CN115261293A

CN115261293A - Genetic engineering bacterium for producing hydroxyadipic acid

Info

Publication number: CN115261293A
Application number: CN202110475909.2A
Authority: CN
Inventors: 谭天伟; 刘猛; 张洋; 王萌
Original assignee: Beijing University of Chemical Technology
Current assignee: Beijing University of Chemical Technology
Priority date: 2021-04-29
Filing date: 2021-04-29
Publication date: 2022-11-01
Anticipated expiration: 2041-04-29
Also published as: CN115261293B

Abstract

The invention relates to a genetic engineering bacterium for producing hydroxy adipic acid. The genetic engineering bacterium is obtained by adopting escherichia coli as a host cell, introducing genes for encoding lysine dehydrogenase, aminoadipate semialdehyde dehydrogenase, aminoadipate transaminase and ketoadipate reductase, and carrying out chassis microbial transformation on the escherichia coli by strengthening a precursor synthesis path and knocking out or weakening genes related to a competitive metabolic path, and is the genetic engineering bacterium for high-yield hydroxyl adipic acid.

Description

Genetic engineering bacterium for producing hydroxyadipic acid

Technical Field

The invention belongs to the technical field of biology, and relates to a genetic engineering bacterium for producing hydroxyadipic acid, in particular to a genetic engineering bacterium for producing hydroxyadipic acid and application thereof in producing hydroxyadipic acid.

Background

The molecular formula of 2-hydroxyadipic acid (2-hydroxyadipic acid) is C₆H₁₀O₅The formula weight is 162.14. It is an adipic acid derivative with 2-hydroxy substitution, which can be used as adipic acid (precursor of nylon 6, 6)Synthetic intermediates. Due to the need of technological development in recent years, hydroxyadipic acid has attracted much attention as a small molecule compound that plays an important role in scientific research fields such as medicine, chemical synthesis, and biosynthesis.

Currently, hydroxyadipic acid is produced mainly by chemical synthesis. However, the traditional chemical synthesis method has the defects of large pollution, high cost, low yield and the like. With the rapid development of synthetic biology, the biological synthesis of hydroxyadipic acid becomes the most potential strategy for replacing chemical synthesis.

Therefore, research and development of a biosynthesis technology of hydroxyadipic acid, which has high conversion rate and good economical efficiency and is easy for industrial production, are needed.

Disclosure of Invention

The invention aims to provide a genetic engineering bacterium for producing hydroxyadipic acid, which is a genetic engineering bacterium for producing the hydroxyadipic acid with high yield, has high conversion rate and good economy and is easy for industrial production.

Therefore, the invention provides a genetic engineering bacterium for producing hydroxy adipic acid.

According to an embodiment of the first aspect of the present invention, the genetically engineered bacterium producing hydroxyadipic acid is a recombinant host bacterium comprising a gene encoding lysine dehydrogenase, a gene encoding aminoadipate semialdehyde dehydrogenase, a gene encoding aminoadipate aminotransferase and a gene encoding ketoadipate reductase.

In some embodiments of the invention, the gene encoding lysine dehydrogenase is selected from the group consisting of a lysine dehydrogenase-encoding gene lysDH-Am derived from bacillus 12AMOR1 or a codon-optimized lysine dehydrogenase-encoding gene lysDH-Am derived from bacillus 12AMOR1, a lysine dehydrogenase-encoding gene lysDH-Bt derived from bacillus thermophilus 1A1 or a codon-optimized lysine dehydrogenase-encoding gene lysDH-Bt derived from bacillus thermophilus 1A1, a lysine dehydrogenase-encoding gene lysDH-Ab derived from acidic bacterium adurb.

In further embodiments of the invention, the gene encoding an aminoadipate semialdehyde dehydrogenase is selected from the group consisting of the gene Psefu _1272 encoding an aminoadipate semialdehyde dehydrogenase derived from pseudomonas 12-X or the gene Psefu _1272 encoding an aminoadipate semialdehyde dehydrogenase derived from pseudomonas 12-X and codon-optimized, the gene ALDH7A1 encoding an aminoadipate semialdehyde dehydrogenase derived from human or the gene ALDH7A1 encoding an aminoadipate semialdehyde dehydrogenase derived from human and codon-optimized, the gene PMI27 encoding an aminoadipate semialdehyde dehydrogenase derived from pseudomonas GM41 (2012) or the gene PMI27 encoding an aminoadipate semialdehyde dehydrogenase derived from pseudomonas GM41 (2012) and codon-optimized, preferably the gene Psefu _1272 encoding an aminoadipate semialdehyde dehydrogenase derived from pseudomonas 12-X and codon-optimized.

In further embodiments of the invention, the gene encoding an aminoadipate transaminase is selected from the group consisting of the gene ARO8 encoding an aminoadipate transaminase derived from S288c of saccharomyces cerevisiae or the codon-optimized gene ARO8 encoding an aminoadipate transaminase derived from S288c of saccharomyces cerevisiae, the gene lysN encoding an aminoadipate transaminase derived from thermus thermophilus HB27 or the gene lysN encoding an aminoadipate transaminase derived from thermus thermophilus HB27 and codon-optimized, preferably the gene ARO8 encoding an aminoadipate transaminase derived from S288c of saccharomyces cerevisiae and codon-optimized.

In still further embodiments of the invention, the gene encoding ketoadipate reductase is selected from the group consisting of a ketoadipate reductase-encoding gene AeLDH derived from Chlorella eutrophic H16 or a codon-optimized ketoadipate reductase-encoding gene AeLDH derived from Chlorella eutrophic H16, a ketoadipate reductase-encoding gene HgdH derived from Micrococcus fermentans, or a codon-optimized ketoadipate reductase-encoding gene HgdH derived from Micrococcus fermentans, preferably a ketoadipate reductase-encoding gene AeLDH derived from Chlorella eutrophic H16.

According to an embodiment of the second aspect of the present invention, the genetically engineered bacterium is a genetically engineered bacterium which is modified by a chassis microorganism and produces hydroxyadipic acid; preferably, the chassis microbial engineering comprises the intensification of precursor synthetic pathways and the knock-out or attenuation of genes associated with competing metabolic pathways.

In the present invention, the reinforcement of the precursor synthesis pathway comprises overexpression of a key gene of the precursor synthesis pathway in the genetically engineered bacterium.

In some embodiments of the invention, the key genes of the precursor synthesis pathway include a gene aspC encoding aspartate aminotransferase, a gene lysC encoding aspartate kinase, a gene dapA encoding dihydrodipicolinate synthase, and a gene lysA encoding diaminopimelate decarboxylase.

In the present invention, the competitive metabolic pathway-related genes include lysine degradation pathway-related genes, lactate pathway-related genes, and acetate pathway-related genes.

In some embodiments of the invention, the lysine degradation pathway-related genes comprise the genes cadA and ldcC encoding lysine decarboxylase.

In other embodiments of the present invention, the lactate pathway-associated gene comprises a gene ldh encoding lactate dehydrogenase.

In other embodiments of the invention, the acetate pathway-related genes include a gene pta encoding phosphoacetyltransferase, a gene ackA encoding acetate kinase, and a gene poxB encoding pyruvate dehydrogenase.

According to the invention, the host bacteria comprise Escherichia coli, corynebacterium glutamicum, yeast, and modified bacteria and fungi.

In some preferred embodiments of the invention, the host bacterium is E.coli.

In some further preferred embodiments of the invention, the host bacterium is Escherichia coli JM109 (DE 3).

The invention also provides an application of the genetic engineering bacteria in the production of the hydroxy adipic acid.

According to the invention, the application comprises the steps of inoculating the genetic engineering bacteria for producing the hydroxy adipic acid into a fermentation culture medium, carrying out fermentation culture, and then separating and purifying the obtained fermentation culture solution to prepare the hydroxy adipic acid.

In some embodiments of the invention, the fermentation culture conditions are: the fermentation culture time is 72h, when the thallus grows to OD600= 0.6-0.8, adding IPTG, after adding IPTG, the fermentation temperature is changed from 37 ℃ to 30 ℃, and the induction concentration of IPTG is 0.8-1.2mM; further preferably, lysine is added in vitro, and the amount of lysine added is 2 to 10g/L, more preferably 5 to 10g/L.

In some embodiments of the present invention, the isolation and purification of the obtained fermentation broth comprises:

step S1, carrying out first centrifugal separation on a fermentation culture solution to obtain first supernatant;

step S2, diluting the supernatant I by 20 times with ultrapure water, uniformly mixing to obtain a solution A, diluting the solution A by 10 times with methanol containing 0.1% formic acid, uniformly mixing, and performing centrifugal separation for the second time to obtain a supernatant II;

and S3, filtering the second supernatant by using a 0.22-micron organic phase filter membrane to obtain the hydroxy adipic acid.

The invention adopts Escherichia coli as host cells, carries out chassis microbial transformation on the Escherichia coli by introducing genes for coding lysine dehydrogenase, amino adipic acid semialdehyde dehydrogenase, amino adipic acid transaminase and ketoadipic acid reductase and strengthening a precursor synthesis way and knocking out or weakening genes related to a competitive metabolic way, constructs and obtains the gene engineering bacteria of the hydroxy adipic acid, and the bacteria are the gene engineering bacteria for producing the hydroxy adipic acid with high yield.

Drawings

The invention is described in further detail below with reference to the attached drawing figures:

FIG. 1 shows the reaction mechanism for the biosynthesis of hydroxyadipic acid;

FIG. 2 is a graph of the production of hydroxyadipic acid by fermentation based on strain 109-H with the addition of 5g/L lysine;

FIG. 3 is a graph of the production of hydroxyadipic acid based on the fermentation of strain 109-H without addition of lysine.

The strain 109-H carries a pET-H plasmid, and the plasmid carries a codon-optimized lysine dehydrogenase encoding gene lysDH-Bt (shown in SEQ No. 4), a codon-optimized aminoadipate semialdehyde dehydrogenase encoding gene Psefu _1272 (shown in SEQ No. 10), a codon-optimized aminoadipate transaminase encoding gene ARO8 (shown in SEQ No. 16) and a codon-optimized ketoadipate reductase encoding gene AeLDH (shown in SEQ No. 20).

Detailed Description

In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to the appended drawings. However, before the present invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

Term of

The term "Chassis microorganism" also referred to as "Chassis microbial cell" as used herein means a functional biological system using a microbial cell as a platform, which is capable of providing a function required by a human being for biosynthesis. It is more likely that the vehicle has a chassis base on which various vehicle bodies can be manufactured and various functional components can be mounted. Therefore, the underpan microbial cells need to be simplified in functions, but have the most basic self-replication and metabolism capabilities, so that the underpan microbial cells become a blank platform capable of continuously adding functions.

The term "genetically engineered bacterium" as used herein refers to a bacterium or fungus, such as Escherichia coli, which is transformed by introducing a desired gene into a host organism (i.e., a host cell or a basal disc microorganism or a bacterial body) to express the gene, or by modifying the bacterial body to produce a desired protein by a basal disc microorganism including the knock-out or attenuation of genes involved in the enhanced and competitive metabolic pathways of the precursor synthesis pathway. The core technology of genetic engineering is the recombination technology of DNA, therefore, the genetic engineering bacteria are also called recombinant microorganisms in the invention.

The term "recombinant" as used herein refers to the construction of a transgenic organism that utilizes the genetic material of a donor organism or an artificially synthesized gene, which is cleaved with restriction enzymes in vitro or ex vivo and then ligated with a suitable vector to form a recombinant DNA molecule, which is then introduced into a recipient cell or a recipient organism to construct a transgenic organism that exhibits a certain property of another organism according to a human blueprint that has been previously designed.

Embodiments II

In order to achieve the above-mentioned objective of biosynthesis of hydroxyadipic acid, the present inventors have conducted extensive studies on a process technique for synthesizing hydroxyadipic acid by a biological method. The inventor finds that escherichia coli is adopted as a host cell, the genes for coding lysine dehydrogenase, aminoadipic semialdehyde dehydrogenase, aminoadipic transaminase and ketoadipic reductase are introduced, a precursor synthesis path is enhanced, and genes related to a competitive metabolic path are knocked out or weakened to carry out chassis microbial transformation on the escherichia coli, so that the genetically engineered bacterium for producing high-yield hydroxyadipic acid is successfully constructed and obtained, and the genetically engineered bacterium for producing the hydroxyadipic acid is high in conversion rate, good in economy and easy for industrial production. The present invention was thus obtained.

Therefore, the invention provides a new way for synthesizing the hydroxyadipic acid, which realizes the high-efficiency synthesis of the hydroxyadipic acid by using the lysine as the precursor through a genetic engineering bacterium for high-yield production of the hydroxyadipic acid.

As used herein, "hydroxyadipic acid" refers to an adipic acid derivative having 2-hydroxy substitution, i.e., 2-hydroxyadipic acid.

In order to realize the technical scheme, the invention provides a host strain capable of producing the hydroxyadipic acid, which expresses genes in a hydroxyadipic acid synthesis path in original or modified bacterial and fungal cells to prepare a host capable of synthesizing the hydroxyadipic acid.

In an embodiment of the first aspect of the present invention, the present invention provides a hydroxyadipic acid-producing genetically engineered bacterium, which is a recombinant host bacterium expressing a gene in a hydroxyadipic acid synthesis pathway.

The reaction mechanism for biosynthesis of hydroxyadipic acid in the present invention is shown in FIG. 1, and it can be understood from FIG. 1 that the genes in the synthesis pathway of hydroxyadipic acid include a gene encoding lysine dehydrogenase, a gene encoding aminoadipate semialdehyde dehydrogenase, a gene encoding aminoadipate transaminase, and a gene encoding ketoadipate reductase.

Based on the above, it can be easily understood that the genetically engineered bacterium producing hydroxyadipic acid referred to in the present invention is a recombinant host bacterium comprising genes encoding lysine dehydrogenase, aminoadipate semialdehyde dehydrogenase, aminoadipate transaminase, and ketoadipate reductase.

In some embodiments of the invention, the nucleotide sequence of lysDH-Am (GenBank: AKM 17750.1) of the gene encoding lysine dehydrogenase derived from Bacillus 12AMOR1 is shown in SEQ No. 1.

In other embodiments of the invention, the codon-optimized lysDH-Am gene encoding a lysine dehydrogenase derived from Bacillus 12AMOR1 has the nucleotide sequence shown in SEQ No. 2.

In some embodiments of the invention, the nucleotide sequence of the gene lysDH-Bt (GenBank: CEE 01557.1) encoding lysine dehydrogenase derived from Bacillus thermophilus 1A1 is shown in SEQ No. 3.

In other embodiments of the invention, the nucleotide sequence of the codon-optimized gene lysDH-Bt encoding lysine dehydrogenase derived from Bacillus thermophilus 1A1 is shown in SEQ No. 4.

In some embodiments of the invention, the nucleotide sequence of the gene lysDH-Ab (GenBank: OQC 42178.1) encoding lysine dehydrogenase derived from acid bacterium ADurb. Bin051 is shown in SEQ No. 5.

In other embodiments of the invention, the codon-optimized lysDH-Ab gene from acid bacterium ADurb. Bin051 has the nucleotide sequence shown in SEQ No. 6.

In some embodiments of the invention, the nucleotide sequence of the gene lysDH-Bb (GenBank: OQB 61741.1) encoding lysine dehydrogenase derived from Bacteroides bacterium ADurb. Bin 141 is shown in SEQ No. 7.

In other embodiments of the invention, the codon-optimized nucleotide sequence of lysDH-Bb gene encoding lysine dehydrogenase derived from Bacteroides bacterium ADurb. Bin 141 is shown in SEQ No. 8.

In some embodiments of the invention, the nucleotide sequence of the gene Psefu _1272 (GenBank: AEF 21248.1) encoding aminoadipate semialdehyde dehydrogenase derived from Pseudomonas 12-X is shown in SEQ No. 9.

In other embodiments of the invention, the codon-optimized gene Psefu _1272 encoding aminoadipate semialdehyde dehydrogenase derived from Pseudomonas 12-X has the nucleotide sequence shown in SEQ No. 10.

In some embodiments of the invention, the nucleotide sequence of the human-derived gene ALDH7A1 (GenBank: NM-001182.5) encoding aminoadipate semialdehyde dehydrogenase is shown as SEQ No. 11.

In some specific embodiments of the invention, the nucleotide sequence of the human-derived and codon-optimized gene ALDH7A1 encoding aminoadipate semialdehyde dehydrogenase is shown in SEQ No. 12.

In some embodiments of the invention, the nucleotide sequence of the gene PMI27 (GenBank: EUB 77087.1) encoding aminoadipate semialdehyde dehydrogenase derived from Pseudomonas GM41 (2012) is shown as SEQ No. 13.

In some specific embodiments of the invention, the codon-optimized nucleotide sequence of the gene PMI27 derived from pseudomonas GM41 (2012) and encoding aminoadipate semialdehyde dehydrogenase is as shown in SEQ No. 14.

In further embodiments of the invention, the gene encoding an aminoadipate transaminase is selected from the group consisting of the gene ARO8 encoding an aminoadipate transaminase derived from saccharomyces cerevisiae S288c or the codon-optimized gene ARO8 encoding an aminoadipate transaminase derived from saccharomyces cerevisiae S288c, the gene lysN encoding an aminoadipate transaminase derived from thermus thermophilus HB27 or the gene lysN encoding an aminoadipate transaminase derived from thermus thermophilus HB27 and codon-optimized, preferably the gene ARO8 encoding an aminoadipate transaminase derived from saccharomyces cerevisiae S288 c.

In some embodiments of the invention, the nucleotide sequence of the gene ARO8 (GenBank: CAA 73946.1) encoding aminoadipate transaminase derived from Saccharomyces cerevisiae S288c is shown as SEQ No. 15.

In other embodiments of the invention, the codon-optimized nucleotide sequence of the gene ARO8 encoding aminoadipate transaminase derived from saccharomyces cerevisiae S288c is shown in SEQ No. 16.

In some embodiments of the invention, the nucleotide sequence of the gene lysN (GenBank: AAS 80391.1) encoding aminoadipate aminotransferase, derived from Thermus thermophilus HB27, is shown in SEQ No. 17.

In other embodiments of the invention, the codon-optimized nucleotide sequence of lysN, a gene encoding aminoadipate aminotransferase, which is derived from Thermus thermophilus HB27, is shown in SEQ No. 18.

In some embodiments of the invention, the nucleotide sequence of the gene AeLDH (GenBank: QCB 99744.1) encoding ketoadipate reductase derived from Chlorella eutrophic H16 is shown in SEQ No. 19.

In other embodiments of the invention, the codon-optimized nucleotide sequence of the gene AeLDH encoding ketoadipate reductase derived from Chlorella eutrophic H16 is shown in SEQ No. 20.

In some embodiments of the invention, the nucleotide sequence of the gene HgdH (GenBank: ADB 47349.1) encoding ketoadipate reductase from Zymococcus amino acid is shown in SEQ No. 21.

In other embodiments of the invention, the codon-optimized nucleotide sequence of the gene HgdH encoding ketoadipate reductase, derived from s.fermentum, is shown in SEQ No. 22.

In some embodiments of the invention, the synthesis of hydroxy-adipate as precursor is achieved by efficiently expressing genes of enzymes involved in the hydroxy-adipate synthesis pathway in a host bacterium (e.g., original or modified bacteria, fungi), preferably a gene lysDH-Bt (GenBank: CEE 01557.1) encoding lysine dehydrogenase derived from Bacillus thermophilus 1A1 (Bacillus thermoaminogenes 1A 1), a gene Psefu _1272 (GenBank: AEF 21248.1) encoding aminoadipate semialdehyde dehydrogenase derived from Pseudomonas 12-X (Pseudomonas fulva 12-X), a gene ARO8 (GenBank: CAA 73946.1) encoding aminoadipate transaminase derived from Saccharomyces cerevisiae S288c (Saccharomyces cerevisiae S288 c), and a gene AecataLDH (GenBank: QC9999B.744) encoding ketoadipate reductase derived from Chlorella vulgaris H16 (Cupriavidiner H16), in a host bacterium, preferably lysine.

In some particularly preferred embodiments of the present invention, the lysine-precursor hydroxyadipic acid synthesis is achieved by efficiently expressing a gene of a relevant enzyme in the hydroxyadipic acid synthesis pathway in a host bacterium (e.g., original or modified bacterium, fungus), preferably lysDH-Bt (nucleotide sequence shown in SEQ No. 4) which is derived from Bacillus thermophilus 1A1 and is codon-optimized for a lysine dehydrogenase gene, psefu _1272 (nucleotide sequence shown in SEQ No. 10) which is derived from Pseudomonas 12-X and is codon-optimized for an aminoadipate semialdehyde dehydrogenase gene, ARO8 (nucleotide sequence shown in SEQ LDH No. 16) which is derived from Saccharomyces cerevisiae S288c and is codon-optimized for an aminoadipate transaminase gene, and Aeketoreductase (nucleotide sequence shown in SEQ No. 20) which is derived from Chlorella eutrophic H16.

According to the invention, the host bacteria comprise escherichia coli, corynebacterium glutamicum, yeast, and modified bacteria and fungi; preferably, the host bacterium is escherichia coli; specifically, the host bacterium is Escherichia coli JM109 (DE 3).

In the invention, the type of the expression plasmid has no special requirement, and can be correspondingly adjusted according to the selection of a host, and the construction method for expressing the target gene in the escherichia coli can adopt various methods commonly used in the field, for example, the target gene and the expression vector are connected after enzyme digestion treatment, and the details are not repeated.

In some particularly preferred embodiments, the E.coli strain Trans10 is used for vector construction and E.coli JM109 (DE 3) is used as the fermentation strain.

The genetically engineered bacterium producing hydroxyadipic acid according to the embodiment of the first aspect of the present invention is Escherichia coli JM109 (DE 3) expressing a gene encoding lysine dehydrogenase, a gene encoding aminoadipate semialdehyde dehydrogenase, a gene encoding aminoadipate transaminase, and a gene encoding ketoadipate reductase.

In some specific preferred examples, the gene engineering bacteria are constructed by using codon-optimized genes lysDH-Bt, psefu _1272, ARO8 and AeLDH, the relevant primers for constructing the recombinant plasmids are shown in Table 1, and the corresponding sequences are shown in SEQ Nos. 23-30.

TABLE 1 primers related to the construction of recombinant plasmids

In an embodiment of the second aspect of the present invention, the genetically engineered bacterium is a genetically engineered bacterium that produces hydroxyadipic acid and is subjected to chassis microbial modification, wherein the chassis microbial modification comprises strengthening of a precursor synthesis pathway and knockout or attenuation of a gene associated with a competitive metabolic pathway.

In the invention, the reinforcement of the precursor synthesis pathway comprises the overexpression of key genes of the precursor synthesis pathway in genetic engineering bacteria, and the efficient synthesis of the hydroxyadipic acid from simple carbon sources such as glucose or xylose is realized by enhancing the synthesis of precursor lysine.

In some particularly preferred embodiments of the invention, the nucleotide sequence of the gene aspC (GenBank: QPA 14779.1) encoding aspartate aminotransferase is shown in SEQ ID No. 31.

In some particularly preferred embodiments of the invention, the nucleotide sequence of the gene lysC (GenBank: QPA 17672.1) encoding aspartate kinase is shown in SEQ No. 32.

In some particularly preferred embodiments of the present invention, the nucleotide sequence of the gene dapA (GenBank: QPA 16247.1) encoding dihydrodipicolinate synthase is set forth in SEQ No. 33.

In some particularly preferred embodiments of the invention, the nucleotide sequence of the gene lysA (GenBank: QPA 16577.1) encoding diaminopimelate decarboxylase is shown as SEQ No. 34.

In the invention, the chassis microorganism transformation is carried out by knocking out or weakening genes related to competitive metabolic pathways, so that the carbon metabolic flux can be regulated and controlled, more metabolic flux flows to the synthesis of the hydroxy adipic acid, more substrates flow to the synthesis of the hydroxy adipic acid, and the Escherichia coli genetic engineering bacteria with high yield of the hydroxy adipic acid is obtained.

In some embodiments of the invention, the competing metabolic pathway-related genes include lysine degradation pathway-related genes, lactate pathway-related genes, and acetate pathway-related genes; wherein the lysine degradation pathway-related genes include genes encoding lysine decarboxylase cadA and ldcC; the lactate pathway-associated gene is preferably a gene ldh encoding lactate dehydrogenase; the acetate pathway-related genes include a gene pta encoding phosphoacetyltransferase, a gene ackA encoding acetate kinase, and a gene poxB encoding pyruvate dehydrogenase.

In some embodiments of the invention, the nucleotide sequence of the gene cadA (GenBank: QPA 17778.1) encoding lysine decarboxylase is shown in SEQ No. 35.

In some embodiments of the invention, the nucleotide sequence of the gene ldcC (GenBank: QHB 66407.1) encoding lysine decarboxylase is shown in SEQ No. 36.

In some embodiments of the invention, the nucleotide sequence of the gene ldh (GenBank: QPA 15202.1) encoding lactate dehydrogenase is shown in SEQ No. 37.

In some embodiments of the invention, the nucleotide sequence of the gene pta encoding phosphotransacetylase (GenBank: QPA 16074.1) is shown in SEQ No. 38.

In some embodiments of the invention, the nucleotide sequence of the gene ackA (GenBank: QPA 16073.1) encoding acetate kinase is shown in SEQ No. 39.

In some embodiments of the invention, the nucleotide sequence of the gene poxB (GenBank: QPA 14724.1) encoding pyruvate dehydrogenase is shown in SEQ No. 40.

The invention utilizes the embodiments of the first and second aspects to regulate the carbon metabolic flow, thereby achieving the purpose of regulating the synthesis of the hydroxyadipic acid.

The invention adopts escherichia coli as a host cell, and successfully constructs and obtains the genetic engineering bacteria for high-yield hydroxyl adipic acid by introducing a gene for coding lysine dehydrogenase, a gene for coding amino adipic acid semialdehyde dehydrogenase, a gene for coding amino adipic acid transaminase, a gene for coding ketoadipic acid reductase and a reinforced precursor synthesis way, knocking out or weakening genes related to a competitive metabolic way to carry out chassis microbial transformation on the escherichia coli.

Actually, the enzyme activities of lysine dehydrogenase, aminoadipic semialdehyde dehydrogenase, aminoadipic transaminase and ketoadipic reductase coded in escherichia coli or genetic engineering bacteria producing hydroxyadipic acid are regulated and controlled by means of promoter engineering, RBS (RBS) regulation strategy or enzyme modification and the like, so that a novel genetic engineering bacteria capable of producing hydroxyadipic acid at high yield can be further constructed and obtained; for example, by promoter engineering, a highly efficient promoter was selected to enhance transcription of the gene lysDH-Bt encoding lysine dehydrogenase, the gene Psefu _1272 encoding aminoadipate semialdehyde dehydrogenase, the gene ARO8 encoding aminoadipate aminotransferase and the gene AeLDH encoding ketoadipate reductase, thereby enhancing the conversion of lysine into hydroxyadipate.

The present invention relates to the use of the genetically engineered bacteria as described above in the production of hydroxyadipic acid, which can be understood as a method for producing hydroxyadipic acid by using the genetically engineered bacteria as described above.

In some embodiments of the present invention, inoculating the genetically engineered bacterium producing hydroxyadipic acid into a fermentation medium for fermentation culture comprises: inoculating the genetic engineering bacteria producing the hydroxy adipic acid into a fermentation culture medium, performing fermentation culture at 200rpm, adding IPTG (isopropyl-beta-D-thiogalactoside) with the final concentration of 0.8-1.2mM, preferably 1-1.2mM and more preferably 1mM when OD600 is between 0.6-0.8, inducing, and after adding IPTG, changing the fermentation temperature from 37 ℃ to 30 ℃, and culturing for 72 hours to obtain a fermentation culture solution; lysine is added to the medium as needed, and the amount of lysine added is 2-10g/L, preferably 5-10g/L, and more preferably 5g/L.

In other embodiments of the present invention, the isolation and purification of the obtained fermentation broth comprises:

step S1, carrying out first centrifugal separation on a fermentation culture solution at the rotating speed of 12000rpm to obtain first supernatant;

step S2, diluting the supernatant fluid I by 20 times by using ultrapure water, uniformly mixing to obtain a solution A, diluting the solution A by 10 times by using methanol containing 0.1% (volume) formic acid, uniformly mixing, and performing centrifugal separation for the second time at the rotating speed of 15000rpm to obtain a supernatant fluid II;

The fermentation medium in the present invention is not particularly limited as long as it is a fermentation medium for producing hydroxyadipic acid, and preferably, the M9 fermentation medium formulation is: glucose 10g/L, yeast powder 3g/L, M9 salt (10X) 100mL,1M MgSO 4₄ 1mL，1M CaCl₂0.3mL,1g/L biotin 1mL,1g/L thiamine 1mL, M9 trace elements (100X) 10mL (1L system); wherein, the M9 salt (10 x) component is: na (Na)₂HPO₄ 67.8g/L，KH₂PO₄ 30g/L，NaCl 5g/L，NH₄Cl 10g/L; the trace elements of M9 (100 ×) were: EDTA 5g/L, feCl₂·6H₂O 0.83g/L,ZnCl₂84mg/L，CuCl₂·2H₂O 13mg/L，CoCl₂·2H₂O 10mg/L，H₃BO₃ 10mg/L MnCl₂·4H₂O 1.6mg/L。

III example

The present invention will be specifically described below with reference to specific examples. The experimental methods described below are, unless otherwise specified, all routine laboratory procedures. The experimental materials described below, unless otherwise specified, are commercially available.

Example 1:

the primers used in this example are shown in Table 1 above.

The shihuada gene was assigned to lysDH-Bt (GenBank: CEE01557.1, the codon optimized nucleotide sequence is shown as SEQ No. 4), the gene Psefu _1272 (GenBank: AEF21248.1, the codon optimized nucleotide sequence is shown as SEQ No. 10) which is derived from Pseudomonas 12-X (Pseudomonas fulva 12-X) and codes for aminoadipate semialdehyde dehydrogenase, the gene ARO8 (GenBank: CAA73946.1, the codon optimized nucleotide sequence is shown as SEQ No. 16) which is derived from Saccharomyces cerevisiae S288c and codes for aminoadipate transaminase, and the gene AeLDH (GenBank: QCB99744.1, the codon optimized nucleotide sequence is shown as SEQ No. 20) which is derived from Chlorella eutrophic (Cupriavidus necator H16) and codes for ketoadipate reductase are subjected to whole gene pUC synthesis to obtain all the plasmids of the genes Psefu _ 1278, the AefO gene and the codon optimized gene AefO 57-57 (the codon optimized genes of AefO _ 12757, aefO _1272, aefO _ 57 and AefO _ 57-7, wherein the genes are respectively. Amplification of the genes of interest, lysDH-Bt, psefu _1272, ARO8, and pUC57-AeLDH, was carried out using lysDH-Bt-F/lysDH-Bt-R, psefu _1272-F/Psefu _1272-R, ARO8-F/ARO8-R, and AeLDH-F/AeLDH-R as primers and pUC57-lysDH-Bt, pUC57-Psefu _1272, pUC57-ARO8, and pUC57-AeLDH as templates, respectively. The vector was then digested with the corresponding restriction enzymes, the digested fragments were recovered by gel cutting, and the 4 target genes were inserted into E.coli expression vector pETDuet-1 by Gibson ligation to obtain plasmid pET-H (see Table 2).

Example 2:

competent cells of Escherichia coli JM109 (DE 3) were prepared and dispensed in 100. Mu.L into 1.5mL centrifuge tubes for electrotransformation. 2-4. Mu.L of the constructed pET-H recombinant plasmid is added into a 1.5mL centrifuge tube containing 100. Mu.L of competent cells and mixed evenly. The plasmid is then electrotransferred into competent cells using an electrotransfer instrument. After the electrotransfer was completed, LB medium [ peptone 10g/L, yeast powder 5g/L, naCl 5g/L ] was added rapidly, and the mixture was transferred to a 1.5mL centrifuge tube and allowed to resuscitate at 37 ℃ for 1h. Then, the bacterial liquid is coated on a plate containing corresponding antibiotics, and cultured for 12-24h at 37 ℃. Strain 109-H (see Table 2) was prepared which produced hydroxyadipic acid.

TABLE 2 plasmids and strains

In Table 2, the T7 promoter is the original promoter of pETDuet-1 plasmid; the ColE1 replicon is a replicon of pETDuet-1 plasmid; the pETDuet-1 plasmid is commercially available.

Example 3:

(1) Shake flask culture of genetic engineering strain for producing hydroxy adipic acid

A single colony is picked from a plate of a strain 109-H (the strain carries pET-H plasmid) for producing hydroxyl adipic acid, the single colony is inoculated into 4mL of liquid LB with resistance, the liquid LB with resistance is cultured for 12H at 37 ℃, then the bacterial liquid is inoculated into 20mL of LB seed culture medium (10 g/L of peptone, 5g/L of yeast powder, 5g/L of NaCl, and sterilized for 25min at 116 ℃), the bacterial liquid is inoculated into 50mL of fermentation medium according to 10 percent of inoculum size, lysine with corresponding concentration is added into the culture medium when needed, when the bacterial grows to OD600= 0.6-0.8, IPTG with final concentration of 1mM is added for induction, after the IPTG is added, the fermentation temperature is changed from 37 ℃ to 30 ℃, the rotation speed is 200rpm, and the culture time is 72H.

(2) Biological quantity measurement

Adding appropriate amount of sterile distilled water into the fermentation liquid, diluting until OD600 is 0.2-0.8, placing 200 μ L diluted fermentation liquid into 96-well plate, and measuring absorbance at 600nm wavelength with microplate reader (Thermo).

(3) Sample processing and detection

The fermentation broth was centrifuged at 12000rpm for 10min at 4 ℃. mu.L of the supernatant was diluted 20 times with 950. Mu.L of ultrapure water to obtain a solution A, and 100. Mu.L of the 20-fold diluted solution (i.e., solution A) was diluted 10 times with 900. Mu.L of methanol containing 0.1 vol% of formic acid to obtain a 200-fold diluted solution. After mixing, the mixture was centrifuged at 15000rpm for 15min at 4 ℃. The supernatant was filtered through a 0.22 μm organic phase filter. Product identification was then performed by gas chromatography-mass spectrometry (7890B-5977A, agilent Technologies) and quantitative detection was performed by liquid chromatography-mass spectrometry (QTRAP 5500, AB SCIEX) or high performance liquid chromatography (UltMate 3000, thermo). The final yield of strain 109-H (which carries the pET-H plasmid) is shown in FIGS. 2 and 3, with 5g/L lysine added in vitro and de novo synthesis yields of 268mg/L and 13mg/L, respectively.

It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention, and any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Beijing university of chemical industry

<120> gene engineering bacterium for producing hydroxy adipic acid

<130> RB2100681-FF

<160> 40

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1161

<212> DNA

<213> (Gene lysDH-Am encoding lysine dehydrogenase)

<400> 1

atgaaagtgc tcgtgcttgg agcggggctg atgggaaaag aagcggcgcg cgatttagtg 60

caaagccaag atgttgaggc ggtgacgttg gcggatgtcg atttggccaa ggcggagcag 120

acggtgcggc agcttcattc cgaaaagctt gccgctgtgc gggtggatgc cggcgatccg 180

caacaactgg cagcggccat gcaagggcat gatgtcgtcg tcaatgcctt gttttaccgc 240

ttcaatgaaa cggtggcgaa aacagcgatc gaaacgggtg ttcattccgt tgatttaggc 300

ggccatatcg gccatattac cgatcgggtg cttgaaatgc acgaggaggc tcagaaagcg 360

ggggtgacga tcattccgga tcttggcgtc gcgccgggga tgatcaacat tttatccggc 420

tatggggcga gtcaactcga tgaggtggaa tccatcttgc tgtatgttgg cggcatcccc 480

gtccgccctg agccgccgct cgagtacaac catgtgtttt cgctcgaggg gctgcttgac 540

cattacaccg atccgtctct cattatccgc gacggccaaa agcaggaagt gccgtcgctt 600

tcggaagtcg agccgattta tttcgaccgg ttcgggccgc ttgaagcgtt tcacacctca 660

ggcgggacgt cgacgctctc gcgctcgttt ccgaacttga agcggctcga gtacaaaacg 720

atccgctacc gcggccatgc agaaaaattt aagctgctcg tcgatttgaa cttgacgcgc 780

cacgatgtgg aagtggaggt caatggatgc aaagtcaaac cgcgcgatgt gctgctttcc 840

gtcctgaagc cgctgcttga tttgaaaggg aaagatgatg tggtgttgct tcgggtcatc 900

gtcggcggta gaaaagatgg aaaagaaacg gtgctggaat acgaaaccgt cacgttcaat 960

gaccgcgaaa ataaggtgac ggcgatggcg cgtacgacgg cctacaccat ttccgctgtc 1020

gcccagctca tcggccgtgg ggtgatcaca aagcgcggcg tctatccgcc ggagcaagcc 1080

gtgccaggag aggtgtatat tgaggaaatg aaaaggcgcg gggtcgtgat tagcgagaaa 1140

caaacgattc gcccttgcta a 1161

<210> 2

<211> 1161

<212> DNA

<213> (codon-optimized lysine dehydrogenase-encoding gene lysDH-Am)

<400> 2

atgaaagttc tggttctggg tgctggtctg atgggtaaag aagctgctcg tgacctggtt 60

cagtctcagg acgttgaagc tgttaccctg gctgacgttg acctggctaa agctgaacag 120

accgttcgtc agctgcactc tgaaaaactg gctgctgttc gtgttgacgc tggtgacccg 180

cagcagctgg ctgctgctat gcagggtcac gacgttgttg ttaacgctct gttctaccgt 240

ttcaacgaaa ccgttgctaa aaccgctatc gaaaccggtg ttcactctgt tgacctgggt 300

ggtcacatcg gtcacatcac cgaccgtgtt ctggaaatgc acgaagaagc tcagaaagct 360

ggtgttacca tcatcccgga cctgggtgtt gctccgggta tgatcaacat cctgtctggt 420

tacggtgctt ctcagctgga cgaagttgaa tctatcctgc tgtacgttgg tggtatcccg 480

gttcgtccgg aaccgccgct ggaatacaac cacgttttct ctctggaagg tctgctggac 540

cactacaccg acccgtctct gatcatccgt gacggtcaga aacaggaagt tccgtctctg 600

tctgaagttg aaccgatcta cttcgaccgt ttcggtccgc tggaagcttt ccacacctct 660

ggtggtacct ctaccctgtc tcgttctttc ccgaacctga aacgtctgga atacaaaacc 720

atccgttacc gtggtcacgc tgaaaaattc aaactgctgg ttgacctgaa cctgacccgt 780

cacgacgttg aagttgaagt taacggttgc aaagttaaac cgcgtgacgt tctgctgtct 840

gttctgaaac cgctgctgga cctgaaaggt aaagacgacg ttgttctgct gcgtgttatc 900

gttggtggtc gtaaagacgg taaagaaacc gttctggaat acgaaaccgt taccttcaac 960

gaccgtgaaa acaaagttac cgctatggct cgtaccaccg cttacaccat ctctgctgtt 1020

gctcagctga tcggtcgtgg tgttatcacc aaacgtggtg tttacccgcc ggaacaggct 1080

gttccgggtg aagtttacat cgaagaaatg aaacgtcgtg gtgttgttat ctctgaaaaa 1140

cagaccatcc gtccgtgcta a 1161

<210> 3

<211> 1149

<212> DNA

<213> (Gene lysDH-Bt encoding lysine dehydrogenase)

<400> 3

atgaaaatcg gtgtgttagg ctctggactt atgggcaaag aagcagcccg ggacttggtg 60

gaaagttccg gtgtaacgac tgtcggattg gctgatcttg attttaaccg tgcaaaaaca 120

gtatgtgaac aaattcaatc tcaaaaattg actccctttc aagtcgatgc tcgtaatgaa 180

gaagaactgg cgaattttat gagtcaattt gattgtatta ttaatgcact attttactcg 240

tttaatgaaa tcgtcgcaaa aacagcaatt aaagtgggtg ttcattctgt agatttaggt 300

ggtcatatcg gtcatatgac ggataaagta cttgcactaa aaaatgatgc gaaaaacgcc 360

ggagttacca ttatccctga tttaggggta gctccgggta tgattaatat tttatcaggt 420

tatggataca gtaagcttga taaagcaaca gcggtaaaat tgtacgtggg cggaattccg 480

gtaagaccgg atccaccatt agaatacaat catgtgttct caatggaagg tttacttgat 540

cactacactg atccttcttt aattattcgt aatggaaaat taatggttgt accatcgtta 600

tctgaaatag aaccgattta ttttgaaaaa tttggtccac tggaagcctt tcatacatca 660

ggcggaactt caacattatc tatttcttat ccggaattag aaacattgga atataaaacg 720

attcgttatc caggtcatgc gaaaaagatg aaattacttg ttgatttaaa tcttacaaga 780

acagattatg aagttgaagt tgacggaaaa accattaatc caaggaaagt actattaaag 840

gtcctagatc cgattgttga attaaaagat aaagacgatg tcactttatt acgggtaatc 900

gtttcagggg aaaaagattt aaatccagct acttatacgt atgaaatggt gacatttaag 960

gattcgacta aaaatatcac cgcgatggcg agagcaacag cgaatacgat ttcagttgtt 1020

gcacaaatga ttgcaaatgg tattattaca aaaagaggcg tttatccacc tgaacaaatt 1080

gttcccggtg atatttatat tgaagaaatg aaaaagcgtg gtgtggttat atcggaaaat 1140

ttaagttga 1149

<210> 4

<211> 1149

<212> DNA

<213> (codon-optimized gene lysDH-Bt encoding lysine dehydrogenase)

<400> 4

atgaaaattg gcgtgctggg cagcggcctg atgggcaaag aagcggcgcg tgatttagtt 60

gaaagcagcg gtgttaccac cgtgggtctg gcggatttag attttaatcg cgcgaaaacc 120

gtgtgcgaac agattcagag ccagaaactg accccgtttc aggtggatgc gcgcaatgaa 180

gaagaactgg cgaattttat gagccagttt gattgcatta tcaacgcgct gttttacagc 240

ttcaatgaga tcgtggcgaa aaccgcgatt aaagtgggcg tgcatagcgt ggatctgggc 300

ggccatattg gccatatgac cgataaagtg ctggcgctga aaaatgatgc gaaaaatgcg 360

ggcgtgacca ttattccgga tctgggcgtt gcgccgggca tgattaatat tctgagcggc 420

tatggctata gcaaactgga taaagcgacc gcggtgaaac tgtatgtggg cggcattccg 480

gtgcgcccgg atcctccatt agaatataat catgtgttta gcatggaggg cctgctggat 540

cattataccg atccgagcct gattattcgc aatggcaaac tgatggtggt gccgagcctg 600

agcgaaattg aaccgattta ttttgagaag ttcggcccgc tggaagcgtt tcataccagc 660

ggcggtacca gcaccttaag cattagctat ccggaactgg aaaccctgga atataaaacc 720

attcgctatc cgggccatgc gaaaaaaatg aaactgctgg tggatctgaa tctgacccgc 780

accgattatg aagtggaagt ggatggcaaa accattaatc cgcgcaaagt gctgctgaaa 840

gtgctggatc cgattgtgga actgaaagat aaagatgacg tgaccctgct gcgcgtgatt 900

gtgagcggcg aaaaagatct gaatccggcg acctatacct atgaaatggt gacctttaaa 960

gacagcacca agaatattac cgcgatggcg cgcgcgaccg cgaatactat tagcgttgtt 1020

gcgcaaatga ttgcgaatgg cattattacc aaacgcggcg tgtatccgcc ggaacagatt 1080

gtgccaggtg atatttatat tgaagagatg aaaaagcgcg gcgtggtgat tagcgaaaat 1140

ctgagctaa 1149

<210> 5

<211> 1176

<212> DNA

<213> (Gene lysDH-Ab encoding lysine dehydrogenase)

<400> 5

atgagccgga agaccaggat cgccctgctc ggcgccggcc gcgtcggtgc ggcaatggcg 60

ctcgatctcg ccgccgaccc cgggctcgag gtcgtggtgg ccgaccgcga cgagcgggcg 120

ctcgccccgc tggcggcgcg cgggctcgcc gtgcggaagg ccgatctcgg cggccccggc 180

ctcgacgaaa tcctcgccgc ctgcgatctc gccgtcggcg cggtacccgg ctacctcggc 240

ttcgccaccg cccgccgggt catcgaagcc ggccgggggc tcgtcgacat ctccttcttc 300

ccggaagacg ccttcgcgct cgacgagctc gcgcgcgagc gcggggtcac ggcgctcgtc 360

gactgcggcg tcgcccccgg cctctcgaac ctcctgctcg gcgccgtggc ggcccagatg 420

gagcgcgtcg aacggttcac ctgctacgtc ggcggcctgc cggagcaccc gcagccgccc 480

tgggagtaca aggcaccgtt ctctccggtc gacgtgctcg aggagtacac ccggccggcc 540

cgctacgtgc gcggcggccg gatcgtcacc gagccggcgc tcgccggcct cgaatggcgc 600

gaattcccgg ggatcggctc cctcgaggcg tttctcaccg acggcctccg cagcctgctg 660

accacgatgc cgcagatccc ggagatgcgg gagctgacgc tccgctaccc gggacacgtg 720

gcgaaggtgc agctgctgcg cgacagcggc ttcctctctt ccgaggcgct ggaggtcagc 780

ggcacccggg tgcggccggt cgacctcgcg ttgcggctgc tcctgccgca ctgggagttc 840

gctcccggcg agcccgatct gaccgtcttc gaggtgatcg tcgagggagt ggaaaagggc 900

ggtcgcgtgc gccgggtctt cttcttcctc gaccgtttcg acgccgcgac cggtacgctc 960

tcgatggcgc gcaccacggg ttataccggc accgccatcg tccggctcgt cgcccgcggc 1020

ctcttccacc gcccggggat ccatcctccc gagcacctcg cgaccgcgcc cggctgcgtc 1080

gacttcgtcc tgggcgagct cgccgcccgc ggcatccgcc tggcggaaag gagggagtcc 1140

ctcccgcccg cggcgactca ctcctcgtcg agatag 1176

<210> 6

<211> 1176

<212> DNA

<213> (codon-optimized lysine dehydrogenase-encoding gene lysDH-Ab)

<400> 6

atgtctcgta aaacccgtat cgctctgctg ggtgctggtc gtgttggtgc tgctatggct 60

ctggacctgg ctgctgaccc gggtctggaa gttgttgttg ctgaccgtga cgaacgtgct 120

ctggctccgc tggctgctcg tggtctggct gttcgtaaag ctgacctggg tggtccgggt 180

ctggacgaaa tcctggctgc ttgcgacctg gctgttggtg ctgttccggg ttacctgggt 240

ttcgctaccg ctcgtcgtgt tatcgaagct ggtcgtggtc tggttgacat ctctttcttc 300

ccggaagacg ctttcgctct ggacgaactg gctcgtgaac gtggtgttac cgctctggtt 360

gactgcggtg ttgctccggg tctgtctaac ctgctgctgg gtgctgttgc tgctcagatg 420

gaacgtgttg aacgtttcac ctgctacgtt ggtggtctgc cggaacaccc gcagccgccg 480

tgggaataca aagctccgtt ctctccggtt gacgttctgg aagaatacac ccgtccggct 540

cgttacgttc gtggtggtcg tatcgttacc gaaccggctc tggctggtct ggaatggcgt 600

gaattcccgg gtatcggttc tctggaagct ttcctgaccg acggtctgcg ttctctgctg 660

accaccatgc cgcagatccc ggaaatgcgt gaactgaccc tgcgttaccc gggtcacgtt 720

gctaaagttc agctgctgcg tgactctggt ttcctgtctt ctgaagctct ggaagtttct 780

ggtacccgtg ttcgtccggt tgacctggct ctgcgtctgc tgctgccgca ctgggaattc 840

gctccgggtg aaccggacct gaccgttttc gaagttatcg ttgaaggtgt tgaaaaaggt 900

ggtcgtgttc gtcgtgtttt cttcttcctg gaccgtttcg acgctgctac cggtaccctg 960

tctatggctc gtaccaccgg ttacaccggt accgctatcg ttcgtctggt tgctcgtggt 1020

ctgttccacc gtccgggtat ccacccgccg gaacacctgg ctaccgctcc gggttgcgtt 1080

gacttcgttc tgggtgaact ggctgctcgt ggtatccgtc tggctgaacg tcgtgaatct 1140

ctgccgccgg ctgctaccca ctcttcttct cgttaa 1176

<210> 7

<211> 1131

<212> DNA

<213> (Gene lysDH-Bb encoding lysine dehydrogenase)

<400> 7

atgagtaaaa ttatagtatt aggctcaggt atggttggca gtgctatggc attggactta 60

gccacaaatc atcaggttac tgttgccgac attaacactg cttccttaga aaaactaaag 120

gcaaagagca acgcaataaa tgtgttgact gctgatgtac gcaactcagc tgaattaata 180

aagatgcttg agccattcga cctggtggta tgtgcagtgc cgggatttct cggatatcaa 240

accttaaaaa acattattga agcaggaaaa aataccgttg atatttcatt cttccctgaa 300

aattgtcttg agctgaatga ccttgctgta caaaaaaatg ttacggcaat tgtagattgc 360

ggagtagctc ccggaatggg aaacattatt ttaggatatt ataatgagaa gtttcaaatt 420

acagattttg aatgtttagt aggcggtttg cctaagataa aaaaatggcc ttttgcttat 480

aaagcacctt tctcacctat tgatgtgata gaagaataca ctcgtcctgc gcgttatgtg 540

gagaatggtt gtatcgtaac aagagaagca ctgagtgatg tagagcaggt ggagtttgaa 600

ggtgttggaa cactggagtc ttttaacagt gatggtttac gttccttgct gttcaccatg 660

ccgcacataa aaaacatgaa agaaaaaaca ctgcggtatc ccggccatgt tgaatatgtc 720

agggtgctca ggtcaacagg ctttttcagt accacacctg tagaaataaa tggaacaaaa 780

atccgtccga tagattttac ctcacgtttg ctttttgatg aatggaaatt gggcgaaaca 840

gaagaagagc gtaccgttat gcgtattacc ataaagggtt ttgatgataa aggacaacaa 900

accatcgttt atcatttgca tgacaattac aaccatgcca ccaacacttc ttcaatggcg 960

cgtacaacag gatatacagc cacagctgct gcacatttaa tcttaaacgg tatgtttgat 1020

aacaaaggag tgtttccacc ggagttggtt ggaaaatatg aaaactgttt tcactccatc 1080

atgcaatacc ttaacgaaag aggtgttcaa tatgtaaaaa cacaatactg a 1131

<210> 8

<211> 1131

<212> DNA

<213> (codon-optimized lysine dehydrogenase-encoding gene lysDH-Bb)

<400> 8

atgtctaaaa tcatcgttct gggttctggt atggttggtt ctgctatggc tctggacctg 60

gctaccaacc accaggttac cgttgctgac atcaacaccg cttctctgga aaaactgaaa 120

gctaaatcta acgctatcaa cgttctgacc gctgacgttc gtaactctgc tgaactgatc 180

aaaatgctgg aaccgttcga cctggttgtt tgcgctgttc cgggtttcct gggttaccag 240

accctgaaaa acatcatcga agctggtaaa aacaccgttg acatctcttt cttcccggaa 300

aactgcctgg aactgaacga cctggctgtt cagaaaaacg ttaccgctat cgttgactgc 360

ggtgttgctc cgggtatggg taacatcatc ctgggttact acaacgaaaa attccagatc 420

accgacttcg aatgcctggt tggtggtctg ccgaaaatca aaaaatggcc gttcgcttac 480

aaagctccgt tctctccgat cgacgttatc gaagaataca cccgtccggc tcgttacgtt 540

gaaaacggtt gcatcgttac ccgtgaagct ctgtctgacg ttgaacaggt tgaattcgaa 600

ggtgttggta ccctggaatc tttcaactct gacggtctgc gttctctgct gttcaccatg 660

ccgcacatca aaaacatgaa agaaaaaacc ctgcgttacc cgggtcacgt tgaatacgtt 720

cgtgttctgc gttctaccgg tttcttctct accaccccgg ttgaaatcaa cggtaccaaa 780

atccgtccga tcgacttcac ctctcgtctg ctgttcgacg aatggaaact gggtgaaacc 840

gaagaagaac gtaccgttat gcgtatcacc atcaaaggtt tcgacgacaa aggtcagcag 900

accatcgttt accacctgca cgacaactac aaccacgcta ccaacacctc ttctatggct 960

cgtaccaccg gttacaccgc taccgctgct gctcacctga tcctgaacgg tatgttcgac 1020

aacaaaggtg ttttcccgcc ggaactggtt ggtaaatacg aaaactgctt ccactctatc 1080

atgcagtacc tgaacgaacg tggtgttcag tacgttaaaa cccagtacta a 1131

<210> 9

<211> 1494

<212> DNA

<213> (Gene Psefu _1272 encoding aminoadipate semialdehyde dehydrogenase)

<400> 9

atggtcaact cgctactcga acgtctcggt gtcagcgcca gcgcctacca gaacggcagt 60

cacgcggttc atacgccgat cgacggcagc cagatcggca gcctgaccct tgagggcgca 120

gacgccgtgc gtgccaagat caccgccggc cacgacgcct ttctggcctg gcgcaaggtg 180

ccggcgccgc ggcgtggcga gctggtgcgt ctgttcggcg aggtgctgcg tgagcacaag 240

gccgatctcg gcgagctggt gtccatcgaa gccggcaaga tcactcagga aggcctgggc 300

gaagtgcagg aaatgatcga catctgcgac ttcgccgtcg gcctgtcgcg ccagctctac 360

ggcctgacca tcgcctccga gcgctcgggc caccatatgc gtgaaacctg gcacccgctg 420

ggcgtggtcg gcgtgatcag cgccttcaac ttcccggtcg ccgtgtgggc gtggaacacc 480

accctggccc tggtcgccgg caacgcggtg atctggaagc cgtcggaaaa gaccccgctg 540

accgccctgg cctcccaggc actgttcgac aaggccctcg agcgcttcgg cagcgacgcc 600

ccgcaaggcc tggcgcaact ggtgatcggt gatcgcgaag ccggcgaagt gctggtcgac 660

gacccgcgcg tgccgctgat cagcgcgacc ggcagcaccc gcatgggccg cgaagtcgcc 720

ccgcgggtgg ctgcccgctt cggccgcagc attctggaac tgggcggcaa caacgccatg 780

atcctcgccc ccagcgccga cctcgacctg gccgtgcgcg gcatcctgtt cagcgccgtc 840

ggcaccgccg gccagcgttg caccaccctg cgccgcctga tcgtccatcg ttcgatcaag 900

gacgaggtgg tcgcccgcgt caaagccgcc tacgccaagg tacgcatcgg cgacccgcgc 960

cagggcaacc tgatcggccc gctgatcgac aagcaggcgt tcagcgccat gcaggacgcc 1020

ctcgccaagg cccgcgacga aggcggccag gtgttcggtg gcgagcgcca gctggccgac 1080

accttcccca acggctacta cgtgagccct gccatcgtcg agatgccggg ccagactgca 1140

gtggtgcgcc atgaaacctt cgcgccgatc ctctacgtgc tcgcctacga cgacttcgaa 1200

gaggcgctgc gcctgaacaa cgaagtgccc cagggcctgt cctcgtgcat cttcaccacc 1260

gacgtgcgtg aagccgaagc cttccagggc gcggccggca gcgactgcgg catcgccaac 1320

gtcaacatcg gcaccagcgg tgcggaaatc ggcggcgcct ttggcggcga gaaggaaacc 1380

ggtggcggtc gcgagtccgg ctccgatgcc tggaaggcct acatgcgccg ccagaccaat 1440

accgtcaact actcccgaga gttgccgctg gcccagggca tcgtgttcga ctga 1494

<210> 10

<211> 1494

<212> DNA

<213> (codon-optimized Gene Psefu _1272 encoding aminoadipate semialdehyde dehydrogenase)

<400> 10

atggtgaata gcctgctgga acgcctgggc gtgagcgcaa gcgcgtatca aaatggcagc 60

catgcggttc ataccccgat tgatggcagc cagattggca gcctgaccct ggaaggtgcg 120

gatgcggttc gtgcaaaaat taccgcgggc catgatgcgt ttctggcgtg gcgtaaagtg 180

ccggcgccaa gacgtggtga attagtgcgt ttatttggtg aagtgctgcg cgaacataaa 240

gcggatctgg gcgaactggt gagcattgaa gcgggcaaaa ttacccagga aggcctgggc 300

gaagtgcagg aaatgattga tatttgcgat ttcgcggtgg gcctgagccg ccaattgtat 360

ggcttgacca ttgcgagcga acgcagcggc catcatatgc gcgaaacctg gcatccgctg 420

ggcgttgttg gtgtgattag cgcgtttaat tttccggtgg cggtgtgggc gtggaatacc 480

actttagcgt tagttgcggg caatgcggtt atttggaaac cgagcgaaaa aaccccgctg 540

accgcgctgg cgagtcaagc gttatttgat aaagcgctgg aacgctttgg cagcgatgcg 600

ccgcaaggtt tagcgcaatt ggttattggt gatcgcgaag cgggtgaagt gctggtggat 660

gatccgcgcg ttccgttaat tagcgcgacc ggcagcaccc gtatgggtcg tgaagtggcg 720

ccaagagttg cggcgcgttt tggtcgtagt attttggaat taggcggcaa taatgcgatg 780

attctggcgc cgagcgcgga tctggatctg gcggttcgtg gtattctgtt tagcgcggtg 840

ggcaccgcgg gccaacgttg tactacctta cgtcgcttaa ttgtgcatcg cagcattaaa 900

gatgaagtgg tggcgcgcgt gaaagcggcg tatgcgaaag ttcgtattgg cgatccgcgc 960

cagggtaatc tgattggccc attaattgat aaacaggcgt ttagcgcgat gcaggatgcg 1020

ctggcgaaag cgagagatga aggtggccaa gtttttggcg gcgaacgcca actggcggat 1080

acctttccaa atggctatta tgtgagcccg gcgattgtgg aaatgccggg ccaaaccgca 1140

gttgtgcgtc atgaaacctt tgcgccgatt ctgtatgtgc tggcgtatga tgattttgag 1200

gaagcgctgc gcctgaataa tgaagtgccg cagggcctga gcagctgcat ttttaccacc 1260

gatgtgcgcg aagcggaagc gtttcagggc gcggcgggta gtgattgtgg tattgcgaat 1320

gttaatattg gcaccagcgg cgcggaaatt ggcggcgcat ttggtggtga aaaagaaacc 1380

ggtggcggcc gcgaaagcgg cagcgatgcg tggaaagcgt atatgcgccg ccagaccaat 1440

accgtgaatt atagccgcga actgccgctg gcgcagggca ttgtttttga ttaa 1494

<210> 11

<211> 1620

<212> DNA

<213> (Gene ALDH7A1 encoding aminoadipate semialdehyde dehydrogenase)

<400> 11

atgtggcgcc ttcctcgcgc gctgtgtgtg cacgctgcaa agaccagcaa gctctctgga 60

ccttggagca ggcctgccgc cttcatgtcc actctcctca tcaatcagcc ccagtatgcg 120

tggctgaaag agctggggct ccgcgaggaa aacgagggcg tgtataatgg aagctgggga 180

ggccggggag aggttattac gacctattgc cctgctaaca acgagccaat agcaagagtc 240

cgacaggcca gtgtggcaga ctatgaagaa actgtaaaga aagcaagaga agcatggaaa 300

atctgggcag atattcctgc tccaaaacga ggagaaatag taagacagat tggcgatgcc 360

ttgcgggaga agatccaagt actaggaagc ttggtgtctt tggagatggg gaaaatctta 420

gtggaaggtg tgggtgaagt tcaggagtat gtggatatct gtgactatgc tgttggttta 480

tcaaggatga ttggaggacc tatcttgcct tctgaaagat ctggccatgc actgattgag 540

cagtggaatc ccgtaggcct ggttggaatc atcacggcat tcaatttccc tgtggcagtg 600

tatggttgga acaacgccat cgccatgatc tgtggaaatg tctgcctctg gaaaggagct 660

ccaaccactt ccctcattag tgtggctgtc acaaagataa tagccaaggt tctggaggac 720

aacaagctgc ctggtgcaat ttgttccttg acttgtggtg gagcagatat tggcacagca 780

atggccaaag atgaacgagt gaacctgctg tccttcactg ggagcactca ggtgggaaaa 840

caggtgggcc tgatggtgca ggagaggttt gggagaagtc tgttggaact tggaggaaac 900

aatgccatta ttgcctttga agatgcagac ctcagcttag ttgttccatc agctctcttc 960

gctgctgtgg gaacagctgg ccagaggtgt accactgcga ggcgactgtt tatacatgaa 1020

agcatccatg atgaggttgt aaacagactt aaaaaggcct atgcacagat ccgagttggg 1080

aacccatggg accctaatgt tctctatggg ccactccaca ccaagcaggc agtgagcatg 1140

tttcttggag cagtggaaga agcaaagaaa gaaggtggca cagtggtcta tgggggcaag 1200

gttatggatc gccctggaaa ttatgtagaa ccgacaattg tgacaggtct tggccacgat 1260

gcgtccattg cacacacaga gacttttgct ccgattctct atgtctttaa attcaagaat 1320

gaagaagagg tctttgcatg gaataatgaa gtaaaacagg gactttcaag tagcatcttt 1380

accaaagatc tgggcagaat ctttcgctgg cttggaccta aaggatcaga ctgtggcatt 1440

gtaaatgtca acattccaac aagtggggct gagattggag gtgcctttgg aggagaaaag 1500

cacactggtg gtggcaggga gtctggcagt gatgcctgga aacagtacat gagaaggtct 1560

acttgtacta tcaactacag taaagacctt cctctggccc aaggaatcaa gtttcagtaa 1620

<210> 12

<211> 1620

<212> DNA

<213> (codon-optimized Gene ALDH7A1 encoding aminoadipate semialdehyde dehydrogenase)

<400> 12

atgtggcgtc tgccgcgtgc tctgtgcgtt cacgctgcta aaacctctaa actgtctggt 60

ccgtggtctc gtccggctgc tttcatgtct accctgctga tcaaccagcc gcagtacgct 120

tggctgaaag aactgggtct gcgtgaagaa aacgaaggtg tttacaacgg ttcttggggt 180

ggtcgtggtg aagttatcac cacctactgc ccggctaaca acgaaccgat cgctcgtgtt 240

cgtcaggctt ctgttgctga ctacgaagaa accgttaaaa aagctcgtga agcttggaaa 300

atctgggctg acatcccggc tccgaaacgt ggtgaaatcg ttcgtcagat cggtgacgct 360

ctgcgtgaaa aaatccaggt tctgggttct ctggtttctc tggaaatggg taaaatcctg 420

gttgaaggtg ttggtgaagt tcaggaatac gttgacatct gcgactacgc tgttggtctg 480

tctcgtatga tcggtggtcc gatcctgccg tctgaacgtt ctggtcacgc tctgatcgaa 540

cagtggaacc cggttggtct ggttggtatc atcaccgctt tcaacttccc ggttgctgtt 600

tacggttgga acaacgctat cgctatgatc tgcggtaacg tttgcctgtg gaaaggtgct 660

ccgaccacct ctctgatctc tgttgctgtt accaaaatca tcgctaaagt tctggaagac 720

aacaaactgc cgggtgctat ctgctctctg acctgcggtg gtgctgacat cggtaccgct 780

atggctaaag acgaacgtgt taacctgctg tctttcaccg gttctaccca ggttggtaaa 840

caggttggtc tgatggttca ggaacgtttc ggtcgttctc tgctggaact gggtggtaac 900

aacgctatca tcgctttcga agacgctgac ctgtctctgg ttgttccgtc tgctctgttc 960

gctgctgttg gtaccgctgg tcagcgttgc accaccgctc gtcgtctgtt catccacgaa 1020

tctatccacg acgaagttgt taaccgtctg aaaaaagctt acgctcagat ccgtgttggt 1080

aacccgtggg acccgaacgt tctgtacggt ccgctgcaca ccaaacaggc tgtttctatg 1140

ttcctgggtg ctgttgaaga agctaaaaaa gaaggtggta ccgttgttta cggtggtaaa 1200

gttatggacc gtccgggtaa ctacgttgaa ccgaccatcg ttaccggtct gggtcacgac 1260

gcttctatcg ctcacaccga aaccttcgct ccgatcctgt acgttttcaa attcaaaaac 1320

gaagaagaag ttttcgcttg gaacaacgaa gttaaacagg gtctgtcttc ttctatcttc 1380

accaaagacc tgggtcgtat cttccgttgg ctgggtccga aaggttctga ctgcggtatc 1440

gttaacgtta acatcccgac ctctggtgct gaaatcggtg gtgctttcgg tggtgaaaaa 1500

cacaccggtg gtggtcgtga atctggttct gacgcttgga aacagtacat gcgtcgttct 1560

acctgcacca tcaactactc taaagacctg ccgctggctc agggtatcaa attccagtaa 1620

<210> 13

<211> 1491

<212> DNA

<213> (Gene PMI27 encoding aminoadipate semialdehyde dehydrogenase)

<400> 13

atggttgccg cattgcttga tcgtcttggt gtgaacccgg ccctgtacca gaacggcaaa 60

gtgccggtgc attcgccgat cgatggcagc cagatcgccg ccgtgaactg ggaaggcgcc 120

gctgaagtcg agcagcacat cagtcgtgca gatcatgcgt tcgaattgtg gcgcaaggtc 180

ccggccccgc gccgtggtga actggtccgc caactgggcg atatcctgcg tgaatacaag 240

gccgaccttg gcgagctggt gtcctgggaa gccggcaaga tcactcagga aggcctgggt 300

gaagttcagg aaatgatcga catctgcgat ttcgccgtcg gcctgtcccg ccagctgtac 360

ggtttgacca tcgcctccga gcgtccgggc caccacatgc gcgaaacctg gcacccgctg 420

ggcgtcgttg gcgtcatcag cgcattcaac ttcccggttg ccgtctgggc ctggaacacc 480

gcgctggcgc tggtctgcgg caacccggtg atctggaaac cgtcggagaa aacgccactg 540

accgcactgg cctgtcaggc actgttcgac cgtgtactga agaacttcag cgatgcacct 600

ccgcacctga gtcaggtgat tatcggcggt cgcgatgccg gcgaagccct ggtcgatgac 660

ccgcgtgtcg cgctgatcag cgccaccggc agcacccgca tgggccgtga agtggcgccg 720

aaaatcgctg cacgtttcgc tcgcagcatt ctggaactgg gcggtaacaa cgccatgatc 780

ctcggcccaa gcgccgacct ggacatggcc gtacgagcca tcctgttcag cgccgtcggc 840

actgccggtc agcgttgcac cacgttgcgt cgcctgattg cccatgaatc ggtgaaagaa 900

gaaatcgtca cccgcctgaa agccgcgtac tccaaagtgc gtatcggcaa ccctctggaa 960

ggcaatctga tcggtccgct gatcgacaaa cacagcttcg aaaacatgca ggatgcgctt 1020

gagcaggcct tgagcgaagg cggccgggtg ttcggcggca agcgccaact ggaagacaaa 1080

ttccctaacg cttattacgt ctcgccggcc atcgttgaaa tgccggagca gagcgatgtg 1140

gtttgccacg aaaccttcgc accgattctg tacgtggtcg gttacaagga cttcgacgaa 1200

gcgctgcgcc tgaacaacgc cgtgccacaa ggcctgtcgt cgtgcatctt caccactgac 1260

gtgcgtgaag ccgagcagtt catgtcggcg gtgggcagcg actgcggcat cgccaacgtc 1320

aacatcggcc cgagcggcgc ggaaatcggc ggggcgtttg gcggtgagaa agaaacgggc 1380

ggcggtcgtg agtccggttc cgatgcatgg cgcggctaca tgcgccgtca gaccaacacc 1440

gtgaactatt cgctggagtt gccgttggct cagggtatta ccttcgactg a 1491

<210> 14

<211> 1491

<212> DNA

<213> (codon-optimized Gene PMI27 encoding aminoadipate semialdehyde dehydrogenase)

<400> 14

atggttgctg ctctgctgga ccgtctgggt gttaacccgg ctctgtacca gaacggtaaa 60

gttccggttc actctccgat cgacggttct cagatcgctg ctgttaactg ggaaggtgct 120

gctgaagttg aacagcacat ctctcgtgct gaccacgctt tcgaactgtg gcgtaaagtt 180

ccggctccgc gtcgtggtga actggttcgt cagctgggtg acatcctgcg tgaatacaaa 240

gctgacctgg gtgaactggt ttcttgggaa gctggtaaaa tcacccagga aggtctgggt 300

gaagttcagg aaatgatcga catctgcgac ttcgctgttg gtctgtctcg tcagctgtac 360

ggtctgacca tcgcttctga acgtccgggt caccacatgc gtgaaacctg gcacccgctg 420

ggtgttgttg gtgttatctc tgctttcaac ttcccggttg ctgtttgggc ttggaacacc 480

gctctggctc tggtttgcgg taacccggtt atctggaaac cgtctgaaaa aaccccgctg 540

accgctctgg cttgccaggc tctgttcgac cgtgttctga aaaacttctc tgacgctccg 600

ccgcacctgt ctcaggttat catcggtggt cgtgacgctg gtgaagctct ggttgacgac 660

ccgcgtgttg ctctgatctc tgctaccggt tctacccgta tgggtcgtga agttgctccg 720

aaaatcgctg ctcgtttcgc tcgttctatc ctggaactgg gtggtaacaa cgctatgatc 780

ctgggtccgt ctgctgacct ggacatggct gttcgtgcta tcctgttctc tgctgttggt 840

accgctggtc agcgttgcac caccctgcgt cgtctgatcg ctcacgaatc tgttaaagaa 900

gaaatcgtta cccgtctgaa agctgcttac tctaaagttc gtatcggtaa cccgctggaa 960

ggtaacctga tcggtccgct gatcgacaaa cactctttcg aaaacatgca ggacgctctg 1020

gaacaggctc tgtctgaagg tggtcgtgtt ttcggtggta aacgtcagct ggaagacaaa 1080

ttcccgaacg cttactacgt ttctccggct atcgttgaaa tgccggaaca gtctgacgtt 1140

gtttgccacg aaaccttcgc tccgatcctg tacgttgttg gttacaaaga cttcgacgaa 1200

gctctgcgtc tgaacaacgc tgttccgcag ggtctgtctt cttgcatctt caccaccgac 1260

gttcgtgaag ctgaacagtt catgtctgct gttggttctg actgcggtat cgctaacgtt 1320

aacatcggtc cgtctggtgc tgaaatcggt ggtgctttcg gtggtgaaaa agaaaccggt 1380

ggtggtcgtg aatctggttc tgacgcttgg cgtggttaca tgcgtcgtca gaccaacacc 1440

gttaactact ctctggaact gccgctggct cagggtatca ccttcgacta a 1491

<210> 15

<211> 1503

<212> DNA

<213> (Gene encoding aminoadipate transaminase ARO 8)

<400> 15

atgactttac ctgaatcaaa agacttttct tacttgtttt cggatgaaac caatgctcgt 60

aaaccatccc cattgaaaac ctgcatccat cttttccaag atcctaacat tatctttttg 120

ggtggtggcc tgccattaaa agattatttc ccatgggata atctatctgt agattcaccc 180

aagcctcctt ttccccaggg tattggagct ccaattgacg agcagaattg cataaaatac 240

accgtcaaca aagattacgc tgataaaagt gccaatcctt ccaacgatat tcctttgtca 300

agagctttgc aatacgggtt cagtgctggt caacctgaac tattaaactt cattagagat 360

cataccaaga ttatccacga tttgaagtat aaggactggg acgttttagc cactgcaggt 420

aacacaaatg cctgggaatc tactttaaga gtcttttgta accgaggtga tgtcatctta 480

gttgaggcac attctttttc ctcttcattg gcttctgcag aggctcaagg tgtcattacc 540

ttccccgtgc caattgacgc tgatggtatc attcctgaaa aattagctaa agtcatggaa 600

aactggacac ctggtgctcc taaaccaaag ttgttataca ctattccaac gggccaaaat 660

ccaactggta cttccattgc agaccataga aaggaggcaa tttacaagat cgctcaaaag 720

tacgacttcc taattgtgga agatgaacct tattatttct tacaaatgaa tccctacatc 780

aaagacttga aggaaagaga gaaggcacaa agttctccaa agcaggacca tgacgaattt 840

ttgaagtcct tggcaaacac tttcctttcc ttggatacag aaggccgtgt tattagaatg 900

gattcctttt caaaagtttt ggccccaggg acaagattgg gttggattac tggttcatcc 960

aaaatcttga agccttactt gagtttgcat gaaatgacga ttcaagcccc agcaggtttt 1020

acacaagttt tggtcaacgc tacgctatcc aggtggggtc aaaagggtta cttggactgg 1080

ttgcttggcc tgcgtcatga atacactttg aaacgtgact gtgccatcga tgccctttac 1140

aagtatctac cacaatctga tgctttcgtg atcaatcctc caattgcagg tatgtttttc 1200

accgtgaaca ttgacgcatc tgtccaccct gagtttaaaa caaaatacaa ctcagaccct 1260

taccagctag aacagagtct ttaccacaaa gtggttgaac gtggtgtttt agtggttccc 1320

ggttcttggt tcaagagtga gggtgagacg gaacctcctc aacccgctga atctaaagaa 1380

gtcagtaatc caaacataat tttcttcaga ggtacctatg cagctgtctc tcctgagaaa 1440

ctgactgaag gtctgaagag attaggtgat actttatacg aagaatttgg tatttccaaa 1500

tag 1503

<210> 16

<211> 1503

<212> DNA

<213> (codon-optimized Gene encoding aminoadipate transaminase ARO 8)

<400> 16

atgaccctgc cggaaagcaa agattttagc tatctgttta gcgacgagac caatgcgcgc 60

aaaccgagcc cactgaaaac ctgcattcat ctgtttcagg atccgaatat tatcttcctg 120

ggcggcggcc tgccgctgaa agattatttt ccgtgggata atctgagcgt ggatagcccg 180

aaaccgccgt ttccgcaggg tattggtgcg ccaattgatg aacagaattg cattaaatac 240

accgtgaaca aggactacgc ggataaaagc gcgaatccga gcaatgatat tccgctgagc 300

cgcgcgctgc agtatggctt tagcgcaggt caaccagaat tgctgaattt tattcgcgat 360

cacaccaaga ttatccacga cctgaaatac aaggactggg acgtgctggc gaccgcgggc 420

aataccaatg cgtgggaaag caccctgcgc gtgttttgca atcgcggcga tgtgattctg 480

gtggaagcgc atagctttag cagcagcctg gcgagcgcgg aagcgcaagg tgttattacc 540

tttccggtgc cgattgatgc ggatggcatt attccggaaa aactggcgaa agtgatggaa 600

aattggaccc cgggcgcgcc gaaaccgaaa ttattatata ccattccgac cggccagaat 660

ccgaccggca ccagtattgc ggatcatcgc aaagaagcga tttataaaat tgcgcagaaa 720

tacgacttcc tgatcgtgga ggatgaaccg tattatttcc tgcagatgaa tccgtatatt 780

aaggacctga aagaacgcga gaaagcgcag agcagcccga aacaggatca tgatgaattt 840

ctgaaaagcc tggcgaatac ctttctgagc ctggataccg aaggccgcgt gattcgcatg 900

gatagcttta gcaaagtgct ggcgccgggc acccgtttgg gttggattac cggtagtagc 960

aaaattctga aaccgtatct gagcctgcat gaaatgacca ttcaggcgcc ggcgggcttt 1020

acccaagttc tggttaatgc gaccctgagc cgctggggcc aaaaaggcta tttggattgg 1080

ctgctgggcc tgcgccatga atataccctg aaacgcgatt gcgcgattga tgcgctgtat 1140

aaatatctgc cgcagagcga tgcgtttgtg attaatccgc cgattgcggg catgtttttt 1200

accgtgaata ttgatgcgag cgtgcatccg gaatttaaaa ccaaatataa cagcgacccg 1260

taccagctgg aacagagcct gtatcataaa gtggtggaac gcggcgtgct ggtggtgcca 1320

ggtagctggt ttaaaagcga aggcgaaacc gaaccgccgc agccggcgga aagcaaagaa 1380

gttagcaatc cgaatattat cttcttccgc ggcacctatg cggcggtgag cccagaaaaa 1440

ctgaccgaag gcctgaaacg cctgggcgat actttatatg aagaatttgg cattagcaag 1500

tga 1503

<210> 17

<211> 1194

<212> DNA

<213> (Gene lysN encoding aminoadipate transaminase)

<400> 17

gtgaaaccgc taagctggtc cgaggcgttc ggtaaaggcg cgggaaggat ccaggcctcc 60

accatccggg agcttctcaa gctcacccag cgccccggca tcctgagctt cgccgggggg 120

ctcccggccc ccgagctctt ccccaaggag gaggcggcgg aagccgcggc gcggatcctg 180

cgggagaagg gcgaggtcgc cctccagtac agccccaccg agggctacgc ccccctaagg 240

gccttcgtgg cggagtggat cggcgtgcgc cccgaggagg tcctcatcac caccgggagc 300

cagcaggcct tggacctcgt gggcaaggtc ttcctggacg agggaagccc cgtgctgctg 360

gaggccccaa gctacatggg ggccatccag gccttccgtc tccaaggccc ccgcttcctc 420

acggtgcccg ccggggagga gggcccggac ctggacgccc tggaagaggt cctcaagagg 480

gagcgccccc gcttcctcta cctcatcccc tctttccaga accccacggg gggcctcacg 540

ccccttcccg cccggaagcg gctcttgcag atggtgatgg agcggggcct cgtggtggtg 600

gaggacgacg cctaccggga gctctacttc ggggaggcgc gcctcccgag cctctttgag 660

ctcgcccggg aggcgggcta ccccggggtc atctacctgg gaagcttctc caaggttctc 720

tcccccgggc ttcgcgtggc cttcgccgtg gcccacccgg aggccctgca gaagctcgtc 780

caggccaagc agggggccga cctccacacc cccatgctca accagatgct ggtccacgag 840

ctcttaaagg agggcttctc cgagcgcttg gaacgggtcc gcagggtcta ccgggaaaag 900

gcccaggcca tgctccatgc cctggaccgg gaggtgccca aggaggtgcg ctacacaagg 960

cccaagggcg ggatgttcgt ctggatggag ctccccaagg gcctctccgc cgagggcctc 1020

ttccggaggg ccctcgagga gaacgtggcc ttcgtgccgg gaggtccttt cttcgccaac 1080

gggggcgggg agaacaccct gaggctctcc tacgccaccc tggaccggga ggggatcgcg 1140

gagggcgtgc ggcggctggg gcgggcgcta aaggggcttt tggccttggt ctag 1194

<210> 18

<211> 1194

<212> DNA

<213> (codon-optimized gene lysN encoding aminoadipate transaminase)

<400> 18

atgaagccac tgtcctggtc cgaagctttc ggtaagggag caggtcgtat ccaggcatcc 60

accatccgcg aactgctgaa gctgacccag cgcccaggca tcctgtcctt cgccggcggc 120

ctgccagcac cagagctgtt cccaaaggaa gaagcagctg aagcagcagc tcgcatcctg 180

cgcgagaagg gcgaagtggc tctgcagtac tccccaaccg agggctacgc acctctgcgc 240

gcattcgtgg cagaatggat cggcgtgcgc ccagaagagg tgctgatcac caccggctcc 300

cagcaggctc tggatctcgt gggcaaggtc ttcctggatg aaggctctcc agtgctgctg 360

gaagcaccat cctacatggg cgctatccag gcattccgcc tgcaaggccc acgcttcttg 420

accgtgccgg ctggcgagga aggcccagat ctggatgcac tggaagaagt gctgaagcgc 480

gaacgcccaa gattcctgta cctgatccca agcttccaga acccaaccgg cggcctgacc 540

ccactgccag ctcgcaagcg ccttctgcag atggttatgg agcgtggtct ggtcgtggtt 600

gaggatgatg cataccgcga gctgtacttc ggcgaagcac gtctgccctc cctgttcgag 660

ctggctcgcg aggcaggcta cccaggcgtg atctacctgg gctccttctc caaggtcctg 720

tccccaggcc tgcgtgtggc attcgcagtc gcccacccag aggcactgca gaagctggtt 780

caggcaaagc agggcgcgga tttgcacacc ccaatgctga accagatgct ggtgcacgaa 840

ctgctcaagg aaggcttctc cgaacgcctg gaacgcgttc gccgcgtgta ccgcgagaag 900

gcacaggcaa tgctccacgc actggatcgc gaagtgccta aggaagtgcg ctatacccgc 960

cctaaaggtg gaatgttcgt ttggatggaa ctgcctaaag gcctttctgc cgaaggcttg 1020

ttccgtcgtg ctcttgaaga aaacgttgct ttcgttcctg gcggtccatt cttcgctaac 1080

ggtggtggtg aaaataccct tagattgagc tacgcaaccc tggaccgtga gggtattgct 1140

gaaggtgttc gacgccttgg tcgcgcactg aaaggcttgt tagctttagt ttaa 1194

<210> 19

<211> 1050

<212> DNA

<213> (Gene AeLDH encoding ketoadipate reductase)

<400> 19

atgaagatct ccctcaccag cgcccgccag cttgcccgcg acatcctcgc cgcgcagcag 60

gtgcccgccg acatcgctga cgacgtggcc gagcacctgg tcgaatccga ccgctgcggc 120

tatatcagcc acggcctgtc gatcctgccc aactaccgca ccgccctcga cggccacagc 180

gtcaacccgc aaggccgcgc caaatgcgtg ctggaccagg gcacgctgat ggtgttcgac 240

ggcgacggcg gcttcggcca gcacgtgggc aagtccgtga tgcaagcagc gatcgagcgc 300

gtgcgccagc atggccactg catcgtcact ctgcgccgct cgcaccatct cggccgcatg 360

ggccactacg gcgagatggc ggccgccgcc ggctttgtgc tgctgagctt caccaacgtg 420

atcaaccgcg cgccggtggt ggcgccgttc ggcggccgcg tggcgcggct caccaccaac 480

ccgctgtgtt tcgccggccc gatgcccaac gggcggccgc ctctggtggt ggacatcgcc 540

accagcgcga ttgccatcaa caaggcccgt gtgctggccg agaaaggcga gccggcgccc 600

gaaggcagca tcatcggcgc cgacggcaac cccaccaccg acgcgtcaac catgttcggc 660

gaacaccccg gcgcgctgct gccctttggc ggccacaagg gctacgcact gggcgttgtg 720

gccgagctgc tggcgggcgt gctgtccggc ggcggtacca tccagccaga caatccgcgc 780

ggcggcgtgg ccaccaacaa cctgttcgcg gtgctgctca atcccgcgct ggacctgggc 840

ctggactggc agagcgccga ggtcgaggcg ttcgtgcgct acctgcacga cacaccgccg 900

gcgccgggcg tcgaccgcgt gcagtacccc ggcgagtacg aggccgccaa ccgggcgcag 960

gccagcgaca cgctaaacat caacccggcc atctggcgca atcttgagcg cctggcgcag 1020

tcgctcaacg tggccgtccc cacggcctga 1050

<210> 20

<211> 1050

<212> DNA

<213> (codon-optimized Gene AeLDH encoding ketoadipate reductase)

<400> 20

atgaaaatta gcctgaccag cgcgcgccag ctggcgcgcg atattttagc ggcgcaacaa 60

gttccagcgg atattgcgga tgatgtggcg gaacatctgg tggaaagcga tcgctgcggc 120

tatattagcc atggcctgag cattctgccg aattatcgca ccgcgctgga tggccatagc 180

gtgaatccac aaggccgcgc gaaatgcgtt ttggatcaag gcaccctgat ggtgtttgat 240

ggcgatggcg gctttggcca gcatgtgggt aaaagcgtta tgcaggcggc gattgaacgc 300

gtgcgccaac atggtcattg cattgtgacc ctgcgccgca gccatcatct gggccgtatg 360

ggtcattatg gcgaaatggc ggcggcggcg ggttttgtat tattaagctt taccaatgtg 420

atcaaccgcg cgccggtggt ggcaccattt ggtggtcgtg ttgcgcgttt gaccaccaat 480

ccgttatgtt ttgcgggtcc gatgccgaat ggccgcccac cattagttgt tgatattgcg 540

accagcgcga ttgcgattaa taaagcgcgc gtgctggcgg aaaaaggcga accggcgcca 600

gaaggtagta ttattggcgc ggatggtaat ccgaccaccg atgcgagcac catgtttggc 660

gaacatccgg gcgcgttact gccatttggt ggtcataaag gctatgcgct gggcgtggtt 720

gcggaattgt tagcgggtgt gttaagcggt ggtggcacca ttcaaccaga taatccgcgc 780

ggcggcgtgg cgactaataa tctgtttgcg gtgctgctga atccggcgct ggatctgggt 840

ttagattggc aaagcgcgga agtggaagcg tttgtgcgct atctgcatga taccccgccg 900

gcgccaggtg ttgatcgtgt tcaatatccg ggtgaatatg aagcggcgaa tcgcgcgcaa 960

gcgagcgata ccttaaatat taatccggcg atttggcgca atctggaacg cctggcgcaa 1020

agcttgaatg tggcggttcc aaccgcgtaa 1050

<210> 21

<211> 996

<212> DNA

<213> (Gene HgdH encoding ketoadipate reductase)

<400> 21

atgaaggttt tatgttatgg tgtaagagat gtagaactgc cgatttttga agcctgcaac 60

aaagaatttg gttacgacat caaatgtgtc cctgattatc tgaacacgaa agaaaccgcc 120

gaaatggctg ctggctttga tgcggttatc ctgcgcggca actgcttcgc caataaacag 180

aacctggaca tttacaaaaa actgggcgta aaatacatcc tgacccgtac cgccggcacg 240

gatcatatcg ataaggaata tgccaaggaa ctgggcttcc ccatggcttt cgttccccgt 300

tattccccca acgccattgc tgaactggct gtaacccagg ccatgatgct gctgcgtcat 360

accgcttaca ccacttcccg cactgccaag aagaacttca aggttgatgc cttcatgttc 420

tccaaagaag tccgcaactg caccgtgggt gttgttggtc tgggccggat cggccgtgtg 480

gctgcccaga tcttccatgg catgggcgct accgttatcg gggaagacgt tttcgaaatc 540

aaagggatcg aagattactg cacccaggtt tccctggatg aagtcctgga aaaatccgac 600

atcatcacca tccatgctcc gtacatcaaa gaaaacggcg ctgtggttac ccgcgatttc 660

ttgaagaaga tgaaagacgg cgccatcctg gtgaactgcg ctcgcggcca gctggttgac 720

accgaagctg tcatcgaagc tgtggaaagc ggtaaactgg gcggctacgg ctgcgacgtt 780

ctggatgggg aagccagcgt attcggcaag gatctggaag gccagaaact ggaaaatccg 840

ctgttcgaaa aactggttga cctgtatccc agagtcctga tcaccccgca tctgggctcc 900

tacaccgacg aagccgtaaa gaacatggtg gaagtttcct accagaacct gaaagatctg 960

gctgaaaccg gcgactgccc caacaagatc aaataa 996

<210> 22

<211> 996

<212> DNA

<213> (codon-optimized Gene HgdH encoding ketoadipate reductase)

<400> 22

atgaaagttc tgtgctacgg tgttcgtgac gttgaactgc cgatcttcga agcttgcaac 60

aaagaattcg gttacgacat caaatgcgtt ccggactacc tgaacaccaa agaaaccgct 120

gaaatggctg ctggtttcga cgctgttatc ctgcgtggta actgcttcgc taacaaacag 180

aacctggaca tctacaaaaa actgggtgtt aaatacatcc tgacccgtac cgctggtacc 240

gaccacatcg acaaagaata cgctaaagaa ctgggtttcc cgatggcttt cgttccgcgt 300

tactctccga acgctatcgc tgaactggct gttacccagg ctatgatgct gctgcgtcac 360

accgcttaca ccacctctcg taccgctaaa aaaaacttca aagttgacgc tttcatgttc 420

tctaaagaag ttcgtaactg caccgttggt gttgttggtc tgggtcgtat cggtcgtgtt 480

gctgctcaga tcttccacgg tatgggtgct accgttatcg gtgaagacgt tttcgaaatc 540

aaaggtatcg aagactactg cacccaggtt tctctggacg aagttctgga aaaatctgac 600

atcatcacca tccacgctcc gtacatcaaa gaaaacggtg ctgttgttac ccgtgacttc 660

ctgaaaaaaa tgaaagacgg tgctatcctg gttaactgcg ctcgtggtca gctggttgac 720

accgaagctg ttatcgaagc tgttgaatct ggtaaactgg gtggttacgg ttgcgacgtt 780

ctggacggtg aagcttctgt tttcggtaaa gacctggaag gtcagaaact ggaaaacccg 840

ctgttcgaaa aactggttga cctgtacccg cgtgttctga tcaccccgca cctgggttct 900

tacaccgacg aagctgttaa aaacatggtt gaagtttctt accagaacct gaaagacctg 960

gctgaaaccg gtgactgccc gaacaaaatc aaataa 996

<210> 23

<211> 40

<212> DNA

<213> （lysDH-Am-F）

<400> 23

actttaagaa ggagatatac catgaaaatt ggcgtgctgg 40

<210> 24

<211> 35

<212> DNA

<213> （lysDH-Am-R）

<400> 24

tatatctcct ttagctcaga ttttcgctaa tcacc 35

<210> 25

<211> 41

<212> DNA

<213> （Psefu_1272-F）

<400> 25

tctgagctaa aggagatata ccatggtgaa tagcctgctg g 41

<210> 26

<211> 33

<212> DNA

<213> （Psefu_1272-R）

<400> 26

tatatctcct ttaatcaaaa acaatgccct gcg 33

<210> 27

<211> 41

<212> DNA

<213> （ARO8-F）

<400> 27

tttttgatta aaggagatat accatgaccc tgccggaaag c 41

<210> 28

<211> 34

<212> DNA

<213> （ARO8-R）

<400> 28

tatatctcct tcacttgcta atgccaaatt cttc 34

<210> 29

<211> 43

<212> DNA

<213> （AeLDH-F）

<400> 29

tagcaagtga aggagatata ccatgaaaat tagcctgacc agc 43

<210> 30

<211> 39

<212> DNA

<213> （AeLDH-R）

<400> 30

tttaccagac tcgagggtac cttacgcggt tggaaccgc 39

<210> 31

<211> 1191

<212> DNA

<213> (Gene encoding aspartate aminotransferase aspC)

<400> 31

atgtttgaga acattaccgc cgctcctgcc gacccgattc tgggcctggc cgatctgttt 60

cgtgccgatg aacgtcccgg caaaattaac ctcgggattg gtgtctataa agatgagacg 120

ggcaaaaccc cggtactgac cagcgtgaaa aaggctgaac agtatctgct cgaaaatgaa 180

accaccaaaa attacctcgg cattgacggc atccctgaat ttggtcgctg cactcaggaa 240

ctgctgtttg gtaaaggtag cgccctgatc aatgacaaac gtgctcgcac ggcacagact 300

ccggggggca ctggcgcact acgcgtggct gccgatttcc tggcaaaaaa taccagcgtt 360

aagcgtgtgt gggtgagcaa cccaagctgg ccgaaccata agagcgtctt taactctgca 420

ggtctggaag ttcgtgaata cgcttattat gatgcggaaa atcacactct tgacttcgat 480

gcactgatta acagcctgaa tgaagctcag gctggcgacg tagtgctgtt ccatggctgc 540

tgccataacc caaccggtat cgaccctacg ctggaacaat ggcaaacact ggcacaactc 600

tccgttgaga aaggctggtt accgctgttt gacttcgctt accagggttt tgcccgtggt 660

ctggaagaag atgctgaagg actgcgcgct ttcgcggcta tgcataaaga gctgattgtt 720

gccagttcct actctaaaaa ctttggcctg tacaacgagc gtgttggcgc ttgtactctg 780

gttgctgccg acagtgaaac cgttgatcgc gcattcagcc aaatgaaagc ggcgattcgc 840

gctaactact ctaacccacc agcacacggc gcttctgttg ttgccaccat cctgagcaac 900

gatgcgttac gtgcgatttg ggaacaagag ctgactgata tgcgccagcg tattcagcgt 960

atgcgtcagt tgttcgtcaa tacgctgcag gaaaaaggcg caaaccgcga cttcagcttt 1020

atcatcaaac agaacggcat gttctccttc agtggcctga caaaagaaca agtgctgcgt 1080

ctgcgcgaag agtttggcgt atatgcggtt gcttctggtc gcgtaaatgt ggccgggatg 1140

acaccagata acatggctcc gctgtgcgaa gcgattgtgg cagtgctgta a 1191

<210> 32

<211> 1350

<212> DNA

<213> (Gene lysC encoding aspartokinase)

<400> 32

atgtctgaaa ttgttgtctc caaatttggc ggtaccagcg tagctgattt tgacgccatg 60

aaccgcagcg ctgatattgt gctttctgat gccaacgtgc gtttagttgt cctctcggct 120

tctgctggta tcactaatct gctggtcgct ttagctgaag gactggaacc tggcgagcga 180

ttcgaaaaac tcgacgctat ccgcaacatc cagtttgcca ttctggaacg tctgcgttac 240

ccgaacgtta tccgtgaaga gattgaacgt ctgctggaga acattactgt tctggcagaa 300

gcggcggcgc tggcaacgtc tccggcgctg acagatgagc tggtcagcca cggcgagctg 360

atgtcgaccc tgctgtttgt tgagatcctg cgcgaacgcg atgttcaggc acagtggttt 420

gatgtacgta aagtgatgcg taccaacgac cgatttggtc gtgcagagcc agatatagcc 480

gcgctggcgg aactggccgc gctgcagctg ctcccacgtc tcaatgaagg cttagtgatc 540

acccagggat ttatcggtag cgaaaataaa ggtcgtacaa cgacgcttgg ccgtggaggc 600

agcgattata cggcagcctt gctggcggag gctttacacg catctcgtgt tgatatctgg 660

accgacgtcc cgggcatcta caccaccgat ccacgcgtag tttccgcagc aaaacgcatt 720

gatgaaatcg cgtttgccga agcggcagag atggcaactt ttggtgcaaa agtactgcat 780

ccggcaacgt tgctacccgc agtacgcagc gatatcccgg tctttgtcgg ctccagcaaa 840

gacccacgcg caggtggtac gctggtgtgc aataaaactg aaaatccgcc gctgttccgc 900

gctctggcgc ttcgtcgcaa tcagactctg ctcactttgc acagcctgaa tatgctgcat 960

tctcgcggtt tcctcgcgga agttttcggc atcctcgcgc ggcataatat ttcggtagac 1020

ttaatcacca cgtcagaagt gagcgtggca ttaacccttg ataccaccgg ttcaacctcc 1080

actggcgata cgttgctgac gcaatctctg ctgatggagc tttccgcact gtgtcgggtg 1140

gaggtggaag aaggtctggc gctggtcgcg ttgattggca atgacctgtc aaaagcctgc 1200

ggcgttggca aagaggtatt cggcgtactg gaaccgttca acattcgcat gatttgttat 1260

ggcgcatcca gccataacct gtgcttcctg gtgcccggcg aagatgccga gcaggtggtg 1320

caaaaactgc atagtaattt gtttgagtaa 1350

<210> 33

<211> 879

<212> DNA

<213> (Gene dapA encoding dihydrodipicolinate synthase)

<400> 33

atgttcacgg gaagtattgt cgcgattgtt actccgatgg atgaaaaagg taatgtctgt 60

cgggctagct tgaaaaaact gattgattat catgtcgcca gcggtacttc ggcgatcgtt 120

tctgttggca ccactggcga gtccgctacc ttaaatcatg acgaacatgc tgatgtggtg 180

atgatgacgc tggatctggc tgatgggcgc attccggtaa ttgccgggac cggcgctaac 240

gctactgcgg aagccattag cctgacgcag cgcttcaatg acagtggtat cgtcggctgc 300

ctgacggtaa ccccttacta caatcgtccg tcgcaagaag gtttgtatca gcatttcaaa 360

gccatcgctg agcatactga cctgccgcaa attctgtata atgtgccgtc ccgtactggc 420

tgcgatctgc tcccggaaac ggtgggccgt ctggcgaaag taaaaaatat tatcggaatc 480

aaagaggcaa cagggaactt aacgcgtgta aaccagatca aagagctggt ttcagatgat 540

tttgttctgc tgagcggcga tgatgcgagc gcgctggact tcatgcaatt gggcggtcat 600

ggggttattt ccgttacggc taacgtcgca gcgcgtgata tggcccagat gtgcaaactg 660

gcagcagaag ggcattttgc cgaggcacgc gttattaatc agcgtctgat gccattacac 720

aacaaactat ttgtcgaacc caatccaatc ccggtgaaat gggcatgtaa ggaactgggt 780

cttgtggcga ccgatacgct gcgcctgcca atgacaccaa tcaccgacag tggtcgtgag 840

acggtcagag cggcgcttaa gcatgccggt ttgctgtaa 879

<210> 34

<211> 1263

<212> DNA

<213> (Gene lysA encoding diaminopimelate decarboxylase)

<400> 34

atgccacatt cactgttcag caccgatacc gatctcaccg ccgaaaatct gctgcgtttg 60

cccgctgaat ttggctgccc ggtgtgggtc tacgatgcgc aaattattcg tcggcagatt 120

gcagcgctga aacagtttga tgtggtgcgc tttgcacaga aagcctgttc caatattcat 180

attttgcgct taatgcgtga gcagggcgtg aaagtggatt ccgtctcgtt aggcgaaata 240

gagcgtgcgt tggcggcggg ttacaatccg caaacgcacc ccgatgatat tgtttttacg 300

gcagatgtta tcgatcaggc gacgcttgaa cgcgtcagtg aattgcaaat tccggtgaat 360

gcgggttctg ttgatatgct cgaccaactg ggccaggttt cgccagggca tcgggtatgg 420

ctgcgcgtta atccggggtt tggtcacgga catagccaaa aaaccaatac cggtggcgaa 480

aacagcaagc acggtatctg gtacaccgat ctgcccgccg cactggacgt gatacaacgt 540

catcatctgc agctggtcgg cattcacatg cacattggtt ctggcgttga ttatgcccat 600

ctggaacagg tgtgtggtgc tatggtgcgt caggtcatcg aattcggtca ggatttacag 660

gctatttctg cgggcggtgg gctttctgtt ccttatcaac agggtgaaga ggcggttgat 720

accgaacatt attatggtct gtggaatgcc gcgcgtgagc aaatcgcccg ccatttgggc 780

caccctgtga aactggaaat tgaaccgggt cgcttcctgg tagcgcagtc tggcgtatta 840

attactcagg tgcggagcgt caaacaaatg gggagccgcc actttgtgct ggttgatgcc 900

gggttcaacg atctgatgcg cccggcaatg tacggtagtt accaccatat cagtgccctg 960

gcagctgatg gtcgttctct ggaacacgcg ccaacggtgg aaaccgtcgt cgccggaccg 1020

ttatgtgaat cgggcgatgt ctttacccag caggaagggg gaaatgttga aacccgcgcc 1080

ttgccggaag tgaaggcagg tgattatctg gtactgcatg atacaggggc atatggcgca 1140

tcaatgtcat ccaactacaa tagccgtccg ctgttaccag aagttctgtt tgataatggt 1200

caggcgcggt tgattcgccg tcgccagacc atcgaagaat tactggcgct ggaattgctt 1260

taa 1263

<210> 35

<211> 2148

<212> DNA

<213> (Gene cadA encoding lysine decarboxylase)

<400> 35

atgaacgtta ttgcaatatt gaatcacatg ggggtttatt ttaaagaaga acccatccgt 60

gaacttcatc gcgcgcttga acgtctgaac ttccagattg tttacccgaa cgaccgtgac 120

gacttattaa aactgatcga aaacaatgcg cgtctgtgcg gcgttatttt tgactgggat 180

aaatataatc tcgagctgtg cgaagaaatt agcaaaatga acgagaacct gccgttgtac 240

gcgttcgcta atacgtattc cactctcgat gtaagcctga atgacctgcg tttacagatt 300

agcttctttg aatatgcgct gggtgctgct gaagatattg ctaataagat caagcagacc 360

actgacgaat atatcaacac tattctgcct ccgctgacta aagcactgtt taaatatgtt 420

cgtgaaggta aatatacttt ctgtactcct ggtcacatgg gcggtactgc attccagaaa 480

agcccggtag gtagcctgtt ctatgatttc tttggtccga ataccatgaa atctgatatt 540

tccatttcag tatctgaact gggttctctg ctggatcaca gtggtccaca caaagaagca 600

gaacagtata tcgctcgcgt ctttaacgca gaccgcagct acatggtgac caacggtact 660

tccactgcga acaaaattgt tggtatgtac tctgctccag caggcagcac cattctgatt 720

gaccgtaact gccacaaatc gctgacccac ctgatgatga tgagcgatgt tacgccaatc 780

tatttccgcc cgacccgtaa cgcttacggt attcttggtg gtatcccaca gagtgaattc 840

cagcacgcta ccattgctaa gcgcgtgaaa gaaacaccaa acgcaacctg gccggtacat 900

gctgtaatta ccaactctac ctatgatggt ctgctgtaca acaccgactt catcaagaaa 960

acactggatg tgaaatccat ccactttgac tccgcgtggg tgccttacac caacttctca 1020

ccgatttacg aaggtaaatg cggtatgagc ggtggccgtg tagaagggaa agtgatttac 1080

gaaacccagt ccactcacaa actgctggcg gcgttctctc aggcttccat gatccacgtt 1140

aaaggtgacg taaacgaaga aacctttaac gaagcctaca tgatgcacac caccacttct 1200

ccgcactacg gtatcgtggc gtccactgaa accgctgcgg cgatgatgaa aggcaatgca 1260

ggtaagcgtc tgatcaacgg ttctattgaa cgtgcgatca aattccgtaa agagatcaaa 1320

cgtctgagaa cggaatctga tggctggttc tttgatgtat ggcagccgga tcatatcgat 1380

acgactgaat gctggccgct gcgttctgac agcacctggc acggcttcaa aaacatcgat 1440

aacgagcaca tgtatcttga cccgatcaaa gtcaccctgc tgactccggg gatggaaaaa 1500

gacggcacca tgagcgactt tggtattccg gccagcatcg tggcgaaata cctcgacgaa 1560

catggcatcg ttgttgagaa aaccggtccg tataacctgc tgttcctgtt cagcatcggt 1620

atcgataaga ccaaagcact gagcctgctg cgtgctctga ctgactttaa acgtgcgttc 1680

gacctgaacc tgcgtgtgaa aaacatgctg ccgtctctgt atcgtgaaga tcctgaattc 1740

tatgaaaaca tgcgtattca ggaactggct cagaatatcc acaaactgat tgttcaccac 1800

aatctgccgg atctgatgta tcgcgcattt gaagtgctgc cgacgatggt aatgactccg 1860

tatgctgcat tccagaaaga gctgcacggt atgaccgaag aagtttacct cgacgaaatg 1920

gtaggtcgta ttaacgccaa tatgatcctt ccgtacccgc cgggagttcc tctggtaatg 1980

ccgggtgaaa tgatcaccga agaaagccgt ccggttctgg agttcctgca gatgctgtgt 2040

gaaatcggcg ctcactatcc gggctttgaa accgatattc acggtgcata ccgtcaggct 2100

gatggccgct ataccgttaa ggtattgaaa gaagaaagca aaaaataa 2148

<210> 36

<211> 2142

<212> DNA

<213> (Gene coding for lysine decarboxylase ldcC)

<400> 36

atgaacatca ttgccattat gggaccgcat ggcgtctttt ataaagatga gcccatcaaa 60

gaactggagt cggcgctggt ggcgcaaggc tttcagatta tctggccaca aaacagcgtt 120

gatttgctga aatttatcga gcataaccct cgaatttgcg gcgtgatttt tgactgggat 180

gagtacagtc tcgatttatg tagcgatatc aatcagctta atgaatatct cccgctttat 240

gccttcatca acacccactc gacgatggat gtcagcgtgc aggatatgcg gatggcgctc 300

tggttttttg aatatgcgct ggggcaggcg gaagatatcg ccattcgtat gcgtcagtac 360

accgacgaat atcttgataa cattacaccg ccgttcacga aagccttgtt tacctacgtc 420

aaagagcgga agtacacctt ttgtacgccg gggcatatgg gcggcaccgc atatcaaaaa 480

agcccggttg gctgtctgtt ttatgatttt ttcggcggga atactcttaa ggctgatgtc 540

tctatttcgg tcaccgagct tggttcgttg ctcgaccaca ccgggccaca cctggaagcg 600

gaagagtaca tcgcgcggac ttttggcgcg gaacagagtt atatcgttac caacggaaca 660

tcgacgtcga acaaaattgt gggtatgtac gccgcgccat ccggcagtac gctgttgatc 720

gaccgcaatt gtcataaatc gctggcgcat ctgttgatga tgaacgatgt agtgccagtc 780

tggctgaaac cgacgcgtaa tgcgttgggg attcttggtg ggatcccgcg ccgtgaattt 840

actcgcgaca gcatcgaaga gaaagtcgct gctaccacgc aagcacaatg gccggttcat 900

gcggtgatca ccaactccac ctatgatggc ttgctctaca acaccgactg gatcaaacag 960

acgctggatg tcccgtcgat tcacttcgat tctgcctggg tgccgtacac ccattttcat 1020

ccgatctacc agggtaaaag tggtatgagc ggcgagcgtg ttgcgggaaa agtgatcttc 1080

gaaacgcaat cgacccacaa aatgctggcg gcgttatcgc aggcttcgct gatccacatt 1140

aaaggcgagt atgacgaaga ggcctttaac gaagccttta tgatgcatac caccacctcg 1200

cccagttatc ccattgttgc ttcggttgag acggcggcgg cgatgctgcg tggtaatccg 1260

ggcaaacggc tgattaaccg ttcagtagaa cgagctctgc attttcgcaa agaggtccag 1320

cggctgcggg aagagtctga cggttggttt ttcgatatct ggcaaccgcc gcaggtggat 1380

gaagccgaat gctggcccgt tgcgcctggc gaacagtggc acggctttaa cgatgcggat 1440

gccgatcata tgtttctcga tccggttaaa gtcactattt tgacaccggg gatggacgag 1500

cagggcaata tgagcgagga ggggatcccg gcggcgctgg tagcaaaatt cctcgacgaa 1560

cgtgggatcg tagtagagaa aaccggccct tataacctgc tgtttctctt tagtattggc 1620

atcgataaaa ccaaagcaat gggattattg cgtgggttga cggaattcaa acgctcttac 1680

gatctcaacc tgcggatcaa aaatatgcta cccgatctct atgcagaaga tcccgatttc 1740

taccgcaata tgcgtattca ggatctggca caagggatcc ataagctgat tcgtaaacac 1800

gatcttcccg gtttgatgtt gcgggcattc gatactttgc cggagatgat catgacgcca 1860

catcaggcat ggcaacgaca aattaaaggc gaagtagaaa ccattgcgct ggaacaactg 1920

gtcggtagag tatcggcaaa tatgatcctg ccttatccac cgggcgtacc gctgttgatg 1980

cctggagaaa tgctgaccaa agagagccgc acagtactcg attttctact gatgctttgt 2040

tccgtcgggc aacattaccc cggttttgaa acggatattc acggcgcgaa acaggacgaa 2100

gacggcgttt accgcgtacg agtcctaaaa atggcgggat aa 2142

<210> 37

<211> 990

<212> DNA

<213> (Gene ldh encoding lactate dehydrogenase)

<400> 37

atgaaactcg ccgtttatag cacaaaacag tacgacaaga agtacctgca acaggtgaac 60

gagtcctttg gctttgagct ggaatttttt gactttctgc tgacggaaaa aaccgctaaa 120

actgccaatg gctgcgaagc ggtatgtatt ttcgtaaacg atgacggcag ccgcccggtg 180

ctggaagagc tgaaaaagca cggcgttaaa tatatcgccc tgcgctgtgc cggtttcaat 240

aacgtcgacc ttgacgcggc aaaagaactg gggctgaaag tagtccgtgt tccagcctat 300

gatccagagg ccgttgctga acacgccatc ggtatgatga tgacgctgaa ccgccgtatt 360

caccgcgcgt atcagcgtac ccgtgatgct aacttctctc tggaaggtct gaccggcttt 420

actatgtatg gcaaaacggc aggcgttatc ggtaccggta aaatcggtgt ggcgatgctg 480

cgcattctga aaggttttgg tatgcgtctg ctggcgttcg atccgtatcc aagtgcagcg 540

gcgctggaac tcggtgtgga gtatgtcgat ctgccaaccc tgttctctga atcagacgtt 600

atctctctgc actgcccgct gacaccggaa aactatcatc tgttgaacga agccgccttc 660

gaacagatga aaaatggcgt gatgatcgtc aataccagtc gcggtgcatt gattgattct 720

caggcagcaa ttgaagcgct gaaaaatcag aaaattggtt cgttgggtat ggacgtgtat 780

gagaacgaac gcgatctatt ctttgaagat aaatccaacg acgtgatcca ggatgacgta 840

ttccgtcgcc tgtctgcctg ccacaacgtg ctgtttaccg ggcaccaggc attcctgaca 900

gcagaagctc tgaccagtat ttctcagact acgctgcaaa acttaagcaa tctggaaaaa 960

ggcgaaacct gcccgaacga actggtttaa 990

<210> 38

<211> 2130

<212> DNA

<213> (Gene pta encoding Phosphoacetyltransferase)

<400> 38

atgctgatcc ctaccggaac cagcgtcggt ctgaccagcg tcagccttgg cgtgatccgt 60

gcaatggaac gcaaaggcgt tcgtctgagc gttttcaaac ctatcgctca gccgcgtacc 120

ggtggcgatg cgcccgatca gactacgact atcgtgcgtg cgaactcttc caccacgacg 180

gccgctgaac cgctgaaaat gagctacgtt gaaggtctgc tttccagcaa tcagaaagat 240

gtgctgatgg aagagatcgt cgcaaactac cacgctaaca ccaaagacgc tgaagtcgtt 300

ctggttgaag gtctggtccc gacacgtaag caccagtttg cccagtctct gaactacgaa 360

atcgctaaaa cgctgaatgc ggaaatcgtc ttcgttatgt ctcagggcac tgacaccccg 420

gaacagctga aagagcgtat cgaactgacc cgcaacagct tcggcggtgc caaaaacacc 480

aacatcaccg gcgttatcgt taacaaactg aacgcaccgg ttgatgaaca gggtcgtact 540

cgcccggatc tgtccgagat tttcgacgac tcttccaaag ctaaagtaaa caatgttgat 600

ccggcgaagc tgcaagaatc cagcccgctg ccggttctcg gcgctgtgcc gtggagcttt 660

gacctgatcg cgactcgtgc gatcgatatg gctcgccacc tgaatgcgac catcatcaac 720

gaaggcgaca tcaatactcg ccgcgttaaa tccgtcactt tctgcgcacg cagcattccg 780

cacatgctgg agcacttccg tgccggttct ctgctggtga cttccgcaga ccgtcctgac 840

gtgctggtgg ccgcttgcct ggcagccatg aacggcgtag aaatcggtgc cctgctgctg 900

actggcggtt acgaaatgga cgcgcgcatt tctaaactgt gcgaacgtgc tttcgctacc 960

ggcctgccgg tatttatggt gaacaccaac acctggcaga cctctctgag cctgcagagc 1020

ttcaacctgg aagttccggt tgacgatcac gaacgtatcg agaaagttca ggaatacgtt 1080

gctaactaca tcaacgctga ctggatcgaa tctctgactg ccacttctga gcgcagccgt 1140

cgtctgtctc cgcctgcgtt ccgttatcag ctgactgaac ttgcgcgcaa agcgggcaaa 1200

cgtatcgtac tgccggaagg tgacgaaccg cgtaccgtta aagcagccgc tatctgtgct 1260

gaacgtggta tcgcaacttg cgtactgctg ggtaatccgg cagagatcaa ccgtgttgca 1320

gcgtctcagg gtgtagaact gggtgcaggg attgaaatcg ttgatccaga agtggttcgc 1380

gaaagctatg ttggtcgtct ggtcgaactg cgtaagaaca aaggcatgac cgaaaccgtt 1440

gcccgcgaac agctggaaga caacgtggtg ctcggtacgc tgatgctgga acaggatgaa 1500

gttgatggtc tggtttccgg tgctgttcac actaccgcaa acaccatccg tccgccgctg 1560

cagctgatca aaactgcacc gggcagctcc ctggtatctt ccgtgttctt catgctgctg 1620

ccggaacagg tttacgttta cggtgactgt gcgatcaacc cggatccgac cgctgaacag 1680

ctggcagaaa tcgcgattca gtccgctgat tccgctgcgg ccttcggtat cgaaccgcgc 1740

gttgctatgc tctcctactc caccggtact tctggtgcag gtagcgacgt agaaaaagtt 1800

cgcgaagcaa ctcgtctggc gcaggaaaaa cgtcctgacc tgatgatcga cggtccgctg 1860

cagtacgacg ctgcggtaat ggctgacgtt gcgaaatcca aagcgccgaa ctctccggtt 1920

gcaggtcgcg ctaccgtgtt catcttcccg gatctgaaca ccggtaacac cacctacaaa 1980

gcggtacagc gttctgccga cctgatctcc atcgggccga tgctgcaggg tatgcgcaag 2040

ccggttaacg acctgtcccg tggcgcactg gttgacgata tcgtctacac catcgcgctg 2100

actgcgattc agtctgcaca gcagcagtaa 2130

<210> 39

<211> 1203

<212> DNA

<213> (Gene encoding acetate kinase, ackA)

<400> 39

atgtcgagta agttagtact ggttctgaac tgcggtagtt cttcactgaa atttgccatc 60

atcgatgcag taaatggtga agagtacctt tctggtttag ccgaatgttt ccacctgccc 120

gaagcacgta tcaaatggaa aatggacggc aataaacagg aagcggcttt aggtgcaggc 180

gccgctcaca gcgaagcgct caactttatc gttaatacta ttctggcaca aaaaccagaa 240

ctgtctgcgc agctgactgc tatcggtcac cgtatcgtac acggcggcga aaagtatacc 300

agctccgtag tgatcgatga gtctgttatt cagggtatca aagatgcagc ttcttttgca 360

ccgctgcaca acccggctca cctgatcggt atcgaagaag ctctgaaatc tttcccacag 420

ctgaaagaca aaaacgttgc tgtatttgac accgcgttcc accagactat gccggaagag 480

tcttacctct acgccctgcc ttacaacctg tacaaagagc acggcatccg tcgttacggc 540

gcgcacggca ccagccactt ctatgtaacc caggaagcgg caaaaatgct gaacaaaccg 600

gtagaagaac tgaacatcat cacctgccac ctgggcaacg gtggttccgt ttctgctatc 660

cgcaacggta aatgcgttga cacctctatg ggcctgaccc cgctggaagg tctggtcatg 720

ggtacccgtt ctggtgatat cgatccggcg atcatcttcc acctgcacga caccctgggc 780

atgagcgttg acgcaatcaa caaactgctg accaaagagt ctggcctgct gggtctgacc 840

gaagtgacca gcgactgccg ctatgttgaa gacaactacg cgacgaaaga agacgcgaag 900

cgcgcaatgg acgtttactg ccaccgcctg gcgaaataca tcggtgccta cactgcgctg 960

atggatggtc gtctggacgc tgttgtattc actggtggta tcggtgaaaa tgccgcaatg 1020

gttcgtgaac tgtctctggg caaactgggc gtgctgggct ttgaagttga tcatgaacgc 1080

aacctggctg cacgtttcgg caaatctggt ttcatcaaca aagaaggtac ccgtcctgcg 1140

gtggttatcc caaccaacga agaactggtt atcgcgcaag acgcgagccg cctgactgcc 1200

tga 1203

<210> 40

<211> 1719

<212> DNA

<213> (Gene poxB encoding pyruvate dehydrogenase)

<400> 40

atgaaacaaa cggttgcagc ttatatcgcc aaaacactcg aatcggcagg ggtgaaacgc 60

atctggggag tcacaggcga ctctctgaac ggtcttagtg acagtcttaa tcgcatgggc 120

accatcgagt ggatgtccac ccgccacgaa gaagtggcgg cctttgccgc tggcgctgaa 180

gcacaactta gcggagaact ggcggtctgc gccggatcgt gcggccccgg caacctgcac 240

ttaatcaacg gcctgttcga ttgccaccgc aatcacgttc cggtactggc gattgccgct 300

catattccct ccagcgaaat tggcagcggc tatttccagg aaacccaccc acaagagcta 360

ttccgcgaat gtagtcacta ttgcgagctg gtttccagcc cggagcagat cccacaagta 420

ctggcgattg ccatgcgcaa agcggtgctt aaccgtggcg tttcggttgt cgtgttacca 480

ggcgacgtgg cgttaaaacc tgcgccagaa ggggcaacca tgcactggta tcatgcgcca 540

caaccagtcg tgacgccgga agaagaagag ttacgcaaac tggcgcaact gctgcgttat 600

tccagcaata tcgccctgat gtgtggcagc ggctgcgcgg gggcgcataa agagttagtt 660

gagtttgccg ggaaaattaa agcgcctatt gttcatgccc tgcgcggtaa agaacatgtc 720

gaatacgata atccgtatga tgttggaatg accgggttaa tcggcttctc gtcaggtttc 780

cataccatga tgaacgccga cacgttagtg ctactcggca cgcaatttcc ctaccgcgcc 840

ttctacccga ccgatgccaa aatcattcag attgatatca acccagccag catcggcgct 900

cacagcaagg tggatatggc actggtcggc gatatcaagt cgactctgcg tgcattgctt 960

ccattggtgg aagaaaaagc cgatcgcaag tttctggata aagcgctgga agattaccgc 1020

gacgcccgca aagggctgga cgatttagct aaaccgagcg agaaagccat tcacccgcaa 1080

tatctggcgc agcaaattag tcattttgcc gccgatgacg ctattttcac ctgtgacgtt 1140

ggtacgccaa cggtgtgggc ggcacgttat ctaaaaatga acggcaagcg tcgcctgtta 1200

ggttcgttta accacggttc gatggctaac gccatgccgc aggcgctggg tgcgcaggcg 1260

acagagccag aacgtcaggt ggtcgccatg tgcggcgatg gcggttttag catgttgatg 1320

ggcgatttcc tctcagtagt gcagatgaaa ctgccagtga aaattgtcgt ctttaacaac 1380

agcgtgctgg gctttgtggc gatggagatg aaagctggtg gctatttgac tgacggcacc 1440

gaactacacg acacaaactt tgcccgcatt gccgaagcgt gcggcattac gggtatccgt 1500

gtagaaaaag cgtctgaagt tgatgaagcc ctgcaacgcg ccttctccat cgacggtccg 1560

gtgttggtgg atgtggtggt cgccaaagaa gagttagcca ttccaccgca gatcaaactc 1620

gaacaggcca aaggtttcag cctgtatatg ctgcgcgcaa tcatcagcgg acgcggtgat 1680

gaagtgatcg aactggcgaa aacaaactgg ctaaggtaa 1719

Claims

1. A genetically engineered bacterium producing hydroxyadipic acid, which is a recombinant host bacterium comprising a gene encoding lysine dehydrogenase, a gene encoding aminoadipate semialdehyde dehydrogenase, a gene encoding aminoadipate transaminase, and a gene encoding ketoadipate reductase.

2. The genetically engineered bacterium of claim 1, wherein the gene encoding lysine dehydrogenase is selected from the group consisting of a lysine dehydrogenase-encoding gene lysDH-Am from Bacillus 12AMOR1 or a codon-optimized lysine dehydrogenase-encoding gene lysDH-Am from Bacillus 12AMOR1, a lysine dehydrogenase-encoding gene lysDH-Bt from Bacillus thermophilus 1A1 or a codon-optimized lysine dehydrogenase-encoding gene lysDH-Bt from Bacillus thermophilus 1A1, a lysine dehydrogenase-encoding gene lysDH-Ab from acidic bacterium ADurb.Bin051 or a codon-optimized lysine dehydrogenase-encoding gene lysDH-Ab from acidic bacterium ADurb.051Bin141, a lysine dehydrogenase-encoding gene lysDH-Bb from Bacteroides ADurb.Bin 141 or a codon-optimized lysine dehydrogenase-encoding gene lysDH-Bb from thermophilic bacterium ADurb.Bin 141, preferably the codon-optimized lysine dehydrogenase-encoding gene lysDH-Bt 1.

3. The genetically engineered bacterium of claim 1, wherein the gene encoding aminoadipate semialdehyde dehydrogenase is selected from the group consisting of a gene Psefu _1272 encoding aminoadipate semialdehyde dehydrogenase derived from pseudomonas 12-X or a gene Psefu _1272 encoding aminoadipate semialdehyde dehydrogenase derived from pseudomonas 12-X and codon-optimized, a gene ALDH7A1 encoding aminoadipate semialdehyde dehydrogenase derived from human or a gene ALDH7A1 encoding aminoadipate semialdehyde dehydrogenase derived from human and codon-optimized, PMI27 encoding aminoadipate semialdehyde dehydrogenase derived from pseudomonas GM41 (2012), or a gene PMI27 encoding aminoadipate semialdehyde dehydrogenase derived from pseudomonas GM41 (2012) and codon-optimized, preferably a gene Psefu _1272 encoding aminoadipate semialdehyde dehydrogenase derived from pseudomonas 12-X and codon-optimized.

4. The genetically engineered bacterium of claim 1, wherein the gene encoding an aminoadipate aminotransferase is selected from the group consisting of an aminoadipate aminotransferase-encoding gene ARO8 from saccharomyces cerevisiae S288c or a codon-optimized aminoadipate aminotransferase-encoding gene ARO8 from saccharomyces cerevisiae S288c, an aminoadipate aminotransferase-encoding gene lysN from thermus thermophilus HB27 or a codon-optimized aminoadipate aminotransferase-encoding gene lysN from thermus thermophilus HB27, preferably from saccharomyces cerevisiae S288c and a codon-optimized aminoadipate aminotransferase-encoding gene ARO8.

5. The genetically engineered bacterium of claim 1, wherein the gene encoding ketoadipate reductase is selected from the group consisting of a gene AeLDH encoding ketoadipate reductase derived from chlorella vulgaris H16 that is enriched or a gene AeLDH encoding ketoadipate reductase derived from chlorella vulgaris H16 that is enriched and codon-optimized, a gene HgdH encoding ketoadipate reductase derived from zymococcus amino acids that is fermented, or a gene HgdH encoding ketoadipate reductase derived from zymococcus amino acids that is codon-optimized, preferably a gene AeLDH encoding ketoadipate reductase derived from chlorella vulgaris H16 that is enriched and codon-optimized.

6. The genetically engineered bacterium of claims 1-5, wherein the genetically engineered bacterium is a genetically engineered bacterium that is modified by a chassis microorganism to produce hydroxyadipic acid; preferably, the chassis microbial engineering comprises the intensification of precursor synthetic pathways and the knock-out or attenuation of genes associated with competing metabolic pathways.

7. The genetically engineered bacterium of claim 6,

the strengthening of the precursor synthesis pathway comprises over-expressing key genes of the precursor synthesis pathway in genetically engineered bacteria; preferably, the key genes of the precursor synthesis pathway include a gene aspC encoding aspartate aminotransferase, a gene lysC encoding aspartate kinase, a gene dapA encoding dihydrodipicolinate synthase, and a gene lysA encoding diaminopimelate decarboxylase;

and/or, the competing metabolic pathway-related genes include lysine degradation pathway-related genes, lactate pathway-related genes, and acetate pathway-related genes; preferably, the lysine degradation pathway-related genes include genes encoding lysine decarboxylase cadA and ldcC; and/or, the lactate pathway-associated gene comprises a gene ldh encoding lactate dehydrogenase; and/or, the acetate pathway-associated genes include a gene pta encoding phosphoacetyltransferase, a gene ackA encoding acetate kinase, and a gene poxB encoding pyruvate dehydrogenase.

8. The genetically engineered bacterium of any one of claims 1 to 7, wherein the host bacterium comprises Escherichia coli, corynebacterium glutamicum, yeast, and engineered bacteria, fungi; preferably, the host bacterium is escherichia coli; further preferably, the host bacterium is Escherichia coli JM109 (DE 3).

9. The application of the genetically engineered bacterium of any one of 1 to 8 in producing hydroxy adipic acid; preferably, the application comprises the steps of inoculating the genetic engineering bacteria for producing the hydroxyadipic acid into a fermentation culture medium, carrying out fermentation culture, and then separating and purifying the obtained fermentation culture solution to prepare the hydroxyadipic acid; further preferably, the fermentation culture conditions are: the fermentation culture time is 72h, when the thallus grows to OD₆₀₀When the concentration is 0.6-0.8, adding IPTG, and after adding the IPTG, changing the fermentation temperature from 37 ℃ to 30 ℃ and controlling the IPTG induction concentration to be 0.8-1.2mM; particularly preferably, lysine is added in vitro, and the amount of lysine added is 2 to 10g/L, more particularly preferably 5 to 10g/L.

10. The use according to claim 9, wherein the isolation and purification of the obtained fermentation broth comprises:

step S2, diluting the supernatant fluid I by 20 times with ultrapure water, and uniformly mixing to obtain a solution A; diluting the solution A by 10 times by using methanol containing 0.1% formic acid, uniformly mixing, and performing centrifugal separation for the second time to obtain supernatant of the second time;