CN112280722B

CN112280722B - Recombinant bacterium for producing optically pure 1, 3-butanediol and application thereof

Info

Publication number: CN112280722B
Application number: CN201910664906.6A
Authority: CN
Inventors: 陈振; 刘德华; 刘煜
Original assignee: Guangdong Tsinghua Smart Biotech Co ltd; Tsinghua University
Current assignee: Guangdong Tsinghua Smart Biotech Co ltd; Tsinghua University
Priority date: 2019-07-23
Filing date: 2019-07-23
Publication date: 2022-03-08
Anticipated expiration: 2039-07-23
Also published as: CN112280722A

Abstract

The invention provides a recombinant bacterium for producing optically pure 1, 3-butanediol and application thereof. The recombinant strain is formed by introducing phaA, phaB, bld and yqhD genes into a microorganism through plasmids or integrating the genes on a microorganism chromosome through a genetic engineering means. The escherichia coli engineering bacteria are used for fermenting cheap organic carbon sources such as glucose, sucrose or glycerol under the condition of micro-oxygen, the yield of the 1, 3-butanediol relative to a substrate can reach more than 0.4g/g, the purity of the R-type 1, 3-butanediol reaches more than 99 percent, and the escherichia coli engineering bacteria have important industrial application potential.

Description

Recombinant bacterium for producing optically pure 1, 3-butanediol and application thereof

Technical Field

The invention belongs to the technical field of biochemical engineering, and particularly relates to a recombinant bacterium for producing optically pure 1, 3-butanediol and application thereof.

Background

1, 3-butanediol is a diol of great industrial applicability and can be used as a solvent for cosmetics and at the same time as a monomer for the synthesis of polyesters, polyurethanes and bioplasticizers. Meanwhile, the optically pure 1, 3-butanediol can be used as a medical intermediate to synthesize a plurality of medicines. However, 1, 3-butanediol synthesized by the chemical method is a racemate and cannot be directly used as a medical intermediate. Therefore, the biological method for producing the optically pure 1, 3-butanediol has important application value.

Cn201610481598.x and cn201080029715.x disclose two methods for the production of 1, 3-butanediol, respectively, essentially expressing at least one exogenous nucleic acid encoding a 1,3-BDO pathway enzyme expressed in a sufficient amount to produce 1, 3-butanediol under anaerobic conditions. However, the process has low production efficiency, and the highest yield of 1, 3-butanediol is lower than 2mM, so that the process is not industrially feasible.

Disclosure of Invention

The invention aims to provide a recombinant bacterium for producing optically pure 1, 3-butanediol and application thereof.

The invention has the following conception: provided is a recombinant E.coli which can greatly increase the intracellular NADPH gene to satisfy the NADPH reducing power required for the synthesis pathway of 1, 3-butanediol by up-regulating the pntAB gene of E.coli and simultaneously down-regulating the sthA gene, thereby increasing the yield of 1, 3-butanediol. The invention constructs butyraldehyde dehydrogenase mutant of Clostridium saccharoperbutylacetonicum by enzyme design, which is formed by mutating leucine at 273 bit of butyraldehyde dehydrogenase into threonine, and the mutant can greatly improve the yield of 1, 3-butanediol by expressing in escherichia coli. According to the invention, the adhE gene, ldhA gene, pta gene and ackA gene are knocked out, so that the metabolic flux of acetyl-CoA flowing to a 1, 3-butanediol synthesis pathway is improved, and the yield of 1, 3-butanediol is further improved.

In order to achieve the object of the present invention, in a first aspect, the present invention provides a recombinant bacterium for producing optically pure 1, 3-butanediol, which is obtained by introducing phaA, phaB, bld and yqhD genes into a microorganism through a plasmid or integrating the genes into a chromosome of the microorganism through genetic engineering means.

Wherein the phaA gene is acetyl CoA acyltransferase gene, is derived from cupriavidius necator, and is a gene coding the following protein (a) or (b):

(a) a protein consisting of the amino acid sequence shown in SEQ ID NO. 3;

(b) 3, protein which is derived from (a) and has the same function by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 3.

The phaB gene is a 3-oxoacyl- (acyl carrier protein) reductase gene derived from cupriavidius necator, which is a gene encoding the following protein (c) or (d):

(c) a protein consisting of an amino acid sequence shown as SEQ ID NO. 4;

(d) 4, protein which is derived from (c) and has the same function by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 4.

The bld gene is derived from Clostridium saccharoperbutylacetonicum, and the amino acid sequence of the coded protein is shown as SEQ ID NO. 1 or 2.

The yqhD gene is an alcohol dehydrogenase gene, is derived from Escherichia coli (Escherichia coli), and is a gene encoding the following protein (e) or (f):

(e) a protein consisting of an amino acid sequence shown as SEQ ID NO. 6;

(f) and (b) protein which is derived from (e) and has the same function and is obtained by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 6.

The microorganism is selected from the species in the genus Escherichia (Escherichia), Klebsiella (Klebsiella), Corynebacterium (Corynebacterium), Brevibacterium (Brevibacterium), etc.; escherichia coli is preferred.

The recombinant strain can be constructed by the following method: after the phaA and phaB genes are optimized by a codon, the genes are constructed on an expression vector together with bld and yqhD genes, the recombinant vector is used for transforming escherichia coli, and positive transformants are screened.

Preferably, the sequence of the codon-optimized phaA-phaB operon is shown in SEQ ID NO 5.

More preferably, the recombinant bacterium is constructed as follows: the phaAB-bld2-yqhD tandem gene expression cassette is constructed on ptrc99a plasmid, and Escherichia coli (such as W3110) is transformed with the recombinant plasmid, and positive transformants are selected.

Wherein, the sequences of the bld-yqhD-phaAB tandem gene expression cassettes are shown in SEQ ID NO. 9 and 10.

In a second aspect, the invention provides a recombinant escherichia coli for producing optically pure 1, 3-butanediol, wherein the recombinant escherichia coli is an engineering bacterium with enhanced intracellular NADPH supply, which is obtained by taking the recombinant bacterium as an initial strain and modifying the initial strain by using a genetic engineering means. The method comprises the following scheme:

scheme I: the supply of intracellular NADPH is increased by enhancing genes involved in the NADPH biosynthetic pathway in the starting strain.

Preferably, the gene associated with NADPH biosynthetic pathway is pntAB gene, and the nucleotide sequence is as follows:

i) the nucleotide sequence shown as SEQ ID NO. 7;

ii) a nucleotide sequence which is obtained by substituting, deleting and/or adding one or more nucleotides into the nucleotide sequence shown in SEQ ID NO. 7 and expresses the same functional protein;

iii) a nucleotide sequence which hybridizes with the sequence shown in SEQ ID NO. 7 under stringent conditions in a 0.1 XSSPE containing 0.1% SDS or a 0.1 XSSC solution containing 0.1% SDS at 65 ℃ and washing the membrane with the solution and expresses the same functional protein; or

iv) a nucleotide sequence which has more than 90% homology with the nucleotide sequence of i), ii) or iii) and expresses the same functional protein.

In the present invention, the enhanced pathway is selected from the following 1) to 6), or an optional combination:

1) enhanced by introduction of a plasmid having the gene;

2) enhanced by increasing the copy number of the gene on the chromosome;

3) enhanced by altering the promoter sequence of the gene on the chromosome;

4) enhanced by operably linking a strong promoter to the gene;

5) enhanced by the introduction of enhancers;

6) enhanced by using genes or alleles having the corresponding enzymes or proteins which encode high activities.

Scheme II: weakening sthA gene in original strain to increase supply of NADPH in cell;

the nucleotide sequence of sthA gene is as follows:

i) the nucleotide sequence shown as SEQ ID NO. 8;

ii) a nucleotide sequence which is obtained by substituting, deleting and/or adding one or more nucleotides into the nucleotide sequence shown in SEQ ID NO. 8 and expresses the same functional protein;

iii) a nucleotide sequence which hybridizes to the sequence shown in SEQ ID NO. 8 under stringent conditions in which hybridization is performed at 65 ℃ in a 0.1 XSSPE containing 0.1% SDS or a 0.1 XSSC containing 0.1% SDS solution and the membrane is washed with the solution and expresses the same functional protein; or

In the present invention, the attenuation includes knocking out or reducing the expression of the gene.

Scheme III: the intracellular supply of NADPH was increased by enhancing the pntAB gene in the starting strain (E.coli) and attenuating the sthA gene.

In a third aspect, the invention provides an engineering bacterium for high yield of 1, 3-butanediol, wherein the engineering bacterium is an engineering bacterium obtained by further modifying an original strain by using the recombinant bacterium or the recombinant escherichia coli as the original strain by using a genetic engineering means and enhancing the metabolic flux of intracellular acetyl CoA flowing to a 1, 3-butanediol synthesis pathway.

Preferably, at least one of the adhE, ldhA, pta, ackA genes in the original strain is attenuated to increase the metabolic flux of intracellular acetyl CoA towards the 1, 3-butanediol synthesis pathway; the attenuation includes knocking out or reducing expression of the gene.

The amino acid sequences of the proteins coded by the adhE, ldhA, pta and ackA genes are respectively shown in SEQ ID NO. 11-14.

Preferably, the adhE, ldhA, pta and ackA genes in the original strain (E.coli) are attenuated to increase the metabolic flux of intracellular acetyl CoA to the 1, 3-butanediol synthesis pathway.

In a fourth aspect, the invention provides a butyraldehyde dehydrogenase mutant, which is obtained by mutating the 273 rd leucine of butyraldehyde dehydrogenase (SEQ ID NO:1) into threonine, wherein the amino acid sequence of the mutant is shown as SEQ ID NO: 2.

In a fifth aspect, the invention provides a biological material containing the butyraldehyde dehydrogenase mutant encoding gene, wherein the biological material comprises recombinant DNA, an expression cassette, a transposon, a plasmid vector, a phage vector, a viral vector or an engineering bacterium.

In a sixth aspect, the invention provides the use of the above mentioned biological material in the microbial fermentation production of 1, 3-butanediol.

In a seventh aspect, the invention provides the recombinant bacterium, the recombinant escherichia coli, or the engineering bacterium for use in fermentation production of 1, 3-butanediol.

In an eighth aspect, the present invention provides a method for producing optically pure 1, 3-butanediol, comprising culturing the above recombinant bacterium, or the above recombinant Escherichia coli, or the above engineered bacterium in a fermentation medium to produce 1, 3-butanediol.

Optionally, the fermentation medium is M9Y: glucose 20g/L, Na₂HPO₄ 6g/L，KH₂PO₄ 3g/L，NaCl 0.5g/L，NH₄Cl 1g/L，MgSO₄ 0.5g/L,CaCl₂15mg/L, 2g/L yeast powder, 100mg/L ampicillin and water.

The culture conditions were: the dissolved oxygen was controlled to be less than 10% of the saturated dissolved oxygen value at 30 ℃ and 200 rpm.

The escherichia coli engineering bacteria are used for fermenting cheap organic carbon sources such as glucose, sucrose or glycerol under the micro-aerobic condition, the yield of the 1, 3-butanediol relative to a substrate can reach more than 0.4g/g, the purity of the R-type 1, 3-butanediol reaches more than 99 percent, and the escherichia coli engineering bacteria have important industrial application potential.

Drawings

FIG. 1 is a map of plasmid ptrc99a-bld2-yqhD-phaAB of the present invention.

FIG. 2 is a map of plasmid ptrc99a-bld-yqhD-phaAB of the present invention.

FIG. 3 is a map of plasmid ptrc99a-pduP-yqhD-phaAB of the present invention.

FIG. 4 is a map of plasmid ptrc99a-eutE-yqhD-phaAB of the present invention.

FIG. 5 is a map of plasmid ptrc99a-bld (L273T) -yqhD-phaAB of the present invention.

FIG. 6 is a map of plasmid ptrc99a-bld (L273T) -yqhD-phaAB-pntAB according to the present invention.

Detailed Description

The specific implementation mode of the invention comprises the following steps: (1) constructing 1, 3-butanediol synthetic plasmid; (2) enhancing the supply of NADPH; (3) synthesis of knock-out by-products.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

Example 1 construction of biosynthetic pathway for 1, 3-butanediol in E.coli

The escherichia coli can not directly synthesize the 1, 3-butanediol, in order to introduce a synthesis way of the 1, 3-butanediol, the invention carries out a great deal of screening and transformation on key genes in the synthesis process of the 1, 3-butanediol, and the yield of the optimized 1, 3-butanediol expression module is greatly improved.

First, a plasmid ptrc99a-bld2-yqhD-phaAB comprising phaA and phaB genes derived from cupriavidius necator, an aldehyde dehydrogenase gene bld2 gene (gene No. Q9X681 in uniprot) derived from Clostridium beijerinckii, and a yqhD gene (SEQ ID NO:6) derived from Escherichia coli was constructed. The codon-optimized phaA-phaB operon (SEQ ID NO:5) was synthesized artificially based on the amino acid sequences of phaA (SEQ ID NO:3) and phaB (SEQ ID NO: 4). Ptrc99a (purchased from Addgene) was double-digested with EcoRI/XbaI, and the artificially synthesized phaA-phaB operon, bld2 gene, and yqhD gene were inserted between EcoRI/XbaI double-digested sites on the backbone of ptrc99a plasmid using Gibson's assembly kit, and the resulting plasmid was named ptrc99a-bld2-yqhD-phaAB (plasmid map is shown in FIG. 1).

Accordingly, ptrc99a (available from Addgene) was double-digested with EcoRI/XbaI, and the synthetic phaA-phaB operon, aldehyde dehydrogenase gene bld gene derived from Clostridium saccharoperbutylacetonicum (amino acid sequence of encoded protein is shown in SEQ ID NO:1), yqhD gene were inserted between EcoRI/XbaI double-digestion sites on the backbone of ptrc99a using Gibson assembly kit, and the resulting plasmid was named ptrc99a-bld-yqhD-phaAB (plasmid map is shown in FIG. 2).

Accordingly, ptrc99a (purchased from Addge company) was double-digested with EcoRI/XbaI, and the artificially synthesized phaA-phaB operon, the pduP gene of the aldehyde dehydrogenase gene derived from Klebsiella pneumoniae (gene No. B5XUS 2in uniprot), and the yqhD gene were inserted between EcoRI/XbaI double-digested sites on the plasmid backbone of ptrc99a using a Gibson assembly kit, and the resulting plasmid was named ptrc99a-pduP-yqhD-phaAB (plasmid map is shown in FIG. 3).

Accordingly, ptrc99a (purchased from Addge Inc.) was double-digested with EcoRI/XbaI, and the artificially synthesized phaA-phaB operon, the aldehyde dehydrogenase gene eutE gene derived from Salmonella typhimurium (gene No. P41793 in uniprot), and the yqhD gene were inserted between EcoRI/XbaI double-cleavage sites on the plasmid backbone of ptrc99a using the Gibson assembly kit, and the resulting plasmid was named ptrc99a-eutE-yqhD-phaAB (plasmid map is shown in FIG. 4).

The four plasmids are respectively transformed into Escherichia coli W3110, and the obtained recombinant bacteria are named as E.coli/ptrc99a-bld2-yqhD-phaAB, E.coli/ptrc99a-bld-yqhD-phaAB, E.coli/ptrc99a-pduP-yqhD-phaAB, and E.coli/ptrc99 a-eutE-yqhD-phaAB. The four strains were cultured in 500mL baffleless shake flasks in 200mL of medium M9Y (glucose 20g/L, Na)₂HPO₄ 6g/L，KH₂PO₄ 3g/L，NaCl 0.5g/L，NH₄Cl 1g/L，MgSO₄ 0.5g/L,CaCl₂15mg/L, 2g/L yeast powder, 100mg/L ampicillin), the culture temperature is 30 ℃, the rotation speed is 200rpm, and the OD of the bacterial liquid is₆₀₀When the concentration is 0.6, 1mM IPTG is added for induction, sampling is carried out when fermentation is carried out for 72h, and the condition that four strains produce 1, 3-butanediol is detected by high performance liquid chromatography. The results showed that only E.coli/ptrc99a-bld-yqhD-phaAB produced 0.3g/L of 1, 3-butanediol, while the other three strains did not produce 1, 3-butanediol. It is shown that the screening of key enzymes in the 1, 3-butanediol pathway is crucial for the synthesis of 1, 3-butanediol.

Further, the present invention finds that aldehyde dehydrogenase is a key limiting factor affecting the synthesis of 1, 3-butanediol. Therefore, the invention carries out a great deal of protein engineering modification, and finally discovers that the 273 site amino acid of the bld gene is mutated from leucine to threonine (SEQ ID NO:2) by using a gene mutation kit of Biorad by taking ptrc99a-bld-yqhD-phaAB as a template to obtain a plasmid ptrc99a-bld (L273T) -yqhD-phaAB (a plasmid map is shown in figure 5). The plasmid was transformed into E.coli W3110, and the resulting recombinant strain was named E.coli/ptrc99a-bld (L273T) -yqhD-phaAB. The strain is cultured in an M9Y culture medium under the same culture conditions, sampling is carried out at 72 hours, and the yield of the 1, 3-butanediol is detected by using high performance liquid chromatography. The results showed that the yield of 1, 3-butanediol of E.coli/ptrc99a-bld (L273T) -yqhD-phaAB reached 2.2 g/L.

Therefore, the invention finds that the selection of the 1, 3-butanediol synthetic pathway enzyme is crucial to the production of 1, 3-butanediol, and the 1, 3-butanediol yield is greatly improved by systematically optimizing the pathway of 1, 3-butanediol.

Example 2 increase in 1, 3-butanediol production by enhancing NADPH Synthesis

The invention creatively discovers that the overexpression of the pntAB gene and the knockout of the sthA gene of escherichia coli can obviously improve the yield of 1, 3-butanediol of cells.

The ptrc99a-bld (L273T) -yqhD-phaAB was digested simultaneously with SbfI, and the pntAB gene of E.coli (SEQ ID NO:7) was inserted into the plasmid backbone ptrc99a-bld (L273T) -yqhD-phaAB using the Gibson assembly kit, and the resulting plasmid was named ptrc99a-bld (L273T) -yqhD-phaAB-pntAB (plasmid map is shown in FIG. 6). The plasmid was transformed into E.coli W3110, and the resulting recombinant strain was named E.coli/ptrc99a-bld (L273T) -yqhD-phaAB-pntAB. The strain is cultured in an M9Y culture medium, the fermentation conditions are the same as the above, sampling is carried out at 72h, and the yield of the 1, 3-butanediol is detected by utilizing high performance liquid chromatography. The results showed that E.coli/ptrc99a-bld (L273T) -yqhD-phaAB-pntAB yields 1, 3-butanediol of 4.8 g/L. The detection of the intracellular NADPH content of the strain shows that the NADPH content of the strain is improved by 1.8 times compared with that of E.coli/ptrc99a-bld (L273T) -yqhD-phaAB.

Further, the sthA gene (SEQ ID NO:8) of E.coli W3110 was knocked out to enhance the supply of NADPH and to increase the production of 1, 3-butanediol. The specific method is characterized in that the method is implemented by taking Escherichia coli MG1655 delta sthA: : kan (purchased from Yale university CGSC E.coli mutant library, see Baba et al, Construction of Escherichia coli K-12in-frame, Single-gene knock-out variants: the Keio collection, Mol Syst biol.2006,2:2006.0008.) prepared P1 phage lysates, the lysates were mixed with fresh E.coli W3110 cultures for 30 minutes, 1ml LB medium containing 0.2M sodium citrate was added, incubated at 37 ℃ for 1 hour, plated on LB plates containing 50mg/L kanamycin resistance, and recombinant strains with resistance were selected. Further, a plasmid pCP20 (purchased from the CGSC E.coli mutant library of Yale university, see Datsenko & Wanner, One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products, Proc Natl Acad Sci USA.2000,97(12): 6640. sup. su. 6645.) carrying Flp enzyme was transferred into this strain, and the strain from which the kanamycin resistance marker was removed was obtained was designated E.coli. DELTA. sthA. The plasmid ptrc99a-bld (L273T) -yqhD-phaAB-pntAB was transformed into E.coli W3110, and the resulting recombinant strain was named E.coli. DELTA. sthA/ptrc99a-bld (L273T) -yqhD-phaAB-pntAB. The strain is cultured in an M9Y culture medium, the fermentation conditions are the same as the above, sampling is carried out at 72h, and the yield of the 1, 3-butanediol is detected by utilizing high performance liquid chromatography. The results showed that the yield of 1, 3-butanediol of E.coli. DELTA.sthA/ptrc 99a-bld (L273T) -yqhD-phaAB-pntAB reached 5.8 g/L. The detection of the intracellular NADPH content of the strain shows that the NADPH content of the strain is improved by 2.3 times compared with that of E.coli/ptrc99a-bld (L273T) -yqhD-phaAB.

Therefore, the invention finds that the content of NADPH in Escherichia coli is important for the synthesis of 1, 3-butanediol, and the yield of 1, 3-butanediol is greatly improved by strengthening the content of NADPH in the system.

Example 3 enhancing acetyl-CoA supply to increase 1, 3-butanediol production

The metabolic precursor of the 1, 3-butanediol synthetic pathway is acetyl-CoA, and the present inventors have found that the yield of 1, 3-butanediol can be further increased by reducing the flow of acetyl-CoA to other metabolic branches. The ethanol synthesis gene adhE, the lactate synthesis gene ldhA, and the acetate synthesis genes pta and ackA of E.coli Δ sthA were further knocked out by transfection with P1 phage lysate. Wherein the amino acid sequences of the proteins coded by the adhE, ldhA, pta and ackA genes are respectively shown in SEQ ID NO. 11-14. The obtained strains were designated E.coli. DELTA. sthA. DELTA. adhE, E.coli. DELTA. sthA. DELTA. ldhA, and E.coli. DELTA. sthA. DELTA. pta-ackA, respectively. Meanwhile, a strain containing the three gene mutations is constructed by a method of transfecting a P1 phage lysate and named as E. Plasmids ptrc99a-bld (L273T) -yqhD-phaAB-pntAB were transformed into these four strains, and the obtained strains were named E.coli. DELTA. sthA. DELTA. adhE/ptrc99a-bld (L273T) -yqhD-phaAB-pntAB, E.coli. DELTA. sthA. DELTA. ldhA/ptrc99a-bld (L273T) -yqhD-phaAB-pntAB, E.coli. DELTA.sthA. DELTA. pta-ackA/ptrc99a-bld (L273T) -yqhD-phaAB-pntAB, E.coli. DELTA. sthA. DELTA. adhA. DELTA. qpta-qpcA/pt 99 AB 99a-bld (L273T) -yhD-pntAB, respectively. The strain is cultured in an M9Y culture medium, the fermentation conditions are the same as the above, sampling is carried out at 72h, and the yield of the 1, 3-butanediol is detected by utilizing high performance liquid chromatography. The results showed that yields of 1, 3-butanediol for E.coli. DELTA. sthA. DELTA. adhE/ptrc99a-bld (L273T) -yqhD-phaAB-pntAB, E.coli. DELTA. sthA. DELTA. ldhA/ptrc99a-bld (L273T) -yqhD-phaAB-pntAB, E.coli. DELTA. sthA. DELTA. pta-ackA/ptrc99a-bld (L273T) -yqhD-phaAB-pntAB, E.coli. DELTA. sthA. adhA. DELTA. ldhA. pta-ackA/ptrc99a-bld (L273T) -yqhD-phaAB-pntAB reached 6.1g/L,6.2g/L,6.8g/L,8.1g/L, respectively.

The optical chromatography detection shows that the 1, 3-butanediol produced by the strain is all in R configuration, and the purity is over 99 percent.

Therefore, the invention obtains the high-yield strain of the 1, 3-butanediol through systematic transformation, the yield of the 1, 3-butanediol to a substrate can reach more than 0.4g/g, and the purity of the R type 1, 3-butanediol reaches more than 99 percent, thereby having important industrial application potential.

EXAMPLE 4 production of 1, 3-butanediol by recombinant E.coli Using other carbon sources

The recombinant strain E.coli. DELTA. sthA. DELTA. adhE. DELTA. ldhA. DELTA. pta-ackA/ptrc99a-bld (L273T) -yqhD-phaAB-pntAB was further tested for the production of 1, 3-butanediol using other carbon sources. Using glycerol as carbon source, and using fermentation medium M9Y-G (20G/L glycerol, Na)₂HPO₄ 6g/L，KH₂PO₄ 3g/L，NaCl 0.5g/L，NH₄Cl 1g/L，MgSO₄0.5g/L,CaCl₂15mg/L, 2g/L yeast powder, ampicillin100mg/L of mycin) at the temperature of 30 ℃ and the rotation speed of 200rpm, and culturing until the bacterial liquid OD₆₀₀When the yield of the 1, 3-butanediol produced by the strain is 0.6, adding 1mM IPTG (isopropyl-beta-thiogalactoside) for induction, sampling when fermenting for 72h, and detecting the condition of producing the 1, 3-butanediol by using high performance liquid chromatography, wherein the yield of the 1, 3-butanediol reaches 8.3g/L, which shows that the recombinant strain constructed by the invention can also efficiently utilize glycerol to produce the 1, 3-butanediol at high yield.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Guangdong Qingzhixing Biotech Co., Ltd, Qinghua university

<120> recombinant bacterium for producing optically pure 1, 3-butanediol and application thereof

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Cys Phe Gly Val Phe Glu Asn Val Glu Asn Ala Ile Ser Asn Ala Val

35 40 45

His Ala Gln Lys Ile Leu Ser Leu His Tyr Thr Lys Glu Gln Arg Glu

50 55 60

Lys Ile Ile Thr Glu Ile Arg Lys Ala Ala Leu Glu Asn Lys Glu Ile

65 70 75 80

Leu Ala Thr Met Ile Leu Glu Glu Thr His Met Gly Arg Tyr Glu Asp

85 90 95

Lys Ile Leu Lys His Glu Leu Val Ala Lys Tyr Thr Pro Gly Thr Glu

100 105 110

Asp Leu Thr Thr Thr Ala Trp Ser Gly Asp Asn Gly Leu Thr Val Val

115 120 125

Glu Met Ser Pro Tyr Gly Val Ile Gly Ala Ile Thr Pro Ser Thr Asn

130 135 140

Pro Thr Glu Thr Val Ile Cys Asn Ser Ile Gly Met Ile Ala Ala Gly

145 150 155 160

Asn Thr Val Val Phe Asn Gly His Pro Gly Ala Lys Lys Cys Val Ala

165 170 175

Phe Ala Val Glu Met Ile Asn Lys Ala Ile Ile Ser Cys Gly Gly Pro

180 185 190

Glu Asn Leu Val Thr Thr Ile Lys Asn Pro Thr Met Asp Ser Leu Asp

195 200 205

Ala Ile Ile Lys His Pro Ser Ile Lys Leu Leu Cys Gly Thr Gly Gly

210 215 220

Pro Gly Met Val Lys Thr Leu Leu Asn Ser Gly Lys Lys Ala Ile Gly

225 230 235 240

Ala Gly Ala Gly Asn Pro Pro Val Ile Val Asp Asp Thr Ala Asp Ile

245 250 255

Glu Lys Ala Gly Lys Ser Ile Ile Glu Gly Cys Ser Phe Asp Asn Asn

260 265 270

Thr Pro Cys Ile Ala Glu Lys Glu Val Phe Val Phe Glu Asn Val Ala

275 280 285

Asp Asp Leu Ile Ser Asn Met Leu Lys Asn Asn Ala Val Ile Ile Asn

290 295 300

Glu Asp Gln Val Ser Lys Leu Ile Asp Leu Val Leu Gln Lys Asn Asn

305 310 315 320

Glu Thr Gln Glu Tyr Ser Ile Asn Lys Lys Trp Val Gly Lys Asp Ala

325 330 335

Lys Leu Phe Leu Asp Glu Ile Asp Val Glu Ser Pro Ser Ser Val Lys

340 345 350

Cys Ile Ile Cys Glu Val Ser Ala Arg His Pro Phe Val Met Thr Glu

355 360 365

Leu Met Met Pro Ile Leu Pro Ile Val Arg Val Lys Asp Ile Asp Glu

370 375 380

Ala Ile Glu Tyr Ala Lys Ile Ala Glu Gln Asn Arg Lys His Ser Ala

385 390 395 400

Tyr Ile Tyr Ser Lys Asn Ile Asp Asn Leu Asn Arg Phe Glu Arg Glu

405 410 415

Ile Asp Thr Thr Ile Phe Val Lys Asn Ala Lys Ser Phe Ala Gly Val

420 425 430

Gly Tyr Glu Ala Glu Gly Phe Thr Thr Phe Thr Ile Ala Gly Ser Thr

435 440 445

Gly Glu Gly Ile Thr Ser Ala Arg Asn Phe Thr Arg Gln Arg Arg Cys

450 455 460

Val Leu Ala Gly

465

<210> 3

<211> 393

<212> PRT

<213> Cupriavidus necator

<400> 3

Met Thr Asp Val Val Ile Val Ser Ala Ala Arg Thr Ala Val Gly Lys

1 5 10 15

Phe Gly Gly Ser Leu Ala Lys Ile Pro Ala Pro Glu Leu Gly Ala Val

20 25 30

Val Ile Lys Ala Ala Leu Glu Arg Ala Gly Val Lys Pro Glu Gln Val

35 40 45

Ser Glu Val Ile Met Gly Gln Val Leu Thr Ala Gly Ser Gly Gln Asn

50 55 60

Pro Ala Arg Gln Ala Ala Ile Lys Ala Gly Leu Pro Ala Met Val Pro

65 70 75 80

Ala Met Thr Ile Asn Lys Val Cys Gly Ser Gly Leu Lys Ala Val Met

85 90 95

Leu Ala Ala Asn Ala Ile Met Ala Gly Asp Ala Glu Ile Val Val Ala

100 105 110

Gly Gly Gln Glu Asn Met Ser Ala Ala Pro His Val Leu Pro Gly Ser

115 120 125

Arg Asp Gly Phe Arg Met Gly Asp Ala Lys Leu Val Asp Thr Met Ile

130 135 140

Val Asp Gly Leu Trp Asp Val Tyr Asn Gln Tyr His Met Gly Ile Thr

145 150 155 160

Ala Glu Asn Val Ala Lys Glu Tyr Gly Ile Thr Arg Glu Ala Gln Asp

165 170 175

Glu Phe Ala Val Gly Ser Gln Asn Lys Ala Glu Ala Ala Gln Lys Ala

180 185 190

Gly Lys Phe Asp Glu Glu Ile Val Pro Val Leu Ile Pro Gln Arg Lys

195 200 205

Gly Asp Pro Val Ala Phe Lys Thr Asp Glu Phe Val Arg Gln Gly Ala

210 215 220

Thr Leu Asp Ser Met Ser Gly Leu Lys Pro Ala Phe Asp Lys Ala Gly

225 230 235 240

Thr Val Thr Ala Ala Asn Ala Ser Gly Leu Asn Asp Gly Ala Ala Ala

245 250 255

Val Val Val Met Ser Ala Ala Lys Ala Lys Glu Leu Gly Leu Thr Pro

260 265 270

Leu Ala Thr Ile Lys Ser Tyr Ala Asn Ala Gly Val Asp Pro Lys Val

275 280 285

Met Gly Met Gly Pro Val Pro Ala Ser Lys Arg Ala Leu Ser Arg Ala

290 295 300

Glu Trp Thr Pro Gln Asp Leu Asp Leu Met Glu Ile Asn Glu Ala Phe

305 310 315 320

Ala Ala Gln Ala Leu Ala Val His Gln Gln Met Gly Trp Asp Thr Ser

325 330 335

Lys Val Asn Val Asn Gly Gly Ala Ile Ala Ile Gly His Pro Ile Gly

340 345 350

Ala Ser Gly Cys Arg Ile Leu Val Thr Leu Leu His Glu Met Lys Arg

355 360 365

Arg Asp Ala Lys Lys Gly Leu Ala Ser Leu Cys Ile Gly Gly Gly Met

370 375 380

Gly Val Ala Leu Ala Val Glu Arg Lys

385 390

<210> 4

<211> 246

<212> PRT

<213> Cupriavidus necator

<400> 4

Met Thr Gln Arg Ile Ala Tyr Val Thr Gly Gly Met Gly Gly Ile Gly

1 5 10 15

Thr Ala Ile Cys Gln Arg Leu Ala Lys Asp Gly Phe Arg Val Val Ala

20 25 30

Gly Cys Gly Pro Asn Ser Pro Arg Arg Glu Lys Trp Leu Glu Gln Gln

35 40 45

Lys Ala Leu Gly Phe Asp Phe Ile Ala Ser Glu Gly Asn Val Ala Asp

50 55 60

Trp Asp Ser Thr Lys Thr Ala Phe Asp Lys Val Lys Ser Glu Val Gly

65 70 75 80

Glu Val Asp Val Leu Ile Asn Asn Ala Gly Ile Thr Arg Asp Val Val

85 90 95

Phe Arg Lys Met Thr Arg Ala Asp Trp Asp Ala Val Ile Asp Thr Asn

100 105 110

Leu Thr Ser Leu Phe Asn Val Thr Lys Gln Val Ile Asp Gly Met Ala

115 120 125

Asp Arg Gly Trp Gly Arg Ile Val Asn Ile Ser Ser Val Asn Gly Gln

130 135 140

Lys Gly Gln Phe Gly Gln Thr Asn Tyr Ser Thr Ala Lys Ala Gly Leu

145 150 155 160

His Gly Phe Thr Met Ala Leu Ala Gln Glu Val Ala Thr Lys Gly Val

165 170 175

Thr Val Asn Thr Val Ser Pro Gly Tyr Ile Ala Thr Asp Met Val Lys

180 185 190

Ala Ile Arg Gln Asp Val Leu Asp Lys Ile Val Ala Thr Ile Pro Val

195 200 205

Lys Arg Leu Gly Leu Pro Glu Glu Ile Ala Ser Ile Cys Ala Trp Leu

210 215 220

Ser Ser Glu Glu Ser Gly Phe Ser Thr Gly Ala Asp Phe Ser Leu Asn

225 230 235 240

Gly Gly Leu His Met Gly

245

<210> 5

<211> 1974

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

attgaaagga ctacacaatg actgacgttg tcatcgtatc cgccgcccgc accgcggtcg 60

gcaagtttgg cggctcgctg gccaagatcc cggcaccgga actgggtgcc gtggtcatca 120

aggccgcgct ggagcgcgcc ggcgtcaagc cggagcaggt gagcgaagtc atcatgggcc 180

aggtgctgac cgccggttcg ggccagaacc ccgcacgcca ggccgcgatc aaggccggcc 240

tgccggcgat ggtgccggcc atgaccatca acaaggtgtg cggctcgggc ctgaaggccg 300

tgatgctggc cgccaacgcg atcatggcgg gcgacgccga gatcgtggtg gccggcggcc 360

aggaaaacat gagcgccgcc ccgcacgtgc tgccgggctc gcgcgatggt ttccgcatgg 420

gcgatgccaa gctggtcgac accatgatcg tcgacggcct gtgggacgtg tacaaccagt 480

accacatggg catcaccgcc gagaacgtgg ccaaggaata cggcatcaca cgcgaggcgc 540

aggatgagtt cgccgtcggc tcgcagaaca aggccgaagc cgcgcagaag gccggcaagt 600

ttgacgaaga gatcgtcccg gtgctgatcc cgcagcgcaa gggcgacccg gtggccttca 660

agaccgacga gttcgtgcgc cagggcgcca cgctggacag catgtccggc ctcaagcccg 720

ccttcgacaa ggccggcacg gtgaccgcgg ccaacgcctc gggcctgaac gacggcgccg 780

ccgcggtggt ggtgatgtcg gcggccaagg ccaaggaact gggcctgacc ccgctggcca 840

cgatcaagag ctatgccaac gccggtgtcg atcccaaggt gatgggcatg ggcccggtgc 900

cggcctccaa gcgcgccctg tcgcgcgccg agtggacccc gcaagacctg gacctgatgg 960

agatcaacga ggcctttgcc gcgcaggcgc tggcggtgca ccagcagatg ggctgggaca 1020

cctccaaggt caatgtgaac ggcggcgcca tcgccatcgg ccacccgatc ggcgcgtcgg 1080

gctgccgtat cctggtgacg ctgctgcacg agatgaagcg ccgtgacgcg aagaagggcc 1140

tggcctcgct gtgcatcggc ggcggcatgg gcgtggcgct ggcagtcgag cgcaaataac 1200

ggcgacgata acgaagccaa tcaaggagtg gacatgactc agcgcattgc gtatgtgacc 1260

ggcggcatgg gtggtatcgg aaccgccatt tgccagcggc tggccaagga tggctttcgt 1320

gtggtggccg gttgcggccc caactcgccg cgccgcgaaa agtggctgga gcagcagaag 1380

gccctgggct tcgatttcat tgcctcggaa ggcaatgtgg ctgactggga ctcgaccaag 1440

accgcattcg acaaggtcaa gtccgaggtc ggcgaggttg atgtgctgat caacaacgcc 1500

ggtatcaccc gcgacgtggt gttccgcaag atgacccgcg ccgactggga tgcggtgatc 1560

gacaccaacc tgacctcgct gttcaacgtc accaagcagg tgatcgacgg catggccgac 1620

cgtggctggg gccgcatcgt caacatctcg tcggtgaacg ggcagaaggg ccagttcggc 1680

cagaccaact actccaccgc caaggccggc ctgcatggct tcaccatggc actggcgcag 1740

gaagtggcga ccaagggcgt gaccgtcaac acggtctctc cgggctatat cgccaccgac 1800

atggtcaagg cgatccgcca ggacgtgctc gacaagatcg tcgcgacgat cccggtcaag 1860

cgcctgggcc tgccggaaga gatcgcctcg atctgcgcct ggttgtcgtc ggaggagtcc 1920

ggtttctcga ccggcgccga cttctcgctc aacggcggcc tgcatatggg ctga 1974

<210> 6

<211> 387

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 6

Met Asn Asn Phe Asn Leu His Thr Pro Thr Arg Ile Leu Phe Gly Lys

1 5 10 15

Gly Ala Ile Ala Gly Leu Arg Glu Gln Ile Pro His Asp Ala Arg Val

20 25 30

Leu Ile Thr Tyr Gly Gly Gly Ser Val Lys Lys Thr Gly Val Leu Asp

35 40 45

Gln Val Leu Asp Ala Leu Lys Gly Met Asp Val Leu Glu Phe Gly Gly

50 55 60

Ile Glu Pro Asn Pro Ala Tyr Glu Thr Leu Met Asn Ala Val Lys Leu

65 70 75 80

Val Arg Glu Gln Lys Val Thr Phe Leu Leu Ala Val Gly Gly Gly Ser

85 90 95

Val Leu Asp Gly Thr Lys Phe Ile Ala Ala Ala Ala Asn Tyr Pro Glu

100 105 110

Asn Ile Asp Pro Trp His Ile Leu Gln Thr Gly Gly Lys Glu Ile Lys

115 120 125

Ser Ala Ile Pro Met Gly Cys Val Leu Thr Leu Pro Ala Thr Gly Ser

130 135 140

Glu Ser Asn Ala Gly Ala Val Ile Ser Arg Lys Thr Thr Gly Asp Lys

145 150 155 160

Gln Ala Phe His Ser Ala His Val Gln Pro Val Phe Ala Val Leu Asp

165 170 175

Pro Val Tyr Thr Tyr Thr Leu Pro Pro Arg Gln Val Ala Asn Gly Val

180 185 190

Val Asp Ala Phe Val His Thr Val Glu Gln Tyr Val Thr Lys Pro Val

195 200 205

Asp Ala Lys Ile Gln Asp Arg Phe Ala Glu Gly Ile Leu Leu Thr Leu

210 215 220

Ile Glu Asp Gly Pro Lys Ala Leu Lys Glu Pro Glu Asn Tyr Asp Val

225 230 235 240

Arg Ala Asn Val Met Trp Ala Ala Thr Gln Ala Leu Asn Gly Leu Ile

245 250 255

Gly Ala Gly Val Pro Gln Asp Trp Ala Thr His Met Leu Gly His Glu

260 265 270

Leu Thr Ala Met His Gly Leu Asp His Ala Gln Thr Leu Ala Ile Val

275 280 285

Leu Pro Ala Leu Trp Asn Glu Lys Arg Asp Thr Lys Arg Ala Lys Leu

290 295 300

Leu Gln Tyr Ala Glu Arg Val Trp Asn Ile Thr Glu Gly Ser Asp Asp

305 310 315 320

Glu Arg Ile Asp Ala Ala Ile Ala Ala Thr Arg Asn Phe Phe Glu Gln

325 330 335

Leu Gly Val Pro Thr His Leu Ser Asp Tyr Gly Leu Asp Gly Ser Ser

340 345 350

Ile Pro Ala Leu Leu Lys Lys Leu Glu Glu His Gly Met Thr Gln Leu

355 360 365

Gly Glu Asn His Asp Ile Thr Leu Asp Val Ser Arg Arg Ile Tyr Glu

370 375 380

Ala Ala Arg

385

<210> 7

<211> 2948

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 7

gatggaaggg aatatcatgc gaattggcat accaagagaa cggttaacca atgaaacccg 60

tgttgcagca acgccaaaaa cagtggaaca gctgctgaaa ctgggtttta ccgtcgcggt 120

agagagcggc gcgggtcaac tggcaagttt tgacgataaa gcgtttgtgc aagcgggcgc 180

tgaaattgta gaagggaata gcgtctggca gtcagagatc attctgaagg tcaatgcgcc 240

gttagatgat gaaattgcgt tactgaatcc tgggacaacg ctggtgagtt ttatctggcc 300

tgcgcagaat ccggaattaa tgcaaaaact tgcggaacgt aacgtgaccg tgatggcgat 360

ggactctgtg ccgcgtatct cacgcgcaca atcgctggac gcactaagct cgatggcgaa 420

catcgccggt tatcgcgcca ttgttgaagc ggcacatgaa tttgggcgct tctttaccgg 480

gcaaattact gcggccggga aagtgccacc ggcaaaagtg atggtgattg gtgcgggtgt 540

tgcaggtctg gccgccattg gcgcagcaaa cagtctcggc gcgattgtgc gtgcattcga 600

cacccgcccg gaagtgaaag aacaagttca aagtatgggc gcggaattcc tcgagctgga 660

ttttaaagag gaagctggca gcggcgatgg ctatgccaaa gtgatgtcgg acgcgttcat 720

caaagcggaa atggaactct ttgccgccca ggcaaaagag gtcgatatca ttgtcaccac 780

cgcgcttatt ccaggcaaac cagcgccgaa gctaattacc cgtgaaatgg ttgactccat 840

gaaggcgggc agtgtgattg tcgacctggc agcccaaaac ggcggcaact gtgaatacac 900

cgtgccgggt gaaatcttca ctacggaaaa tggtgtcaaa gtgattggtt ataccgatct 960

tccgggccgt ctgccgacgc aatcctcaca gctttacggc acaaacctcg ttaatctgct 1020

gaaactgttg tgcaaagaga aagacggcaa tatcactgtt gattttgatg atgtggtgat 1080

tcgcggcgtg accgtgatcc gtgcgggcga aattacctgg ccggcaccgc cgattcaggt 1140

atcagctcag ccgcaggcgg cacaaaaagc ggcaccggaa gtgaaaactg aggaaaaatg 1200

tacctgctca ccgtggcgta aatacgcgtt gatggcgctg gcaatcattc tttttggctg 1260

gatggcaagc gttgcgccga aagaattcct tgggcacttc accgttttcg cgctggcctg 1320

cgttgtcggt tattacgtgg tgtggaatgt atcgcacgcg ctgcatacac cgttgatgtc 1380

ggtcaccaac gcgatttcag ggattattgt tgtcggagca ctgttgcaga ttggccaggg 1440

cggctgggtt agcttcctta gttttatcgc ggtgcttata gccagcatta atattttcgg 1500

tggcttcacc gtgactcagc gcatgctgaa aatgttccgc aaaaattaag gggtaacata 1560

tgtctggagg attagttaca gctgcataca ttgttgccgc gatcctgttt atcttcagtc 1620

tggccggtct ttcgaaacat gaaacgtctc gccagggtaa caacttcggt atcgccggga 1680

tggcgattgc gttaatcgca accatttttg gaccggatac gggtaatgtt ggctggatct 1740

tgctggcgat ggtcattggt ggggcaattg gtatccgtct ggcgaagaaa gttgaaatga 1800

ccgaaatgcc agaactggtg gcgatcctgc atagcttcgt gggtctggcg gcagtgctgg 1860

ttggctttaa cagctatctg catcatgacg cgggaatggc accgattctg gtcaatattc 1920

acctgacgga agtgttcctc ggtatcttca tcggggcggt aacgttcacg ggttcggtgg 1980

tggcgttcgg caaactgtgt ggcaagattt cgtctaaacc attgatgctg ccaaaccgtc 2040

acaaaatgaa cctggcggct ctggtcgttt ccttcctgct gctgattgta tttgttcgca 2100

cggacagcgt cggcctgcaa gtgctggcat tgctgataat gaccgcaatt gcgctggtat 2160

tcggctggca tttagtcgcc tccatcggtg gtgcagatat gccagtggtg gtgtcgatgc 2220

tgaactcgta ctccggctgg gcggctgcgg ctgcgggctt tatgctcagc aacgacctgc 2280

tgattgtgac cggtgcgctg gtcggttctt cgggggctat cctttcttac attatgtgta 2340

aggcgatgaa ccgttccttt atcagcgtta ttgcgggtgg tttcggcacc gacggctctt 2400

ctactggcga tgatcaggaa gtgggtgagc accgcgaaat caccgcagaa gagacagcgg 2460

aactgctgaa aaactcccat tcagtgatca ttactccggg gtacggcatg gcagtcgcgc 2520

aggcgcaata tcctgtcgct gaaattactg agaaattgcg cgctcgtggt attaatgtgc 2580

gtttcggtat ccacccggtc gcggggcgtt tgcctggaca tatgaacgta ttgctggctg 2640

aagcaaaagt accgtatgac atcgtgctgg aaatggacga gatcaatgat gactttgctg 2700

ataccgatac cgtactggtg attggtgcta acgatacggt taacccggcg gcgcaggatg 2760

atccgaagag tccgattgct ggtatgcctg tgctggaagt gtggaaagcg cagaacgtga 2820

ttgtctttaa acgttcgatg aacactggct atgctggtgt gcaaaacccg ctgttcttca 2880

aggaaaacac ccacatgctg tttggtgacg ccaaagccag cgtggatgca atcctgaaag 2940

ctctgtaa 2948

<210> 8

<211> 1401

<212> DNA

<213> Escherichia coli (Escherichia coli)

<400> 8

atgccacatt cctacgatta cgatgccata gtaataggtt ccggccccgg cggcgaaggc 60

gctgcaatgg gcctggttaa gcaaggtgcg cgcgtcgcag ttatcgagcg ttatcaaaat 120

gttggcggcg gttgcaccca ctggggcacc atcccgtcga aagctctccg tcacgccgtc 180

agccgcatta tagaattcaa tcaaaaccca ctttacagcg accattcccg actgctccgc 240

tcttcttttg ccgatatcct taaccatgcc gataacgtga ttaatcaaca aacgcgcatg 300

cgtcagggat tttacgaacg taatcactgt gaaatattgc agggaaacgc tcgctttgtt 360

gacgagcata cgttggcgct ggattgcccg gacggcagcg ttgaaacact aaccgctgaa 420

aaatttgtta ttgcctgcgg ctctcgtcca tatcatccaa cagatgttga tttcacccat 480

ccacgcattt acgacagcga ctcaattctc agcatgcacc acgaaccgcg ccatgtactt 540

atctatggtg ctggagtgat cggctgtgaa tatgcgtcga tcttccgcgg tatggatgta 600

aaagtggatc tgatcaacac ccgcgatcgc ctgctggcat ttctcgatca agagatgtca 660

gattctctct cctatcactt ctggaacagt ggcgtagtga ttcgtcacaa cgaagagtac 720

gagaagatcg aaggctgtga cgatggtgtg atcatgcatc tgaagtcggg taaaaaactg 780

aaagctgact gcctgctcta tgccaacggt cgcaccggta ataccgattc gctggcgtta 840

cagaacattg ggctagaaac tgacagccgc ggacagctga aggtcaacag catgtatcag 900

accgcacagc cacacgttta cgcggtgggc gacgtgattg gttatccgag cctggcgtcg 960

gcggcctatg accaggggcg cattgccgcg caggcgctgg taaaaggcga agccaccgca 1020

catctgattg aagatatccc taccggtatt tacaccatcc cggaaatcag ctctgtgggc 1080

aaaaccgaac agcagctgac cgcaatgaaa gtgccatatg aagtgggccg cgcccagttt 1140

aaacatctgg cacgcgcaca aatcgtcggc atgaacgtgg gcacgctgaa aattttgttc 1200

catcgggaaa caaaagagat tctgggtatt cactgctttg gcgagcgcgc tgccgaaatt 1260

attcatatcg gtcaggcgat tatggaacag aaaggtggcg gcaacactat tgagtacttc 1320

gtcaacacca cctttaacta cccgacgatg gcggaagcct atcgggtagc tgcgttaaac 1380

ggtttaaacc gcctgtttta a 1401

<210> 9

<211> 4627

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atgaacaaag atactttaat cccgacgacc aaagatttaa aacttaaaac gaatgtcgaa 60

aatatcaatt tgaaaaatta taaagataat tcctcttgct ttggggtctt cgaaaatgtg 120

gaaaatgcga ttaatagcgc agttcacgct caaaaaatcc tttcgttgca ttacaccaag 180

gaacagcgtg agaaaattat caccgaaatt cgtaaagctg cacttgagaa taaggaggtg 240

cttgctacta tgattttgga ggaaacacat atgggccgct acgaagacaa aatccttaaa 300

catgaactgg ttgcgaaata taccccggga accgaagacc ttacaaccac tgcgtggagt 360

ggtgacaacg gcttgactgt tgtagagatg tcgccctatg gggtaattgg ggcgattacc 420

ccgtctacca acccgacgga gaccgtgatc tgcaactcaa ttggaatgat tgctgcagga 480

aatgcagtcg tattcaacgg ccacccgggt gccaaaaagt gcgttgcatt cgcgattgaa 540

atgattaata aggcgattat ttcttgcggt ggtccggaaa atctggtcac cacgatcaag 600

aacccgacaa tggaaagtct ggatgcgatc atcaaacacc ctttgattaa gcttctttgc 660

gggacgggtg gaccgggtat ggttaagact ttgctgaact caggtaagaa agcgattggt 720

gctggcgcag gtaacccgcc tgtgatcgtc gacgatactg ccgacatcga gaaagcaggg 780

aagtccatta ttgaaggatg tagctttgac aacaatttgc cttgcatcgc agagaaagag 840

gtattcgttt ttgagaacgt cgcggacgat ctgattagta acatgctgaa gaacaacgca 900

gtcattatta acgaggatca ggtgtccaaa ctgatcgact tggtgttgca aaaaaataat 960

gaaactcagg agtacttcat caacaagaaa tgggttggaa aggatgccaa gttattcagt 1020

gatgaaattg atgttgaatc gccctcgaac attaagtgta ttgtctgtga ggtcaacgct 1080

aaccatccat ttgttatgac cgaattaatg atgcctattc tgcccatcgt tcgcgtgaag 1140

gacattgatg aagctgtaaa gtataccaaa atcgcggaac aaaaccgtaa gcacagcgca 1200

tacatctaca gtaaaaacat tgacaacctt aaccgttttg agcgcgaaat tgatacgacg 1260

attttcgtta agaacgcaaa aagtttcgct ggggtagggt atgaggcaga aggatttaca 1320

acttttacaa ttgcgggatc tacgggagaa gggattacct ccgcacgcaa tttcacgcgc 1380

caacgtcgtt gtgtgttggc ggggtgaaac tttaagaagg agatatacat gaacaacttt 1440

aatctgcaca ccccaacccg cattctgttt ggtaaaggcg caatcgctgg tttacgcgaa 1500

caaattcctc acgatgctcg cgtattgatt acctacggcg gcggcagcgt gaaaaaaacc 1560

ggcgttctcg atcaagttct ggatgccctg aaaggcatgg acgtgctgga atttggcggt 1620

attgagccaa acccggctta tgaaacgctg atgaacgccg tgaaactggt tcgcgaacag 1680

aaagtgactt tcctgctggc ggttggcggc ggttctgtac tggacggcac caaatttatc 1740

gccgcagcgg ctaactatcc ggaaaatatc gatccgtggc acattctgca aacgggcggt 1800

aaagagatta aaagcgccat cccgatgggc tgtgtgctga cgctgccagc aaccggttca 1860

gaatccaacg caggcgcggt gatctcccgt aaaaccacag gcgacaagca ggcgttccat 1920

tctgcccatg ttcagccggt atttgccgtg ctcgatccgg tttataccta caccctgccg 1980

ccgcgtcagg tggctaacgg cgtagtggac gcctttgtac acaccgtgga acagtatgtt 2040

accaaaccgg ttgatgccaa aattcaggac cgtttcgcag aaggcatttt gctgacgcta 2100

atcgaagatg gtccgaaagc cctgaaagag ccagaaaact acgatgtgcg cgccaacgtc 2160

atgtgggcgg cgactcaggc gctgaacggt ttgattggcg ctggcgtacc gcaggactgg 2220

gcaacgcata tgctgggcca cgaactgact gcgatgcacg gtctggatca cgcgcaaaca 2280

ctggctatcg tcctgcctgc actgtggaat gaaaaacgcg ataccaagcg cgctaagctg 2340

ctgcaatatg ctgaacgcgt ctggaacatc actgaaggtt ccgatgatga gcgtattgac 2400

gccgcgattg ccgcaacccg caatttcttt gagcaattag gcgtgccgac ccacctctcc 2460

gactacggtc tggacggcag ctccatcccg gctttgctga aaaaactgga agagcacggc 2520

atgacccaac tgggcgaaaa tcatgacatt acgttggatg tcagccgccg tatatacgaa 2580

gccgcccgct aattgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat 2640

aacaatttca cccattgaaa ggactacaca atgactgacg ttgtcatcgt atccgccgcc 2700

cgcaccgcgg tcggcaagtt tggcggctcg ctggccaaga tcccggcacc ggaactgggt 2760

gccgtggtca tcaaggccgc gctggagcgc gccggcgtca agccggagca ggtgagcgaa 2820

gtcatcatgg gccaggtgct gaccgccggt tcgggccaga accccgcacg ccaggccgcg 2880

atcaaggccg gcctgccggc gatggtgccg gccatgacca tcaacaaggt gtgcggctcg 2940

ggcctgaagg ccgtgatgct ggccgccaac gcgatcatgg cgggcgacgc cgagatcgtg 3000

gtggccggcg gccaggaaaa catgagcgcc gccccgcacg tgctgccggg ctcgcgcgat 3060

ggtttccgca tgggcgatgc caagctggtc gacaccatga tcgtcgacgg cctgtgggac 3120

gtgtacaacc agtaccacat gggcatcacc gccgagaacg tggccaagga atacggcatc 3180

acacgcgagg cgcaggatga gttcgccgtc ggctcgcaga acaaggccga agccgcgcag 3240

aaggccggca agtttgacga agagatcgtc ccggtgctga tcccgcagcg caagggcgac 3300

ccggtggcct tcaagaccga cgagttcgtg cgccagggcg ccacgctgga cagcatgtcc 3360

ggcctcaagc ccgccttcga caaggccggc acggtgaccg cggccaacgc ctcgggcctg 3420

aacgacggcg ccgccgcggt ggtggtgatg tcggcggcca aggccaagga actgggcctg 3480

accccgctgg ccacgatcaa gagctatgcc aacgccggtg tcgatcccaa ggtgatgggc 3540

atgggcccgg tgccggcctc caagcgcgcc ctgtcgcgcg ccgagtggac cccgcaagac 3600

ctggacctga tggagatcaa cgaggccttt gccgcgcagg cgctggcggt gcaccagcag 3660

atgggctggg acacctccaa ggtcaatgtg aacggcggcg ccatcgccat cggccacccg 3720

atcggcgcgt cgggctgccg tatcctggtg acgctgctgc acgagatgaa gcgccgtgac 3780

gcgaagaagg gcctggcctc gctgtgcatc ggcggcggca tgggcgtggc gctggcagtc 3840

gagcgcaaat aacggcgacg ataacgaagc caatcaagga gtggacatga ctcagcgcat 3900

tgcgtatgtg accggcggca tgggtggtat cggaaccgcc atttgccagc ggctggccaa 3960

ggatggcttt cgtgtggtgg ccggttgcgg ccccaactcg ccgcgccgcg aaaagtggct 4020

ggagcagcag aaggccctgg gcttcgattt cattgcctcg gaaggcaatg tggctgactg 4080

ggactcgacc aagaccgcat tcgacaaggt caagtccgag gtcggcgagg ttgatgtgct 4140

gatcaacaac gccggtatca cccgcgacgt ggtgttccgc aagatgaccc gcgccgactg 4200

ggatgcggtg atcgacacca acctgacctc gctgttcaac gtcaccaagc aggtgatcga 4260

cggcatggcc gaccgtggct ggggccgcat cgtcaacatc tcgtcggtga acgggcagaa 4320

gggccagttc ggccagacca actactccac cgccaaggcc ggcctgcatg gcttcaccat 4380

ggcactggcg caggaagtgg cgaccaaggg cgtgaccgtc aacacggtct ctccgggcta 4440

tatcgccacc gacatggtca aggcgatccg ccaggacgtg ctcgacaaga tcgtcgcgac 4500

gatcccggtc aagcgcctgg gcctgccgga agagatcgcc tcgatctgcg cctggttgtc 4560

gtcggaggag tccggtttct cgaccggcgc cgacttctcg ctcaacggcg gcctgcatat 4620

gggctga 4627

<210> 10

<211> 4627

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atgatcaagg acaccctggt ttcgattaca aaagacttaa agttgaaaac aaacgtagaa 60

aacgccaacc ttaaaaacta taaagatgac tcgtcttgct tcggggtgtt tgagaacgtg 120

gaaaatgcga tttcgaacgc agtccacgcc cagaagattt tatcgcttca ctacactaag 180

gagcagcgcg aaaagattat cacggagatt cgtaaagctg cacttgagaa caaagagatt 240

ttggcaacca tgatcttgga ggagactcat atggggcgtt atgaagataa aattttaaag 300

catgaacttg tcgctaaata cactccaggg actgaagact taacgaccac ggcatggagc 360

ggagataatg ggcttactgt tgttgaaatg agcccctacg gggtgattgg ggcaatcact 420

cccagcacaa atcctaccga gactgtgatt tgtaattcta ttggcatgat tgcggctggc 480

aatacggtgg tcttcaatgg acatccgggg gccaagaagt gtgttgcatt tgctgttgag 540

atgatcaaca aagccattat ttcatgtggt gggcccgaga atttggttac aaccattaag 600

aatccaacta tggactctct ggacgctatt attaaacacc cgtcgattaa acttttatgc 660

ggaaccggag gaccgggaat ggtgaaaacc cttctgaatt ccgggaaaaa ggcgatcggt 720

gcgggtgccg gcaacccacc tgtcattgtt gatgacacag cagacattga aaaggcgggc 780

aagagcatca tcgagggctg tagttttgat aacaacacgc cctgcattgc tgaaaaggaa 840

gttttcgtct tcgagaatgt tgcggacgac cttatttcca atatgcttaa gaacaacgcg 900

gtaatcatta acgaagacca agtatcaaag ttaatcgatt tggtcctgca gaagaacaat 960

gagactcaag agtatagcat taataaaaaa tgggttggga aggacgcaaa attgtttctt 1020

gatgagatcg atgtggaatc cccttcctct gtcaaatgca tcatttgcga agtatctgcg 1080

cgtcacccat tcgtcatgac agagctgatg atgcccattc tgcctattgt acgcgttaaa 1140

gatatcgacg aagctattga atatgcgaaa atcgccgagc agaaccgtaa gcactcagcg 1200

tatatttatt ccaaaaatat cgataactta aatcgttttg agcgcgaaat cgatacaact 1260

atcttcgtca agaacgccaa aagctttgct ggcgtgggct atgaagctga agggtttacc 1320

acgttcacca tcgcagggag caccggcgaa gggattacaa gtgcgcgcaa tttcactcgt 1380

caacgtcgct gcgtgttagc cggttaaaac tttaagaagg agatatacat gaacaacttt 1440

aatctgcaca ccccaacccg cattctgttt ggtaaaggcg caatcgctgg tttacgcgaa 1500

caaattcctc acgatgctcg cgtattgatt acctacggcg gcggcagcgt gaaaaaaacc 1560

ggcgttctcg atcaagttct ggatgccctg aaaggcatgg acgtgctgga atttggcggt 1620

attgagccaa acccggctta tgaaacgctg atgaacgccg tgaaactggt tcgcgaacag 1680

aaagtgactt tcctgctggc ggttggcggc ggttctgtac tggacggcac caaatttatc 1740

gccgcagcgg ctaactatcc ggaaaatatc gatccgtggc acattctgca aacgggcggt 1800

aaagagatta aaagcgccat cccgatgggc tgtgtgctga cgctgccagc aaccggttca 1860

gaatccaacg caggcgcggt gatctcccgt aaaaccacag gcgacaagca ggcgttccat 1920

tctgcccatg ttcagccggt atttgccgtg ctcgatccgg tttataccta caccctgccg 1980

ccgcgtcagg tggctaacgg cgtagtggac gcctttgtac acaccgtgga acagtatgtt 2040

accaaaccgg ttgatgccaa aattcaggac cgtttcgcag aaggcatttt gctgacgcta 2100

atcgaagatg gtccgaaagc cctgaaagag ccagaaaact acgatgtgcg cgccaacgtc 2160

atgtgggcgg cgactcaggc gctgaacggt ttgattggcg ctggcgtacc gcaggactgg 2220

gcaacgcata tgctgggcca cgaactgact gcgatgcacg gtctggatca cgcgcaaaca 2280

ctggctatcg tcctgcctgc actgtggaat gaaaaacgcg ataccaagcg cgctaagctg 2340

ctgcaatatg ctgaacgcgt ctggaacatc actgaaggtt ccgatgatga gcgtattgac 2400

gccgcgattg ccgcaacccg caatttcttt gagcaattag gcgtgccgac ccacctctcc 2460

gactacggtc tggacggcag ctccatcccg gctttgctga aaaaactgga agagcacggc 2520

atgacccaac tgggcgaaaa tcatgacatt acgttggatg tcagccgccg tatatacgaa 2580

gccgcccgct aattgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcggat 2640

aacaatttca cccattgaaa ggactacaca atgactgacg ttgtcatcgt atccgccgcc 2700

cgcaccgcgg tcggcaagtt tggcggctcg ctggccaaga tcccggcacc ggaactgggt 2760

gccgtggtca tcaaggccgc gctggagcgc gccggcgtca agccggagca ggtgagcgaa 2820

gtcatcatgg gccaggtgct gaccgccggt tcgggccaga accccgcacg ccaggccgcg 2880

atcaaggccg gcctgccggc gatggtgccg gccatgacca tcaacaaggt gtgcggctcg 2940

ggcctgaagg ccgtgatgct ggccgccaac gcgatcatgg cgggcgacgc cgagatcgtg 3000

gtggccggcg gccaggaaaa catgagcgcc gccccgcacg tgctgccggg ctcgcgcgat 3060

ggtttccgca tgggcgatgc caagctggtc gacaccatga tcgtcgacgg cctgtgggac 3120

gtgtacaacc agtaccacat gggcatcacc gccgagaacg tggccaagga atacggcatc 3180

acacgcgagg cgcaggatga gttcgccgtc ggctcgcaga acaaggccga agccgcgcag 3240

aaggccggca agtttgacga agagatcgtc ccggtgctga tcccgcagcg caagggcgac 3300

ccggtggcct tcaagaccga cgagttcgtg cgccagggcg ccacgctgga cagcatgtcc 3360

ggcctcaagc ccgccttcga caaggccggc acggtgaccg cggccaacgc ctcgggcctg 3420

aacgacggcg ccgccgcggt ggtggtgatg tcggcggcca aggccaagga actgggcctg 3480

accccgctgg ccacgatcaa gagctatgcc aacgccggtg tcgatcccaa ggtgatgggc 3540

atgggcccgg tgccggcctc caagcgcgcc ctgtcgcgcg ccgagtggac cccgcaagac 3600

ctggacctga tggagatcaa cgaggccttt gccgcgcagg cgctggcggt gcaccagcag 3660

atgggctggg acacctccaa ggtcaatgtg aacggcggcg ccatcgccat cggccacccg 3720

atcggcgcgt cgggctgccg tatcctggtg acgctgctgc acgagatgaa gcgccgtgac 3780

gcgaagaagg gcctggcctc gctgtgcatc ggcggcggca tgggcgtggc gctggcagtc 3840

gagcgcaaat aacggcgacg ataacgaagc caatcaagga gtggacatga ctcagcgcat 3900

tgcgtatgtg accggcggca tgggtggtat cggaaccgcc atttgccagc ggctggccaa 3960

ggatggcttt cgtgtggtgg ccggttgcgg ccccaactcg ccgcgccgcg aaaagtggct 4020

ggagcagcag aaggccctgg gcttcgattt cattgcctcg gaaggcaatg tggctgactg 4080

ggactcgacc aagaccgcat tcgacaaggt caagtccgag gtcggcgagg ttgatgtgct 4140

gatcaacaac gccggtatca cccgcgacgt ggtgttccgc aagatgaccc gcgccgactg 4200

ggatgcggtg atcgacacca acctgacctc gctgttcaac gtcaccaagc aggtgatcga 4260

cggcatggcc gaccgtggct ggggccgcat cgtcaacatc tcgtcggtga acgggcagaa 4320

gggccagttc ggccagacca actactccac cgccaaggcc ggcctgcatg gcttcaccat 4380

ggcactggcg caggaagtgg cgaccaaggg cgtgaccgtc aacacggtct ctccgggcta 4440

tatcgccacc gacatggtca aggcgatccg ccaggacgtg ctcgacaaga tcgtcgcgac 4500

gatcccggtc aagcgcctgg gcctgccgga agagatcgcc tcgatctgcg cctggttgtc 4560

gtcggaggag tccggtttct cgaccggcgc cgacttctcg ctcaacggcg gcctgcatat 4620

gggctga 4627

<210> 11

<211> 891

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 11

Met Ala Val Thr Asn Val Ala Glu Leu Asn Ala Leu Val Glu Arg Val

1 5 10 15

Lys Lys Ala Gln Arg Glu Tyr Ala Ser Phe Thr Gln Glu Gln Val Asp

20 25 30

Lys Ile Phe Arg Ala Ala Ala Leu Ala Ala Ala Asp Ala Arg Ile Pro

35 40 45

Leu Ala Lys Met Ala Val Ala Glu Ser Gly Met Gly Ile Val Glu Asp

50 55 60

Lys Val Ile Lys Asn His Phe Ala Ser Glu Tyr Ile Tyr Asn Ala Tyr

65 70 75 80

Lys Asp Glu Lys Thr Cys Gly Val Leu Ser Glu Asp Asp Thr Phe Gly

85 90 95

Thr Ile Thr Ile Ala Glu Pro Ile Gly Ile Ile Cys Gly Ile Val Pro

100 105 110

Thr Thr Asn Pro Thr Ser Thr Ala Ile Phe Lys Ser Leu Ile Ser Leu

115 120 125

Lys Thr Arg Asn Ala Ile Ile Phe Ser Pro His Pro Arg Ala Lys Asp

130 135 140

Ala Thr Asn Lys Ala Ala Asp Ile Val Leu Gln Ala Ala Ile Ala Ala

145 150 155 160

Gly Ala Pro Lys Asp Leu Ile Gly Trp Ile Asp Gln Pro Ser Val Glu

165 170 175

Leu Ser Asn Ala Leu Met His His Pro Asp Ile Asn Leu Ile Leu Ala

180 185 190

Thr Gly Gly Pro Gly Met Val Lys Ala Ala Tyr Ser Ser Gly Lys Pro

195 200 205

Ala Ile Gly Val Gly Ala Gly Asn Thr Pro Val Val Ile Asp Glu Thr

210 215 220

Ala Asp Ile Lys Arg Ala Val Ala Ser Val Leu Met Ser Lys Thr Phe

225 230 235 240

Asp Asn Gly Val Ile Cys Ala Ser Glu Gln Ser Val Val Val Val Asp

245 250 255

Ser Val Tyr Asp Ala Val Arg Glu Arg Phe Ala Thr His Gly Gly Tyr

260 265 270

Leu Leu Gln Gly Lys Glu Leu Lys Ala Val Gln Asp Val Ile Leu Lys

275 280 285

Asn Gly Ala Leu Asn Ala Ala Ile Val Gly Gln Pro Ala Tyr Lys Ile

290 295 300

Ala Glu Leu Ala Gly Phe Ser Val Pro Glu Asn Thr Lys Ile Leu Ile

305 310 315 320

Gly Glu Val Thr Val Val Asp Glu Ser Glu Pro Phe Ala His Glu Lys

325 330 335

Leu Ser Pro Thr Leu Ala Met Tyr Arg Ala Lys Asp Phe Glu Asp Ala

340 345 350

Val Glu Lys Ala Glu Lys Leu Val Ala Met Gly Gly Ile Gly His Thr

355 360 365

Ser Cys Leu Tyr Thr Asp Gln Asp Asn Gln Pro Ala Arg Val Ser Tyr

370 375 380

Phe Gly Gln Lys Met Lys Thr Ala Arg Ile Leu Ile Asn Thr Pro Ala

385 390 395 400

Ser Gln Gly Gly Ile Gly Asp Leu Tyr Asn Phe Lys Leu Ala Pro Ser

405 410 415

Leu Thr Leu Gly Cys Gly Ser Trp Gly Gly Asn Ser Ile Ser Glu Asn

420 425 430

Val Gly Pro Lys His Leu Ile Asn Lys Lys Thr Val Ala Lys Arg Ala

435 440 445

Glu Asn Met Leu Trp His Lys Leu Pro Lys Ser Ile Tyr Phe Arg Arg

450 455 460

Gly Ser Leu Pro Ile Ala Leu Asp Glu Val Ile Thr Asp Gly His Lys

465 470 475 480

Arg Ala Leu Ile Val Thr Asp Arg Phe Leu Phe Asn Asn Gly Tyr Ala

485 490 495

Asp Gln Ile Thr Ser Val Leu Lys Ala Ala Gly Val Glu Thr Glu Val

500 505 510

Phe Phe Glu Val Glu Ala Asp Pro Thr Leu Ser Ile Val Arg Lys Gly

515 520 525

Ala Glu Leu Ala Asn Ser Phe Lys Pro Asp Val Ile Ile Ala Leu Gly

530 535 540

Gly Gly Ser Pro Met Asp Ala Ala Lys Ile Met Trp Val Met Tyr Glu

545 550 555 560

His Pro Glu Thr His Phe Glu Glu Leu Ala Leu Arg Phe Met Asp Ile

565 570 575

Arg Lys Arg Ile Tyr Lys Phe Pro Lys Met Gly Val Lys Ala Lys Met

580 585 590

Ile Ala Val Thr Thr Thr Ser Gly Thr Gly Ser Glu Val Thr Pro Phe

595 600 605

Ala Val Val Thr Asp Asp Ala Thr Gly Gln Lys Tyr Pro Leu Ala Asp

610 615 620

Tyr Ala Leu Thr Pro Asp Met Ala Ile Val Asp Ala Asn Leu Val Met

625 630 635 640

Asp Met Pro Lys Ser Leu Cys Ala Phe Gly Gly Leu Asp Ala Val Thr

645 650 655

His Ala Met Glu Ala Tyr Val Ser Val Leu Ala Ser Glu Phe Ser Asp

660 665 670

Gly Gln Ala Leu Gln Ala Leu Lys Leu Leu Lys Glu Tyr Leu Pro Ala

675 680 685

Ser Tyr His Glu Gly Ser Lys Asn Pro Val Ala Arg Glu Arg Val His

690 695 700

Ser Ala Ala Thr Ile Ala Gly Ile Ala Phe Ala Asn Ala Phe Leu Gly

705 710 715 720

Val Cys His Ser Met Ala His Lys Leu Gly Ser Gln Phe His Ile Pro

725 730 735

His Gly Leu Ala Asn Ala Leu Leu Ile Cys Asn Val Ile Arg Tyr Asn

740 745 750

Ala Asn Asp Asn Pro Thr Lys Gln Thr Ala Phe Ser Gln Tyr Asp Arg

755 760 765

Pro Gln Ala Arg Arg Arg Tyr Ala Glu Ile Ala Asp His Leu Gly Leu

770 775 780

Ser Ala Pro Gly Asp Arg Thr Ala Ala Lys Ile Glu Lys Leu Leu Ala

785 790 795 800

Trp Leu Glu Thr Leu Lys Ala Glu Leu Gly Ile Pro Lys Ser Ile Arg

805 810 815

Glu Ala Gly Val Gln Glu Ala Asp Phe Leu Ala Asn Val Asp Lys Leu

820 825 830

Ser Glu Asp Ala Phe Asp Asp Gln Cys Thr Gly Ala Asn Pro Arg Tyr

835 840 845

Pro Leu Ile Ser Glu Leu Lys Gln Ile Leu Leu Asp Thr Tyr Tyr Gly

850 855 860

Arg Asp Tyr Val Glu Gly Glu Thr Ala Ala Lys Lys Glu Ala Ala Pro

865 870 875 880

Ala Lys Ala Glu Lys Lys Ala Lys Lys Ser Ala

885 890

<210> 12

<211> 329

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 12

Met Lys Leu Ala Val Tyr Ser Thr Lys Gln Tyr Asp Lys Lys Tyr Leu

1 5 10 15

Gln Gln Val Asn Glu Ser Phe Gly Phe Glu Leu Glu Phe Phe Asp Phe

20 25 30

Leu Leu Thr Glu Lys Thr Ala Lys Thr Ala Asn Gly Cys Glu Ala Val

35 40 45

Cys Ile Phe Val Asn Asp Asp Gly Ser Arg Pro Val Leu Glu Glu Leu

50 55 60

Lys Lys His Gly Val Lys Tyr Ile Ala Leu Arg Cys Ala Gly Phe Asn

65 70 75 80

Asn Val Asp Leu Asp Ala Ala Lys Glu Leu Gly Leu Lys Val Val Arg

85 90 95

Val Pro Ala Tyr Asp Pro Glu Ala Val Ala Glu His Ala Ile Gly Met

100 105 110

Met Met Thr Leu Asn Arg Arg Ile His Arg Ala Tyr Gln Arg Thr Arg

115 120 125

Asp Ala Asn Phe Ser Leu Glu Gly Leu Thr Gly Phe Thr Met Tyr Gly

130 135 140

Lys Thr Ala Gly Val Ile Gly Thr Gly Lys Ile Gly Val Ala Met Leu

145 150 155 160

Arg Ile Leu Lys Gly Phe Gly Met Arg Leu Leu Ala Phe Asp Pro Tyr

165 170 175

Pro Ser Ala Ala Ala Leu Glu Leu Gly Val Glu Tyr Val Asp Leu Pro

180 185 190

Thr Leu Phe Ser Glu Ser Asp Val Ile Ser Leu His Cys Pro Leu Thr

195 200 205

Pro Glu Asn Tyr His Leu Leu Asn Glu Ala Ala Phe Glu Gln Met Lys

210 215 220

Asn Gly Val Met Ile Val Asn Thr Ser Arg Gly Ala Leu Ile Asp Ser

225 230 235 240

Gln Ala Ala Ile Glu Ala Leu Lys Asn Gln Lys Ile Gly Ser Leu Gly

245 250 255

Met Asp Val Tyr Glu Asn Glu Arg Asp Leu Phe Phe Glu Asp Lys Ser

260 265 270

Asn Asp Val Ile Gln Asp Asp Val Phe Arg Arg Leu Ser Ala Cys His

275 280 285

Asn Val Leu Phe Thr Gly His Gln Ala Phe Leu Thr Ala Glu Ala Leu

290 295 300

Thr Ser Ile Ser Gln Thr Thr Leu Gln Asn Leu Ser Asn Leu Glu Lys

305 310 315 320

Gly Glu Thr Cys Pro Asn Glu Leu Val

325

<210> 13

<211> 714

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 13

Met Ser Arg Ile Ile Met Leu Ile Pro Thr Gly Thr Ser Val Gly Leu

1 5 10 15

Thr Ser Val Ser Leu Gly Val Ile Arg Ala Met Glu Arg Lys Gly Val

20 25 30

Arg Leu Ser Val Phe Lys Pro Ile Ala Gln Pro Arg Thr Gly Gly Asp

35 40 45

Ala Pro Asp Gln Thr Thr Thr Ile Val Arg Ala Asn Ser Ser Thr Thr

50 55 60

Thr Ala Ala Glu Pro Leu Lys Met Ser Tyr Val Glu Gly Leu Leu Ser

65 70 75 80

Ser Asn Gln Lys Asp Val Leu Met Glu Glu Ile Val Ala Asn Tyr His

85 90 95

Ala Asn Thr Lys Asp Ala Glu Val Val Leu Val Glu Gly Leu Val Pro

100 105 110

Thr Arg Lys His Gln Phe Ala Gln Ser Leu Asn Tyr Glu Ile Ala Lys

115 120 125

Thr Leu Asn Ala Glu Ile Val Phe Val Met Ser Gln Gly Thr Asp Thr

130 135 140

Pro Glu Gln Leu Lys Glu Arg Ile Glu Leu Thr Arg Asn Ser Phe Gly

145 150 155 160

Gly Ala Lys Asn Thr Asn Ile Thr Gly Val Ile Val Asn Lys Leu Asn

165 170 175

Ala Pro Val Asp Glu Gln Gly Arg Thr Arg Pro Asp Leu Ser Glu Ile

180 185 190

Phe Asp Asp Ser Ser Lys Ala Lys Val Asn Asn Val Asp Pro Ala Lys

195 200 205

Leu Gln Glu Ser Ser Pro Leu Pro Val Leu Gly Ala Val Pro Trp Ser

210 215 220

Phe Asp Leu Ile Ala Thr Arg Ala Ile Asp Met Ala Arg His Leu Asn

225 230 235 240

Ala Thr Ile Ile Asn Glu Gly Asp Ile Asn Thr Arg Arg Val Lys Ser

245 250 255

Val Thr Phe Cys Ala Arg Ser Ile Pro His Met Leu Glu His Phe Arg

260 265 270

Ala Gly Ser Leu Leu Val Thr Ser Ala Asp Arg Pro Asp Val Leu Val

275 280 285

Ala Ala Cys Leu Ala Ala Met Asn Gly Val Glu Ile Gly Ala Leu Leu

290 295 300

Leu Thr Gly Gly Tyr Glu Met Asp Ala Arg Ile Ser Lys Leu Cys Glu

305 310 315 320

Arg Ala Phe Ala Thr Gly Leu Pro Val Phe Met Val Asn Thr Asn Thr

325 330 335

Trp Gln Thr Ser Leu Ser Leu Gln Ser Phe Asn Leu Glu Val Pro Val

340 345 350

Asp Asp His Glu Arg Ile Glu Lys Val Gln Glu Tyr Val Ala Asn Tyr

355 360 365

Ile Asn Ala Asp Trp Ile Glu Ser Leu Thr Ala Thr Ser Glu Arg Ser

370 375 380

Arg Arg Leu Ser Pro Pro Ala Phe Arg Tyr Gln Leu Thr Glu Leu Ala

385 390 395 400

Arg Lys Ala Gly Lys Arg Ile Val Leu Pro Glu Gly Asp Glu Pro Arg

405 410 415

Thr Val Lys Ala Ala Ala Ile Cys Ala Glu Arg Gly Ile Ala Thr Cys

420 425 430

Val Leu Leu Gly Asn Pro Ala Glu Ile Asn Arg Val Ala Ala Ser Gln

435 440 445

Gly Val Glu Leu Gly Ala Gly Ile Glu Ile Val Asp Pro Glu Val Val

450 455 460

Arg Glu Ser Tyr Val Gly Arg Leu Val Glu Leu Arg Lys Asn Lys Gly

465 470 475 480

Met Thr Glu Thr Val Ala Arg Glu Gln Leu Glu Asp Asn Val Val Leu

485 490 495

Gly Thr Leu Met Leu Glu Gln Asp Glu Val Asp Gly Leu Val Ser Gly

500 505 510

Ala Val His Thr Thr Ala Asn Thr Ile Arg Pro Pro Leu Gln Leu Ile

515 520 525

Lys Thr Ala Pro Gly Ser Ser Leu Val Ser Ser Val Phe Phe Met Leu

530 535 540

Leu Pro Glu Gln Val Tyr Val Tyr Gly Asp Cys Ala Ile Asn Pro Asp

545 550 555 560

Pro Thr Ala Glu Gln Leu Ala Glu Ile Ala Ile Gln Ser Ala Asp Ser

565 570 575

Ala Ala Ala Phe Gly Ile Glu Pro Arg Val Ala Met Leu Ser Tyr Ser

580 585 590

Thr Gly Thr Ser Gly Ala Gly Ser Asp Val Glu Lys Val Arg Glu Ala

595 600 605

Thr Arg Leu Ala Gln Glu Lys Arg Pro Asp Leu Met Ile Asp Gly Pro

610 615 620

Leu Gln Tyr Asp Ala Ala Val Met Ala Asp Val Ala Lys Ser Lys Ala

625 630 635 640

Pro Asn Ser Pro Val Ala Gly Arg Ala Thr Val Phe Ile Phe Pro Asp

645 650 655

Leu Asn Thr Gly Asn Thr Thr Tyr Lys Ala Val Gln Arg Ser Ala Asp

660 665 670

Leu Ile Ser Ile Gly Pro Met Leu Gln Gly Met Arg Lys Pro Val Asn

675 680 685

Asp Leu Ser Arg Gly Ala Leu Val Asp Asp Ile Val Tyr Thr Ile Ala

690 695 700

Leu Thr Ala Ile Gln Ser Ala Gln Gln Gln

705 710

<210> 14

<211> 400

<212> PRT

<213> Escherichia coli (Escherichia coli)

<400> 14

Met Ser Ser Lys Leu Val Leu Val Leu Asn Cys Gly Ser Ser Ser Leu

1 5 10 15

Lys Phe Ala Ile Ile Asp Ala Val Asn Gly Glu Glu Tyr Leu Ser Gly

20 25 30

Leu Ala Glu Cys Phe His Leu Pro Glu Ala Arg Ile Lys Trp Lys Met

35 40 45

Asp Gly Asn Lys Gln Glu Ala Ala Leu Gly Ala Gly Ala Ala His Ser

50 55 60

Glu Ala Leu Asn Phe Ile Val Asn Thr Ile Leu Ala Gln Lys Pro Glu

65 70 75 80

Leu Ser Ala Gln Leu Thr Ala Ile Gly His Arg Ile Val His Gly Gly

85 90 95

Glu Lys Tyr Thr Ser Ser Val Val Ile Asp Glu Ser Val Ile Gln Gly

100 105 110

Ile Lys Asp Ala Ala Ser Phe Ala Pro Leu His Asn Pro Ala His Leu

115 120 125

Ile Gly Ile Glu Glu Ala Leu Lys Ser Phe Pro Gln Leu Lys Asp Lys

130 135 140

Asn Val Ala Val Phe Asp Thr Ala Phe His Gln Thr Met Pro Glu Glu

145 150 155 160

Ser Tyr Leu Tyr Ala Leu Pro Tyr Asn Leu Tyr Lys Glu His Gly Ile

165 170 175

Arg Arg Tyr Gly Ala His Gly Thr Ser His Phe Tyr Val Thr Gln Glu

180 185 190

Ala Ala Lys Met Leu Asn Lys Pro Val Glu Glu Leu Asn Ile Ile Thr

195 200 205

Cys His Leu Gly Asn Gly Gly Ser Val Ser Ala Ile Arg Asn Gly Lys

210 215 220

Cys Val Asp Thr Ser Met Gly Leu Thr Pro Leu Glu Gly Leu Val Met

225 230 235 240

Gly Thr Arg Ser Gly Asp Ile Asp Pro Ala Ile Ile Phe His Leu His

245 250 255

Asp Thr Leu Gly Met Ser Val Asp Ala Ile Asn Lys Leu Leu Thr Lys

260 265 270

Glu Ser Gly Leu Leu Gly Leu Thr Glu Val Thr Ser Asp Cys Arg Tyr

275 280 285

Val Glu Asp Asn Tyr Ala Thr Lys Glu Asp Ala Lys Arg Ala Met Asp

290 295 300

Val Tyr Cys His Arg Leu Ala Lys Tyr Ile Gly Ala Tyr Thr Ala Leu

305 310 315 320

Met Asp Gly Arg Leu Asp Ala Val Val Phe Thr Gly Gly Ile Gly Glu

325 330 335

Asn Ala Ala Met Val Arg Glu Leu Ser Leu Gly Lys Leu Gly Val Leu

340 345 350

Gly Phe Glu Val Asp His Glu Arg Asn Leu Ala Ala Arg Phe Gly Lys

355 360 365

Ser Gly Phe Ile Asn Lys Glu Gly Thr Arg Pro Ala Val Val Ile Pro

370 375 380

Thr Asn Glu Glu Leu Val Ile Ala Gln Asp Ala Ser Arg Leu Thr Ala

385 390 395 400

Claims

1. The recombinant escherichia coli for producing the optically pure 1, 3-butanediol is characterized in that the recombinant escherichia coli takes a recombinant bacterium for producing the optically pure 1, 3-butanediol as an initial strain, the initial strain is modified by a genetic engineering means, and the obtained intracellular NADPH supply-enhanced engineering bacterium;

(1) increasing intracellular supply of NADPH by enhancing genes associated with NADPH biosynthetic pathway in the starting strain; the gene related to the NADPH biosynthetic pathway is pntAB gene, and the nucleotide sequence is shown as SEQ ID NO. 7; and

(2) the supply of NADPH in cells is improved by weakening sthA gene in an original strain; said attenuation comprises knocking out or reducing expression of the gene; the nucleotide sequence of sthA gene is shown in SEQ ID NO. 8;

the recombinant strain for producing the optically pure 1, 3-butanediol is formed by introducing phaA, phaB, bld and yqhD genes into escherichia coli through plasmids or integrating the genes on an escherichia coli chromosome through a genetic engineering means;

wherein the phaA gene is derived fromCupriavidus necatorIt is a gene of a protein shown as SEQ ID NO. 3:

the phaB gene is derived fromCupriavidus necatorIt is a gene of a protein shown as SEQ ID NO. 4:

the bld gene is a coding gene of butyraldehyde dehydrogenase mutant and is derived fromClostridium saccharoperbutylacetonicumThe amino acid sequence of the coded protein is shown as SEQ ID NO. 2;

the yqhD gene is derived from a large intestine rodBacteria (A), (B)Escherichia coli) It is the gene of the protein shown in SEQ ID NO. 6.

2. The recombinant Escherichia coli of claim 1, wherein the recombinant bacterium for producing optically pure 1, 3-butanediol is constructed as follows: after the phaA and phaB genes are optimized by a codon, the phaA and phaB genes and bld and yqhD genes are constructed on an expression vector, the recombinant vector is used for transforming escherichia coli, and a positive transformant is screened;

wherein, the sequence of the codon optimized phaA-phaB operon is shown as SEQ ID NO. 5.

3. The recombinant Escherichia coli of claim 2, wherein the recombinant bacterium for producing optically pure 1, 3-butanediol is constructed as follows: constructing an bld-yqhD-phaAB tandem gene expression cassette on ptrc99a plasmid, transforming escherichia coli by using recombinant plasmid, and screening positive transformants;

4. An engineering bacterium for high yield of 1, 3-butanediol, characterized in that the engineering bacterium is an engineering bacterium which is obtained by further modifying an original strain by using the recombinant escherichia coli of any one of claims 1 to 3 as the original strain by using a genetic engineering means and enhancing the metabolic flux of intracellular acetyl CoA flowing to a 1, 3-butanediol synthesis pathway;

weakening at least one gene of adhE, ldhA, pta and ackA genes in the original strain to improve the metabolic flux of the intracellular acetyl CoA flowing to a 1, 3-butanediol synthetic pathway; said attenuation comprises knocking out or reducing expression of the gene;

5. The engineered bacterium of claim 4, wherein the adhE, ldhA, pta, and ackA genes in the original strain are attenuated to increase the metabolic flux of intracellular acetyl CoA to the 1, 3-butanediol synthesis pathway.

6. Use of the recombinant E.coli of any one of claims 1 to 3, or the engineered bacterium of claim 4 or 5, for the fermentative production of 1, 3-butanediol.

7. The butyraldehyde dehydrogenase mutant is characterized in that the amino acid sequence of the butyraldehyde dehydrogenase mutant is shown as SEQ ID NO. 2.