

As can be seen from the above examples and experimental examples, the present invention provides a recombinant microorganism for high L-tryptophan production, and a construction method and application thereof. The recombinant acid-producing klebsiella (Klebsiella oxytoca) TRP-27 with high L-tryptophan yield is optimized by screening the knocked-out or added genes, the genotype of the recombinant acid-producing klebsiella is K.oxytoca CICC21518ΔbudA::ppsAΔbudB::aroG^fbr(D146N)ΔtnaAΔtrpL::P_budABCΔtrpRΔldhD::aroBΔadhE::aroEΔfrdA::aroCΔpflB::trpE^fbr(S40F)DΔpox::trpDCΔpta::trpBAΔpykF::tktAΔbudC::serA^{fbr(H344A,N346A,N364A)}ΔdhaT::glnAΔgldA::ywkBΔackA::pck., the recombinant acid-producing klebsiella can be used for producing L-tryptophan by fermentation, and experiments prove that the engineering strain can efficiently produce L-tryptophan by metabolizing glucose, the yield reaches 60.0g/L, and the yield reaches 0.23g/g. The invention provides a method for realizing efficient production of L-tryptophan by microorganisms by redirecting 2, 3-butanediol anabolism flux to L-tryptophan synthesis through pyruvic acid, and the L-tryptophan production process is simple and convenient, has low cost and has important economic benefit and social significance.

Claims

1. A recombinant microorganism for producing L-tryptophan is characterized in that an original strain of the recombinant microorganism is selected from Klebsiella strains, and the recombinant microorganism overexpresses genes comprising phosphoenolpyruvate synthase gene ppsA and tryptophan operon partial gene cluster trpE ^fbr D.

2. The recombinant microorganism producing L-tryptophan according to claim 1, wherein the nucleotide sequence of the tryptophan operon-partial gene cluster trpE ^fbr D is shown in SEQ ID NO. 15.

3. The recombinant microorganism producing L-tryptophan according to claim 1, wherein the starting strain is at least one selected from the group consisting of Klebsiella oxytoca, klebsiella terrestris, klebsiella planticola and Klebsiella pneumoniae.

4. A recombinant microorganism producing L-tryptophan according to any one of claims 1 to 3, wherein the recombinant microorganism is constructed by inactivating by-products selected from at least one of acetic acid, formic acid, succinic acid and lactic acid and/or enzyme genes in 2, 3-butanediol synthesis pathway-enhancing genes on the basis of an original strain, and wherein the L-tryptophan synthesis flux-related genes are selected from at least one of L-tryptophan synthesis-related enzyme genes, metabolic pathway-related genes, efflux protein genes and endogenous strong promoter genes.

5. The recombinant microorganism producing L-tryptophan according to claim 4, wherein the byproduct and/or the enzyme gene in the 2, 3-butanediol synthesis pathway is at least one to sixteen selected from the group consisting of a pyruvate kinase encoding gene pykF, a tryptophan enzyme encoding gene tnaA, a tryptophan operon transcription repressor trpR, a tryptophan operon attenuator trpL, a pyruvate oxidase gene pox, a phosphotransacetylase gene pta, an acetate kinase ackA, a fumarate reductase subunit A gene frdA, a lactate dehydrogenase gene ldhD, a pyruvate formate lyase gene pflB, an alcohol dehydrogenase gene adhE, an alpha-acetolactate synthase gene budB, an alpha-acetolactate decarboxylase gene budA, a2, 3-butanediol dehydrogenase gene budC, a glycerol dehydrogenase gene gldA, and a1, 3-propanediol dehydrogenase gene dhaT.

6. The recombinant microorganism producing L-tryptophan according to claim 4, wherein the L-tryptophan synthesis flux-related gene is at least one to thirteen selected from the group consisting of promoter P _budABC of 2, 3-butanediol synthesis gene cluster, 3-deoxy-d-arabinoheptulose 7-phosphate synthase mutein-encoding gene aroG ^fbr, 3-phosphoglycerate dehydrogenase mutein-encoding gene serA ^fbr, 3-dehydroquinic acid synthase-encoding gene aroB, shikimate dehydrogenase-encoding gene aroE, chorismate synthase-encoding gene aroC, tryptophan operon part gene cluster trpDC, tryptophan operon part gene cluster trpBA, transketolase-encoding gene tktA, aromatic amino acid efflux protein-encoding gene ywkB, phosphoenolpyruvate carboxykinase gene pck, glutamine synthase-encoding gene glnA, and L-tryptophan efflux protein gene yddG.

7. The method for constructing a recombinant microorganism producing L-tryptophan according to any one of claims 1 to 6, wherein the method comprises the steps of inactivating by-products and/or enzyme genes in a2, 3-butanediol synthesis pathway based on an original strain, constructing genes related to L-tryptophan synthesis flux enhancement, wherein the by-products are at least one selected from acetic acid, formic acid, succinic acid and lactic acid, and the genes related to L-tryptophan synthesis flux are at least one selected from L-tryptophan synthesis related enzyme genes, metabolic pathway related genes, efflux protein genes and endogenous strong promoter genes.

8. Use of the recombinant microorganism of any one of claims 1-6 for producing L-tryptophan for the production of L-tryptophan.

9. The method according to claim 8, wherein the recombinant microorganism overexpressing the gene cluster trpE ^fbr D comprising the phosphoenolpyruvate synthase gene ppsA and the tryptophan operon is cultured using a fermentation medium containing glucose.

10. The method according to claim 9, wherein the concentration of glucose in the fermentation medium is 80-100g/L;