CN114736841A - Recombinant escherichia coli and preparation method and application thereof - Google Patents

Abstract

The invention discloses recombinant escherichia coli and a preparation method and application thereof. The recombinant escherichia coli disclosed by the invention can use glucose as a raw material to synthesize malonate through an oxaloacetate-aspartate pathway, and is a modularized constitutive overexpression escherichia coli-derived phosphoenolpyruvate carboxylase, aspartate aminotransferase and aspartate-alpha-decarboxylase as well as pseudomonas aeruginosa-derived beta-alanine pyruvate transaminase, escherichia coli-derived succinic semialdehyde dehydrogenase and corynebacterium glutamicum-derived pyruvate carboxylase. The invention makes it possible to synthesize malonic acid by using Escherichia coli total biological method, and the method has simple operation and low cost, and can be used for industrial production.

Description

Recombinant escherichia coli and preparation method and application thereof

Technical Field

The invention belongs to the field of bioengineering, relates to recombinant escherichia coli, a preparation method and application thereof, and particularly relates to a method for synthesizing malonic acid by utilizing the escherichia coli in a full-biological manner.

Background

The malonic acid is mainly used for producing spices, adhesives, resin additives and the like, and can also be used as a surface treating agent for leather products or aluminum products, a foaming agent for foam plastics and a chemical cleaning agent for a nuclear reactor, for example, the malonic acid is a raw material for producing pesticide bactericide Fushiyi, herbicide Suzada and plant growth regulator indolizite; the pharmaceutical industry uses malonic acid to prepare diuretic bensulazolone, anti-inflammatory oxybenzene phenylbutazone, sedative bromomethyloctopine and the like; the malonic acid generates carbon dioxide and water after being heated, has no pollution problem, and can be directly used as an aluminum surface treating agent. In addition, malonic acid and its esters can be widely used as pharmaceutical intermediates, and diethyl malonate, a compound generated by substituting hydroxyl groups in two carboxyl groups of malonic acid by ethoxy groups, is an important organic synthetic raw material and is used for dye and drug synthesis, such as the manufacture of barbiturates, vitamin B1, B6 and the like. With the rapid development of the domestic and international chemical industries at present and the vigorous development of the application of malonic acid, the yield and quality of malonic acid are greatly increased day by day.

The main synthesis method for malonic acid currently used in practical production is chemical synthesis. The method has the characteristics that the process route and the production period are short, three wastes are few, but the hydrolysis process belongs to a reversible reaction, and the malonic acid is easy to generate decarboxylation reaction under high temperature to decompose and generate acetic acid, water and carbon dioxide, so the product yield is low, but the method is easy to control the generation of impurities, so the product purity is higher; secondly, the ester exchange process can prepare high-purity malonic acid with high content, less impurities and low moisture content and obtain a byproduct of preparing ethyl acetate with higher purity as long as the purity of the raw materials is controlled, and the method has the following defects: acetic acid, pungent sour taste, and a mixed solution of ethyl acetate and ethanol as by-products are needed, which is not easy to handle.

The biosynthetic pathway of malonate has not been well developed, and a method for producing malonate from malonyl-CoA by malonyl-CoA hydrolase activity is known (WO 2013134424). However, the malonyl-CoA pathway produces metabolites less efficiently than the aspartate pathway, and thus, the aspartate pathway is a more efficient method for producing malonate.

Disclosure of Invention

In view of the above problems, the present invention provides a recombinant Escherichia coli capable of producing malonic acid, which is capable of synthesizing malonic acid from oxaloacetate-aspartate pathway using glucose as a raw material, wherein the recombinant Escherichia coli is a phosphoenolpyruvate carboxylase ppc derived from Escherichia coli, an aspartate aminotransferase aspC, an aspartate- α decarboxylase panD, and a Pseudomonas aeruginosa (Pseudomonas aeruginosa) derived β -alanine pyruvate aminotransferase pa0132, an Escherichia coli derived succinic semialdehyde dehydrogenase yneI, and a Corynebacterium glutamicum (Corynebacterium glutamicum) derived pyruvate carboxylase pyc, which are constitutively overexpressed. The schematic diagram of the synthesis route of the malonic acid constructed by the invention is shown in figure 1.

In some embodiments, the recombinant e.coli is BL21(TPP), which is host e.coli BL21(DE3), and the modular constitutive overexpression expresses the phosphoenolpyruvate carboxylase ppc, the aspartate aminotransferase aspC, the aspartate-alpha decarboxylase panD, the beta-alanine pyruvate aminotransferase gene pa0132, the succinate semialdehyde dehydrogenase yneI, and the pyruvate carboxylase pyc individually.

In some embodiments, said e.coli BL21(SCR) is e.coli BL21(DE3) as host, said modular constitutive overexpression is fusion expression of said phosphoenolpyruvate carboxylase ppc and said aspartate aminotransferase aspC, expression of said aspartate- α -decarboxylase panD and said pyruvate carboxylase pyc separately, fusion expression of said succinate semialdehyde dehydrogenase yneI and said β -alanine pyruvate aminotransferase pa 0132.

In some embodiments, the recombinant E.coli described above, the promoter used for the modular constitutive overexpression is the constitutive promoter ML2719, the sequence of which is shown in SEQ ID NO: 39.

In some embodiments, any of the above recombinant E.coli cells is a host E.coli BL21(DE 3).

In some embodiments, the recombinant E.coli as described in any of the above, the phosphoenolpyruvate carboxylase ppc, aspartate aminotransferase aspC and aspartate- α -decarboxylase panD are derived from E.coli BL21(DE 3).

In some embodiments, in any of the methods described above, the succinic semialdehyde dehydrogenase ynel is from escherichia coli K12.

In some embodiments, the recombinant Escherichia coli as described in any of the above, wherein the phosphoenolpyruvate carboxylase ppc has a protein sequence as shown in SEQ ID NO 35 at positions 1-883;

the protein sequence of the aspartate aminotransferase aspC is shown as 904-1299 in SEQ ID NO 35;

the protein sequence of the aspartate-alpha-decarboxylase panD is shown as SEQ ID NO 38;

the protein sequence of the beta-alanine pyruvate transaminase pa0132 is shown as 483-930 in SEQ ID NO: 36;

the protein sequence of the succinic semialdehyde dehydrogenase yneI is shown as 1-462 in SEQ ID NO: 36; and/or

The protein sequence of the pyruvate carboxylase pyc is shown as SEQ ID NO: 37.

In some embodiments, in any of the recombinant E.coli described above, the sequence of the linker peptide of the protein expressed by the fusion of phosphoenolpyruvate carboxylase and aspartate aminotransferase is shown in SEQ ID NO 26.

In some embodiments, in any of the recombinant E.coli described above, the sequence of the linker peptide of the protein expressed by fusion of succinate semialdehyde dehydrogenase and the β -alanine pyruvate transaminase is shown in SEQ ID NO 26.

In some embodiments, in any of the recombinant E.coli described above, the phosphoenolpyruvate carboxylase and the aspartate aminotransferase are expressed as a fusion protein having the sequence shown in SEQ ID NO 35.

In some embodiments, in any of the above recombinant Escherichia coli, the succinate semialdehyde dehydrogenase and the beta-alanine pyruvate transaminase are expressed as a fusion protein having the sequence shown in SEQ ID NO: 36.

It is a second object of the present invention to provide a method for constructing a recombinant Escherichia coli as described in any of the above, comprising transferring one or more plasmids containing a gene ppc encoding phosphoenolpyruvate carboxylase derived from Escherichia coli, a gene aspC encoding aspartate aminotransferase, a gene panD encoding aspartate- α -decarboxylase, and a gene pa0132 encoding β -alanine pyruvate aminotransferase derived from Pseudomonas aeruginosa, a gene yneI encoding succinate semialdehyde dehydrogenase derived from Escherichia coli, and a gene pyc encoding pyruvate carboxylase derived from Corynebacterium glutamicum into an Escherichia coli host to perform modular constitutive overexpression.

In some embodiments, in any of the methods described above, the recombinant e.coli BL21(TPP) is e.coli BL21(DE3) as the host, and the modular constitutive overexpression comprises the step of transferring each plasmid comprising the gene ppc encoding phosphoenolpyruvate carboxylase derived from e.coli, the gene aspC of aspartate aminotransferase, the gene panD of aspartate- α -decarboxylase, and the gene pa0132 encoding β -alanine pyruvate aminotransferase derived from pseudomonas aeruginosa, the gene yneI encoding succinate semialdehyde dehydrogenase derived from e.coli, and the gene pyc encoding pyruvate carboxylase derived from corynebacterium glutamicum into the e.coli host for modular constitutive overexpression such that these proteins are expressed individually.

In some embodiments, said e.coli BL21(SCR) is e.coli BL21(DE3) as host, and said modular constitutive overexpression comprises the step of transferring a plasmid fusion expressing phosphoenolpyruvate carboxylase gene ppc and said aspartate aminotransferase gene aspC, a plasmid separately expressing said aspartate- α -dehydrogenase gene panD and said pyruvate carboxylase gene pyc, and a plasmid fusion expressing said succinate semialdehyde dehydrogenase gene yneI and said β -alanine pyruvate aminotransferase gene pa0132 into the e.coli host for modular constitutive overexpression.

In some embodiments, in the above methods, the promoter used for constitutive overexpression of the segment is a constitutive promoter ML2719, the sequence of which is shown in SEQ ID NO: 39.

In some embodiments, in any of the above methods, the e.coli host is e.coli BL21(DE 3).

In some embodiments, in any of the methods described above, the phosphoenolpyruvate carboxylase gene ppc, aspartate aminotransferase gene aspC, and aspartate- α decarboxylase gene panD are from escherichia coli BL21(DE 3).

In some embodiments, in any of the methods described above, the succinic semialdehyde dehydrogenase gene ynel is from escherichia coli K12.

In some embodiments, the method of any one of the above, wherein the phosphoenolpyruvate carboxylase gene ppc has the sequence shown in SEQ ID NO 9;

the sequence of the aspartate aminotransferase gene aspC is shown as SEQ ID NO. 10;

the sequence of the aspartate-alpha-dehydrogenase gene panD is shown as SEQ ID NO 18;

the sequence of the gene pa0132 of the beta-alanine pyruvate transaminase is shown as SEQ ID NO. 2;

the sequence of the gene yneI of the succinic semialdehyde dehydrogenase is shown in SEQ ID NO. 1; and/or

The sequence of the gene pyc of pyruvate carboxylase is shown in SEQ ID NO 17.

In some embodiments, in any of the methods above, the recombinant e.coli is BL21 (SCR);

the plasmid for fusion expression of the phosphoenolpyruvate carboxylase gene ppc and the aspartate aminotransferase gene aspC is pCDF-ppc-linker-aspC;

the plasmid separately expressing the aspartate- α -dehydrogenase gene panD and the pyruvate-carboxylase gene pyc is pTrc 99A-pyc-panD; and/or

The plasmid for fusion expression of the succinic semialdehyde dehydrogenase gene yneI and the beta-alanine pyruvate transaminase gene pa0132 is pRSF-yneI-linker-pa 0132.

In some embodiments, in the above method, the pCDF-ppc-linker-aspC is constructed by homologous recombination of a linker fragment and a pCDF-ppc-aspC plasmid fragment; the linker fragment is obtained by performing PCR amplification on a linker-pCDF-F (SEQ ID NO:27) and a linker-pCDF-R (SEQ ID NO:28) by using a linker gene shown in SEQ ID NO:25 as a template; the pCDF-ppc-aspC plasmid fragment is a linearized pCDF-ppc-aspC plasmid fragment obtained by carrying out PCR amplification by using a primer pCDF-linker-F (SEQ ID NO:29) and a primer pCDF-linker-R (SEQ ID NO:30) as a template;

the pRSF-yneI-linker-pa0132 is constructed by a method of recombining homologous sequences of a linker fragment and a pRSF-yneI-pa0132 plasmid fragment; the linker fragment is obtained by performing PCR amplification on a linker gene shown in SEQ ID NO. 25 as a template and linker-pRSF-F (SEQ ID NO:31) and linker-pRSF-R (SEQ ID NO: 32); the pRSF-yneI-pa0132 plasmid fragment is obtained by taking pRSF-yneI-pa0132 plasmid as a template and carrying out PCR amplification by using primers pRSF-linker-F (SEQ ID NO:33) and pRSF-linker-R (SEQ ID NO:34) to obtain a linearized pRSF-yneI-pa0132 plasmid fragment;

the pTrc99A-pyc-panD was constructed by recombination of homologous sequences of the pyc, panD fragment and pTrc99A plasmid fragment.

In some embodiments, the above method, wherein the pCDF-ppc-aspC plasmid is constructed by recombining homologous sequences of a ppc, an aspC fragment and a pCDFDuet-1 plasmid fragment, wherein the ppc fragment is obtained by PCR amplification using a ppc gene shown in SEQ ID NO:9 as a template and primers ppc-F (SEQ ID NO:11) and ppc-R (SEQ ID NO: 12); the aspC fragment is obtained by using an aspC gene shown in SEQ ID NO. 10 as a template and performing PCR amplification by using primers aspC-F (SEQ ID NO. 13) and aspC-R (SEQ ID NO. 14); the pCDFDuet-1 plasmid fragment is a linearized pCDFDuet-1 plasmid fragment obtained by PCR amplification with primers pCDF-F (SEQ ID NO:15) and pCDF-R (SEQ ID NO:16) using the pCDFDuet-1 plasmid as a template;

the pRSF-yneI-pa0132 plasmid is constructed by recombining the homologous sequences of yneI, pa0132 fragment and pRSFDuet-1 plasmid fragment, wherein the yneI fragment is obtained by taking the yneI gene shown in SEQ ID NO. 1 as a template and carrying out PCR amplification by using primers yneI-F (SEQ ID NO:3) and yneI-R (SEQ ID NO:4) to obtain a yneI fragment; the pa0132 fragment is a pa0132 gene shown in SEQ ID NO. 2 as a template, and primers pa0132-F (SEQ ID NO. 5) and pa0132-R (SEQ ID NO. 6) are used for PCR amplification to obtain a pa0132 fragment; the pRSFDuet-1 plasmid fragment takes pRSFDuet-1 plasmid as a template, and primers pRSF-F (SEQ ID NO:7) and pRSF-R (SEQ ID NO:8) are used for PCR amplification to obtain a linearized pRSFDuet-1 plasmid fragment;

the pyc fragment is obtained by taking a pyc gene shown in SEQ ID NO:17 as a template and carrying out PCR amplification by using primers pyc-F (SEQ ID NO:19) and pyc-R (SEQ ID NO: 20); the panD fragment takes pyc gene shown in SEQ ID NO:18 as a template, and primers panD-F (SEQ ID NO:21) and panD-R (SEQ ID NO:22) are used for PCR amplification to obtain the panD fragment; the pTrc99A plasmid fragment was amplified by PCR using pTrc99A plasmid as a template and primers pTrc99A-F (SEQ ID NO:23) and pTrc99A-R (SEQ ID NO:24) to give a linearized pTrc99A plasmid fragment.

In some embodiments, in any of the methods described above, the recombinant E.coli is BL21(TPP) obtained by transferring pCDF-ppc-aspC, pRSF-yneI-pa0132 and pTrc99A-pyc-panD into BL21(DE 3).

BL21(SCR) recombinant bacteria express the fusion proteins ppc-linker-aspC (shown in SEQ ID NO: 35) and yneI-linker-pa0132 (shown in SEQ ID NO: 36) as well as protein pyc (shown in SEQ ID NO: 37) and panD (shown in SEQ ID NO: 38), wherein the amino acids 1-883 in the SEQ ID NO:35 are ppc protein, the amino acids 884-903 are linkers, the amino acids 904-1299 are aspC protein, the amino acids 1-462 in the SEQ ID NO:36 are yneI protein, the amino acids 463-482 are linkers, and the amino acids 483-930 are pa0132 protein. In contrast, BL21(TPP) recombinant bacteria individually expressed six proteins, ppc, aspC, yneI, pa0132, pyc and panD.

The third object of the present invention is to provide a method for producing malonic acid by using the recombinant Escherichia coli shake flask fermentation, which comprises inoculating any one of the recombinant Escherichia coli described above into an SOB medium containing 4-8g/L glucose, and fermenting at 30-37 ℃ and 300-.

In some embodiments, in the above method, the SoB medium contains peptone 20g/L, yeast extract 5g/L, MgSO 5₄·7H₂O2.47 g/L, NaCl 0.5g/L, KCl 0.186 g/L; the antibiotics ampicillin (Amp), kanamycin (Kan) and streptomycin (Str) were added to the medium at a final concentration of 1 mM.

In some embodiments, in any of the above methods, the rotation speed of 0-3h is 200-300r/min, and the dissolved oxygen and the rotation speed are linked after 3h to ensure that the dissolved oxygen is 25% -35%, and the stirring rotation speed range is 300-800 r/min; the fermentation temperature is 37 ℃ within 0h-12 h; the fermentation temperature is 34 ℃ within 12h-24 h; the fermentation temperature is 32 ℃ within 24h-32 h; the fermentation temperature from 32h to the end of fermentation is 30 ℃; whenever the glucose concentration dropped to 0g/L, glucose was fed to a final concentration of 8 g/L.

In some embodiments, the method of any one of the above, wherein the recombinant E.coli is further cultured in seed broth; the seed liquid culture is to culture the recombinant escherichia coli in an LB culture medium to obtain a seed liquid, then to inoculate the seed liquid to a fermentation culture medium for fermentation, and to obtain an initial OD₆₀₀Is 0.4-0.6. For example, the seed liquid can be prepared by selecting a single colony and inoculating the single colony in a 150mL conical flask containing 25mL of LB liquid medium at 37 ℃Shaking at 230rpm overnight or culturing for 12h, transferring 1mL bacterial solution into 50mL LB liquid culture medium the next day, culturing at 37 deg.C and 230rpm to OD₆₀₀Reach 4-6, transfer to 50mL SOB medium.

The fourth purpose of the invention is to provide the application of any one of the recombinant escherichia coli or any one of the fermentation methods in the preparation of malonic acid and products derived from malonic acid.

In some embodiments, in the above applications, the derivative product includes, but is not limited to, diethyl malonate.

In one embodiment, for the above uses, the derivative product includes, but is not limited to, barbiturates, vitamin B1 or vitamin B6.

The invention has the advantages that:

compared with a chemical method, the Escherichia coli total biological method is used for synthesizing the malonic acid, mainly takes glucose as a substrate, greatly reduces the production cost of the malonic acid, and is simpler and quicker to operate; compared with the previously reported biological method for synthesizing the malonic acid, the synthetic pathway mentioned in the invention has fewer reactions, so the loss of the carbon source is less;

three of six overexpressed genes are from BL21(DE3), so that the stability and activity of the enzyme are better;

the mature fermentation process makes it possible to synthesize malonic acid by using Escherichia coli full-biological method, and the method has simple operation and low cost, and can be used for industrial production;

the constructed strain is a constitutive expression strain, and the used promoter is ML2719 which is 1.4 times of the inducible T7 promoter; IPTG induction is not needed in the fermentation process, and the production cost is saved.

Drawings

FIG. 1 is a schematic diagram of the synthesis route of malonic acid constructed according to the present invention.

FIG. 2 is a map of pRSF-yneI-pa0132 plasmid.

FIG. 3 is a plasmid map of pCDF-ppc-aspC.

FIG. 4 is a map of pTrc99A-pyc-panD plasmid.

FIG. 5 is a plasmid map of pCDF-ppc-linker-aspC.

FIG. 6 is a map of pRSF-yneI-linker-pa0132 plasmid.

FIG. 7 shows the detection result of the shake flask fermentation liquid phase of recombinant bacteria BL21 (TPP).

FIG. 8 shows the results of liquid phase detection of recombinant strain BL21(SCR) in shake flask fermentation.

FIG. 9 shows the results of the fermentation liquid phase detection of recombinant bacteria BL21(SCR) in a 5L fermentation tank under the gradient cooling fermentation strategy.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

The pRSFDuet-1 plasmid is a Youbao biological product, and the catalog number is VT 1238.

The pCDFDuet-1 plasmid is a Youbao biological product, and the catalog number is VT 1239.

The pTrc99a plasmid is a Youbao biological product with the catalog number VT 1294.

Example 1: construction of recombinant plasmid and acquisition of BL21(TPP) recombinant bacteria

1. Construction of recombinant plasmid pRSF-yneI-pa0132

The succinic semialdehyde dehydrogenase gene (yneI) (shown in SEQ ID NO: 1) and the beta-alanine pyruvate transaminase gene (pa0132) (shown in SEQ ID NO: 2) were synthesized by Jinzhi Biotech, Suzhou, Inc.;

using succinic semialdehyde dehydrogenase gene (yneI) as a template, and carrying out PCR amplification by using primers yneI-F (5'-CACACAGGAAACAGACCATGACCATTACTCCGGCAACTC-3', SEQ ID NO:3) and yneI-R (5'-TTCTTTACCAGACTCGAGTCAGATCCGGTCTTTCCACAC-3', SEQ ID NO:4) to obtain a yneI fragment;

PCR amplification was performed using the beta-alanine pyruvate transaminase gene (pa0132) as a template with primers pa0132-F (5'-CACACAGGAAACAGACCATGAATCAGCCCCTGAATGTC-3', SEQ ID NO:5) and pa0132-R (5'-GCCGGATGATTAATTGTCAAAAGCTTTCAGGCAATTCCGTTCAGAG-3', SEQ ID NO:6) to obtain pa0132 fragment;

using pRSFDuet-1 plasmid as template, using primers pRSF-F (5'-CTCGAGTCTGGTAAAGAAACCGC-3', SEQ ID NO:7) and pRSF-R (5'-ATTTCCTAATGCAGGAGTCGCAT-3', SEQ ID NO:8) to make PCR amplification to obtain linearized pRSFDuet-1 plasmid fragment;

and carrying out homologous recombination on the yneI fragment, the pa0132 fragment and the linearized pRSFDuet-1 plasmid fragment by using a homologous recombinase to obtain a recombinant plasmid pRSF-yneI-pa0132, transferring the recombinant plasmid pRSF-yneI-pa0132 into E.coli JM109 competent cells, carrying out colony PCR (polymerase chain reaction) to pick out a positive transformant, carrying out quality plasmid enzyme cutting verification, verifying that the correct plasmid is sent for sequencing, wherein the sequencing result is consistent with an expected sequence, and the plasmid map is shown in figure 2.

2. Construction of recombinant plasmid pCDF-ppc-aspC

Phosphoenolpyruvate carboxylase gene (ppc) (shown in SEQ ID NO: 9) and aspartate aminotransferase gene (aspC) (shown in SEQ ID NO: 10) were synthesized by Kinzhi Biotech, Suzhou;

PCR amplification was performed using phosphoenolpyruvate carboxylase gene (ppc) as a template and the primers ppc-F (5'-AACAGACCCCATGGGCATGAACGAACAATATTCCGCATT-3', SEQ ID NO:11) and ppc-R (5'-GCCGGATGATTAATTGTCAAGAATTCTTAGCCGGTATTACGCATACCTG-3', SEQ ID NO:12) to obtain a ppc fragment;

PCR amplification was performed using the aspartate aminotransferase gene (aspC) as a template and the primers aspC-F (5'-CACACAGGAAACAGACCATGTTTGAGAACATTACCGCCG-3', SEQ ID NO:13) and aspC-R (5'-GCCGCAAGCTTGTCGACTTACAGCACTGCCACAATCGC-3', SEQ ID NO:14) to obtain an aspC fragment;

PCR amplification was performed using pCDFDuet-1 plasmid as a template and primers pCDF-F (5'-GTCGACAAGCTTGCGGCC-3', SEQ ID NO:15) and pCDF-R (5'-ATTTCCTAATGCAGGAGTCGCAT-3', SEQ ID NO:16) to obtain a linearized pCDFDuet-1 plasmid fragment;

homologous recombination is carried out on the ppc fragment, the aspC fragment and the linearized pCDFDuet-1 plasmid fragment by using a homologous recombinase to obtain a recombinant plasmid pCDF-ppc-aspC, the recombinant plasmid pCDF-ppc-aspC is transferred into E.coli JM109 competent cells, a positive transformant is picked by colony PCR, quality improvement and enzyme cutting verification are carried out, the correct plasmid is verified and sent for sequencing, the sequencing result is consistent with the expected sequence, and the plasmid map is shown in figure 3.

3. Construction of the recombinant plasmid pTrc99A-pyc-panD

Pyruvate carboxylase gene (pyc) (sequence shown in SEQ ID NO: 17) and aspartate-alpha decarboxylase gene (panD) (sequence shown in SEQ ID NO: 18) were synthesized by Kingzhi Biotech, Suzhou;

PCR amplification was performed using the pyruvate carboxylase gene (pyc) as a template and primers pyc-F (5'-CACACAGGAAACAGACCGTGTCGACTCACACATCTTCAACG-3', SEQ ID NO:19) and pyc-R (5'-GCCGGATGATTAATTGTCAAGAATTCCTTAGGAAACGACGACGATCAAGTC-3', SEQ ID NO:20) to give a pyc fragment;

PCR amplification was performed using the aspartate- α decarboxylase gene (panD) as a template, and the primers panD-F (5'-GAAACAGACCCTCGAGCAAGAGGTATATATTAATGTTGCGTACTATCC-3', SEQ ID NO:21) and panD-R (5'-GCCAAAACAGCCAAGCTTCTAGATCGAGCGACTGGTTAAAAG-3', SEQ ID NO:22) to obtain a panD fragment;

PCR amplification was performed using pTrc99A plasmid as a template and primers pTrc99A-F (5'-AAGCTTGGCTGTTTTGGCG-3', SEQ ID NO:23) and pTrc99A-R (5'-CAGCTCATTTCAGAATATTTGCCA-3', SEQ ID NO:24) to obtain a linearized pTrc99A plasmid fragment;

homologous recombination is carried out on the pyc fragment, the panD fragment and the linearized pTrc99A plasmid fragment by using a homologous recombinase to obtain a recombinant plasmid pTrc99A-pyc-panD, the recombinant plasmid pTrc 99-pyc-panD is transferred into E.coli JM109 competent cells, colony PCR is used for picking positive transformants and carrying out quality improvement and enzyme cutting verification, the correct plasmids are verified and sent for sequencing, the sequencing result is consistent with the expected sequence, and the plasmid map is shown in figure 4.

4. The pRSF-yneI-pa0132, pCDF-ppc-aspC and pTrc99A-pyc-panD plasmids were electroporated into BL21(DE3) to obtain BL21(TPP) recombinant bacteria.

Example 2: construction of recombinant plasmid and acquisition of BL21(SCR) recombinant bacteria

1. The linker gene (SEQ ID NO:25 in sequence) was synthesized by Kinzhi Biotech, Suzhou, and its encoded amino groupThe sequence is (SSSSSSSG)₄(SEQ ID NO:26) and was codon optimized according to the E.coli preference.

2. Construction of recombinant plasmid pCDF-ppc-linker-aspC

Using linker gene as template, using primer linker-pCDF-F (5'-TAATACCGGCGAATTCTCTTCAAGCTCTGGTAGCTCGTC-3', SEQ ID NO:27) and linker-pCDF-R (5'-CGGCGGTAATGTTCTCAAACATTCCGGAGCTCGAACTGCC-3', SEQ ID NO:28) to make PCR amplification to obtain linker fragment;

taking pCDF-ppc-aspC plasmid as a template, and carrying out PCR amplification by using primers pCDF-linker-F (5'-ATGTTTGAGAACATTACCGCCG-3', SEQ ID NO:29) and pCDF-linker-R (5'-GAATTCGCCGGTATTACGCA-3', SEQ ID NO:30) to obtain a linearized pCDF-ppc-aspC plasmid fragment;

homologous recombination is carried out on the linker fragment and the linearized pCDF-ppc-aspC plasmid fragment by using a homologous recombinase to obtain a recombinant plasmid pCDF-ppc-linker-aspC, the recombinant plasmid pCDF-ppc-linker-aspC is transferred into E.coli JM109 competent cells, a colony PCR (polymerase chain reaction) picks a positive transformant, plasmid cutting verification is carried out, a correct plasmid is verified and sent for sequencing, the sequencing result is consistent with an expected sequence, and a plasmid map is shown in figure 5.

3. Construction of recombinant plasmid pRSF-yneI-linker-pa0132

Using linker gene as template, using primer linker-pRSF-F (5'-ACGGAATTGCCAAGCTTTCTTCAAGCTCTGGTAGCTCGTC-3', SEQ ID NO:31) and linker-pRSF-R (5'-CCGGAGTAATGGTCATTCCGGAGCTCGAACTGCC-3', SEQ ID NO:32) to carry out PCR amplification to obtain linker fragment;

using pRSF-yneI-pa0132 plasmid as a template, and carrying out PCR amplification by using primers pRSF-linker-F (5'-ATGACCATTACTCCGGCAACTC-3', SEQ ID NO:33) and pRSF-linker-R (5'-AAGCTTGGCAATTCCGTTCA-3', SEQ ID NO:34) to obtain a linearized pRSF-yneI-pa0132 plasmid fragment;

homologous recombination is carried out on the linker fragment and the linearized pRSF-yneI-pa0132 plasmid fragment by using a homologous recombinase to obtain a recombinant plasmid pRSF-yneI-linker-pa0132, the recombinant plasmid pRSF-yneI-linker-pa0132 is transferred into E.coli JM109 competent cells, a positive transformant is picked by colony PCR, plasmid-upgrading and enzyme cutting verification are carried out, a correct plasmid is verified and sent for sequencing, the sequencing result is consistent with an expected sequence, and a plasmid map is shown in figure 6.

4. The plasmids pCDF-ppc-linker-aspC, pRSF-yneI-linker-pa0132 and pTrc99A-pyc-panD were electroporated into BL21(DE3) to obtain BL21(SCR) recombinant bacteria.

BL21(SCR) recombinant bacteria express the fusion proteins ppc-linker-aspC (shown in SEQ ID NO: 35) and yneI-linker-pa0132 (shown in SEQ ID NO: 36) as well as protein pyc (shown in SEQ ID NO: 37) and panD (shown in SEQ ID NO: 38), wherein the amino acids 1-883 in the SEQ ID NO:35 are ppc protein, the amino acids 884-903 are linkers, the amino acids 904-1299 are aspC protein, the amino acids 1-462 in the SEQ ID NO:36 are yneI protein, the amino acids 463-482 are linkers, and the amino acids 483-930 are pa0132 protein. In contrast, the BL21(TPP) recombinant strain prepared in example 1 expressed six proteins of ppc, aspC, yneI, pa0132, pyc, and panD, respectively.

The constitutive promoter ML2719 is used to drive the expression of each protein, and the sequence is shown in SEQ ID NO: 39.

Example 3: shake flask fermentation of recombinant escherichia coli

1. Preparing a seed solution: recombinant strains BL21(TPP) and BL21(SCR) obtained by activating an LB solid plate are respectively inoculated in 25mL of liquid LB culture medium, cultured at 37 ℃ at 230r/min for 12h and then inoculated with secondary seed liquid. 1mL of the culture broth was inoculated into 25mL of liquid LB medium and cultured at 37 ℃ at 230r/min for 12 hours.

2. And (3) fermentation: the inoculum size of 2% was inoculated into SOB medium with a liquid loading of 50mL/250mL, and incubated at 37 ℃ and 230 r/min. SOB medium: peptone 20g/L, yeast extract 5g/L, MgSO₄·7H₂O2.47 g/L, NaCl 0.5g/L, KCl 0.186g/L, and the antibiotics ampicillin (Amp), kanamycin (Kan), and streptomycin (Str), all at a final concentration of 1mM, were added to the medium.

And (4) analyzing results: sampling every 12H in the fermentation process, centrifuging for 10min at 13000r/min and passing the supernatant through 0.22 μ L filter membrane for HPLC detection (high performance liquid chromatography, American Bio-Rad Burley Aminex HPX-87H organic acid column with detection parameters of 5mM H)₂SO₄As a mobile phase, the flow rate of 0.6mL/min is used for sample injection, the column temperature is 30 ℃,the sample volume was 20. mu.L, and the temperature of the differential detector was 30 ℃.

The liquid phase detection result of the malonic acid accumulation amount of the recombinant bacterium BL21(TPP) is shown in FIG. 7, and the bacterium accumulates 0.61g/L malonic acid in 48 hours; the results of liquid phase detection of the accumulation of recombinant strain BL21(SCR) on malonic acid are shown in FIG. 8, and the accumulation of 0.83g/L malonic acid in 48h is improved by 36% compared with that in unfused strain BL21(TPP), which indicates that the fusion expression of phosphoenolpyruvate carboxylase and aspartate aminotransferase and the fusion expression of succinate semialdehyde dehydrogenase and beta-alanine pyruvate aminotransferase can reduce the loss of intermediate metabolites and promote the synthesis of malonic acid.

Example 4: 5L fermenter fermentation of recombinant Escherichia coli BL21(SCR)

1. Preparing a seed solution: the recombinant strain BL21(SCR) obtained by activating the LB solid plate is inoculated in 25mL of liquid LB culture medium, and the secondary seed solution is inoculated after the culture is carried out for 12h at 37 ℃ and 230 r/min. Inoculating 1mL of the culture solution into six bottles of 50mL liquid LB culture medium respectively, culturing at 37 ℃ and 230r/min for 12h, OD₆₀₀Up to 4.42.

2. Fermentation conditions are as follows: inoculum size 10% (initial OD) by volume₆₀₀0.46) and initial glucose concentration of 4g/L, wherein the rotating speed of 0-3h is 300r/min, and the dissolved oxygen is linked with the rotating speed after 3h so as to ensure that the dissolved oxygen is about 25 percent and the stirring rotating speed range is 300-800 r/min. The fermentation temperature is 37 ℃ within 0h-12 h; the fermentation temperature is 34 ℃ within 12h-24 h; the fermentation temperature is 32 ℃ between 24h and 32 h; the fermentation temperature from 32h to the end of the fermentation was 30 ℃. Every time the glucose concentration dropped to 0g/L, glucose was fed to a final concentration of 8g/L and sampled.

The fermentation medium is an SOB medium: peptone 20g/L, yeast extract 5g/L, MgSO₄·7H₂O2.47 g/L, NaCl 0.5g/L, KCl 0.186g/L, antibiotics ampicillin (Amp), kanamycin (Kan) and streptomycin (Str), all at a final concentration of 1mM, were added to the medium.

And (4) analyzing results: the procedure was the same as in example 3, and the fermentation results are shown in FIG. 9. The result shows that the recombinant bacterium BL21(SCR) accumulates 5.61g/L of malonic acid in 54 h.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQ ID NO:1：

SEQ ID NO:2：

SEQ ID NO:9：

SEQ ID NO:10：

SEQ ID NO:17：

SEQ ID NO:18：

SEQ ID NO:25

tcttcaagctc tggtagctcg tcgtctgggt cttcaagttc cggcagttcg agctccgga

SEQ ID NO:35

MNEQYSALRSNVSMLGKVLGETIKDALGEHILERVETIRKLSKSSRAGNDANRQELLTTLQNLSNDELLPVARAFSQFLNLANTAEQYHSISPKGEAASNPEVIARTLRKLKNQPELSEDTIKKAVESLSLELVLTAHPTEITRRTLIHKMVEVNACLKQLDNKDIADYEHNQLMRRLRQLIAQSWHTDEIRKLRPSPVDEAKWGFAVVENSLWQGVPNYLRELNEQLEENLGYKLPVEFVPVRFTSWMGGDRDGNPNVTADITRHVLLLSRWKATDLFLKDIQVLVSELSMVEATPELLALVGEEGAAEPYRYLMKNLRSRLMATQAWLEARLKGEELPKPEGLLTQNEELWEPLYACYQSLQACGMGIIANGDLLDTLRRVKCFGVPLVRIDIRQESTRHTEALGELTRYLGIGDYESWSEADKQAFLIRELNSKRPLLPRNWQPSAETREVLDTCQVIAEAPQGSIAAYVISMAKTPSDVLAVHLLLKEAGIGFAMPVAPLFETLDDLNNANDVMTQLLNIDWYRGLIQGKQMVMIGYSDSAKDAGVMAASWAQYQAQDALIKTCEKAGIELTLFHGRGGSIGRGGAPAHAALLSQPPGSLKGGLRVTEQGEMIRFKYGLPEITVSSLSLYTGAILEANLLPPPEPKESWRRIMDELSVISCDLYRGYVRENKDFVPYFRSATPEQELGKLPLGSRPAKRRPTGGVESLRAIPWIFAWTQNRLMLPAWLGAGTALQKVVEDGKQSELEAMCRDWPFFSTRLGMLEMVFAKADLWLAEYYDQRLVDKALWPLGKELRNLQEEDIKVVLAIANDSHLMADLPWIAESIQLRNIYTDPLNVLQAELLHRSRQAEKEGQEPDPRVEQALMVTIAGIAAGMRNTGSSSSGSSSSGSSSSGSSSSGMFENITAAPADPILGLADLFRADERPGKINLGIGVYKDETGKTPVLTSVKKAEQYLLENETTKNYLGIDGIPEFGRCTQELLFGKGSALINDKRARTAQTPGGTGALRVAADFLAKNTSVKRVWVSNPSWPNHKSVFNSAGLEVREYAYYDAENHTLDFDALINSLNEAQAGDVVLFHGCCHNPTGIDPTLEQWQTLAQLSVEKGWLPLFDFAYQGFARGLEEDAEGLRAFAAMHKELIVASSYSKNFGLYNERVGACTLVAADSETVDRAFSQMKAAIRANYSNPPAHGASVVATILSNDALRAIWEQELTDMRQRIQRMRQLFVNTLQEKGANRDFSFIIKQNGMFSFSGLTKEQVLRLREEFGVYAVASGRVNVAGMTPDNMAPLCEAIVAVL

SEQ ID NO:36

MTITPATHAISINPATGEQLSVLPWAGADDIENALQLAAAGFRDWRETNIDYRAEKLRDIGKALRARSEEMAQMITREMGKPINQARAEVAKSANLCDWYAEHGPAMLKAEPTLVENQQAVIEYRPLGTILAIMPWNFPLWQVMRGAVPIILAGNGYLLKHAPNVMGCAQLIAQVFKDAGIPQGVYGWLNADNDGVSQMIKDSRIAAVTVTGSVRAGAAIGAQAGAALKKCVLELGGSDPFIVLNDADLELAVKAAVAGRYQNTGQVCAAAKRFIIEEGIASAFTERFVAAAAALKMGDPRDEENALGPMARFDLRDELHHQVEKTLAQGARLLLGGEKMAGAGNYYPPTVLANVTPEMTAFREEMFGPVAAITIAKDAEHALELANDSEFGLSATIFTTDETQARQMAARLECGGVFINGYCASDARVAFGGVKKSGFGRELSHFGLHEFCNIQTVWKDRISSSSGSSSSGSSSSGSSSSGMNQPLNVAPPVSSELNLRAHWMPFSANRNFQKDPRIIVAAEGSWLTDDKGRKVYDSLSGLWTCGAGHSRKEIQEAVARQLGTLDYSPGFQYGHPLSFQLAEKIAGLLPGELNHVFFTGSGSECADTSIKMARAYWRLKGQPQKTKLIGRARGYHGVNVAGTSLGGIGGNRKMFGQLMDVDHLPHTLQPGMAFTRGMAQTGGVELANELLKLIELHDASNIAAVIVEPMSGSAGVLVPPVGYLQRLREICDQHNILLIFDEVITAFGRLGTYSGAEYFGVTPDLMNVAKQVTNGAVPMGAVIASSEIYDTFMNQALPEHAVEFSHGYTYSAHPVACAAGLAALDILARDNLVQQSAELAPHFEKGLHGLQGAKNVIDIRNCGLAGAIQIAPRDGDPTVRPFEAGMKLWQQGFYVRFGGDTLQFGPTFNARPEELDRLFDAVGEALNGIA

SEQ ID NO:37

MSTHTSSTLPAFKKILVANRGEIAVRAFRAALETGAATVAIYPREDRGSFHRSFASEAVRIGTEGSPVKAYLDIDEIIGAAKKVKADAIYPGYGFLSENAQLARECAENGITFIGPTPEVLDLTGDKSRAVTAAKKAGLPVLAESTPSKNIDEIVKSAEGQTYPIFVKAVAGGGGRGMRFVASPDELRKLATEASREAEAAFGDGAVYVERAVINPQHIEVQILGDHTGEVVHLYERDCSLQRRHQKVVEIAPAQHLDPELRDRICADAVKFCRSIGYQGAGTVEFLVDEKGNHVFIEMNPRIQVEHTVTEEVTEVDLVKAQMRLAAGATLKELGLTQDKIKTHGAALQCRITTEDPNNGFRPDTGTITAYRSPGGAGVRLDGAAQLGGEITAHFDSMLVKMTCRGSDFETAVARAQRALAEFTVSGVATNIGFLRALLREEDFTSKRIATGFIADHPHLLQAPPADDEQGRILDYLADVTVNKPHGVRPKDVAAPIDKLPNIKDLPLPRGSRDRLKQLGPAAFARDLREQDALAVTDTTFRDAHQSLLATRVRSFALKPAAEAVAKLTPELLSVEAWGGATYDVAMRFLFEDPWDRLDELREAMPNVNIQMLLRGRNTVGYTPYPDSVCRAFVKEAASSGVDIFRIFDALNDVSQMRPAIDAVLETNTAVAEVAMAYSGDLSDPNEKLYTLDYYLKMAEEIVKSGAHILAIKDMAGLLRPAAVTKLVTALRREFDLPVHVHTHDTAGGQLATYFAAAQAGADAVDGASAPLSGTTSQPSLSAIVAAFAHTRRDTGLSLEAVSDLEPYWEAVRGLYLPFESGTPGPTGRVYRHEIPGGQLSNLRAQATALGLADRFELIEDNYAAVNEMLGRPTKVTPSSKVVGDLALHLVGAGVDPADFAADPQKYDIPDSVIAFLRGELGNPPGGWPEPLRTRALEGRSEGKAPLTEVPEEEQAHLDADDSKERRNSLNRLLFPKPTEEFLEHRRRFGNTSALDDREFFYGLVEGRETLIRLPDVRTPLLVRLDAISEPDDKGMRNVVANVNGQIRPMRVRDRSVESVTATAEKADSSNKGHVAAPFAGVVTVTVAEGDEVKAGDAVAIIEAMKMEATITASVDGKIDRVVVPAATKVEGGDLIVVVS

SEQ ID NO:38

MLRTILGSKIHRATVTQADLDYVGSVTIDADLVHAAGLIEGEKVAIVDITNGARLETYVIVGDAGTGNICINGAAAHLINPGDLVIIMSYLQATDAEAKAYEPKIVHVDADNRIVALGNDLAEALPGSGLLTSRSI

SEQ ID NO:39

Sequence listing

<110> Wanhua chemical group, Inc

Wan Hua Hua Xue (Sichuan) Co., Ltd.

<120> recombinant escherichia coli, preparation method and application thereof

<130> DSP1F220070ZX

<160> 39

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1389

<212> DNA

<213> Escherichia coli K12(Escherichia coli K-12)

<400> 1

atgaccatta ctccggcaac tcatgcaatt tcgataaatc ctgccacggg tgaacaactt 60

tctgtgctgc cgtgggctgg cgctgacgat atcgaaaacg cacttcagct ggcggcagca 120

ggctttcgcg actggcgcga gacaaatata gattatcgtg ctgaaaaact gcgtgatatc 180

ggtaaggctc tgcgcgctcg tagcgaagaa atggcgcaaa tgatcacccg cgaaatgggc 240

aaaccaatca accaggcgcg cgctgaagtg gcgaaatcgg cgaatttgtg tgactggtat 300

gcagaacatg gtccggcaat gctgaaggcg gaacctacgc tggtggaaaa tcagcaggcg 360

gttattgagt atcgaccgtt ggggacgatt ctggcgatta tgccgtggaa ttttccgtta 420

tggcaggtga tgcgtggcgc tgttcccatc attcttgcag gtaacggcta cttacttaaa 480

catgcgccga atgtgatggg ctgtgcacag ctcattgccc aggtgtttaa agatgcgggt 540

atcccacaag gcgtatatgg ctggctgaat gccgacaacg acggtgtcag tcagatgatt 600

aaagactcgc gcattgctgc tgtcacggtg accggaagtg ttcgtgcggg agcggctatt 660

ggcgcacagg ctggagcggc actgaaaaaa tgcgtactgg aactgggcgg ttcggatccg 720

tttattgtgc ttaacgatgc cgatctggaa ctggcggtga aagcggcggt agccggacgt 780

tatcagaata ccggacaggt atgtgcagcg gcaaaacgct ttattatcga agagggaatt 840

gcttcggcat ttaccgaacg ttttgtggca gctgcggcag ccttgaaaat gggcgatccc 900

cgtgacgaag agaacgctct cggaccaatg gctcgttttg atttacgtga tgagctgcat 960

catcaggtgg agaaaaccct ggcgcagggt gcgcgtttgt tactgggcgg ggaaaagatg 1020

gctggggcag gtaactacta tccgccaacg gttctggcga atgttacccc agaaatgacc 1080

gcgtttcggg aagaaatgtt tggccccgtt gcggcaatca ccattgcgaa agatgcagaa 1140

catgcactgg aactggctaa tgatagtgag ttcggccttt cagcgaccat ttttaccact 1200

gacgaaacac aggccagaca gatggcggca cgtctggaat gcggtggggt gtttatcaat 1260

ggttattgtg ccagcgacgc gcgagtggcc tttggtggcg tgaaaaagag tggctttggt 1320

cgtgagcttt cccatttcgg cttacacgaa ttctgtaata tccagacggt gtggaaagac 1380

cggatctga 1389

<210> 2

<211> 1347

<212> DNA

<213> Pseudomonas aeruginosa (Pseudomonas aeruginosa)

<400> 2

atgaatcagc ccctgaatgt cgctccgccc gtgagctcgg aattaaacct gcgcgcccac 60

tggatgccat tttcggctaa ccgcaatttc caaaaagacc cgcgtattat cgtcgcggcg 120

gagggctcct ggctgaccga cgacaagggc cgtaaagtat acgatagcct gtcaggatta 180

tggacctgcg gtgcggggca tagccgcaag gaaattcagg aagcggttgc tcgtcaactg 240

gggactttgg actattcgcc aggattccaa tatggacatc cattgtcttt ccagttggcc 300

gagaagattg ctgggttatt acctggggaa ttaaaccatg tcttttttac gggatcaggg 360

tcggagtgcg cagacacttc gattaagatg gcccgcgcct actggcgctt aaagggacaa 420

ccccagaaga ctaagctgat tggacgcgca cgcggttacc acggcgtgaa tgtcgcgggc 480

acaagccttg gagggatcgg ggggaaccgc aagatgttcg gacagctgat ggatgtggac 540

catcttcccc atacccttca gccaggtatg gcattcactc gcgggatggc gcagacagga 600

ggcgttgaac tggcgaatga gttattaaag ttaattgaat tgcacgatgc gtctaacatc 660

gcagcggtta ttgtcgagcc catgtccggt tccgcaggag ttttagtgcc acccgtgggc 720

tatctgcagc gtttgcgcga aatctgtgac caacacaata ttctgcttat ctttgatgaa 780

gtgattacgg ctttcgggcg tctgggtact tactcgggag ccgaatactt cggggtcacg 840

ccggacttga tgaatgttgc aaaacaggtc acgaatggtg cagtacctat gggtgctgta 900

atcgcctcta gcgagattta cgatactttc atgaaccagg cgctgcctga acatgcggtt 960

gaattttccc acggttatac atattcagcg cacccagtgg catgtgctgc gggattagca 1020

gcactggaca tcttggcgcg cgataactta gtacagcagt cagcagagtt agctccacac 1080

ttcgagaagg gattgcatgg ccttcaaggt gccaagaatg ttatcgacat tcgcaactgc 1140

ggcttagcag gcgcgatcca gatcgctccc cgtgatgggg atccgacagt tcgccccttt 1200

gaagccggga tgaaactgtg gcaacaaggg ttttacgtcc gtttcggcgg cgacactctg 1260

cagtttgggc caacatttaa tgcacgccca gaggaattgg accgtctttt tgacgctgta 1320

ggtgaggctc tgaacggaat tgcctga 1347

<210> 3

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cacacaggaa acagaccatg accattactc cggcaactc 39

<210> 4

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ttctttacca gactcgagtc agatccggtc tttccacac 39

<210> 5

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cacacaggaa acagaccatg aatcagcccc tgaatgtc 38

<210> 6

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gccggatgat taattgtcaa aagctttcag gcaattccgt tcagag 46

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ctcgagtctg gtaaagaaac cgc 23

<210> 8

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atttcctaat gcaggagtcg cat 23

<210> 9

<211> 2652

<212> DNA

<213> E.coli BL21(DE3) (Escherichia coli BL21(DE3))

<400> 9

atgaacgaac aatattccgc attgcgtagt aatgtcagta tgctcggcaa agtgctggga 60

gaaaccatca aggatgcgtt gggagaacac attcttgaac gcgtagaaac tatccgtaag 120

ttgtcgaaat cttcacgcgc tggcaatgat gctaaccgcc aggagttgct caccacctta 180

caaaatttgt cgaacgacga gctgctgccc gttgcgcgtg cgtttagtca gttcctgaac 240

ctggccaaca ccgccgagca ataccacagc atttcgccga aaggcgaagc tgccagcaac 300

ccggaagtga tcgcccgcac cctgcgtaaa ctgaaaaacc agccggaact gagcgaagac 360

accatcaaaa aagcagtgga atcgctgtcg ctggaactgg tcctcacggc tcacccaacc 420

gaaattaccc gtcgtacact gatccacaaa atggtggaag tgaacgcctg tttaaaacag 480

ctcgataaca aagatatcgc tgactacgaa cacaaccagc tgatgcgtcg cctgcgccag 540

ttgatcgccc agtcatggca taccgatgaa atccgtaagc tgcgtccaag cccggtagat 600

gaagccaaat ggggctttgc cgtagtggaa aacagcctgt ggcaaggcgt accaaattac 660

ctgcgcgaac tgaacgaaca actggaagag aacctcggct acaaactgcc cgtcgaattt 720

gttccggtcc gttttacttc gtggatgggc ggcgaccgcg acggcaaccc gaacgtcact 780

gccgatatca cccgccacgt cctgctactc agccgctgga aagccaccga tttgttcctg 840

aaagatattc aggtgctggt ttctgaactg tcgatggttg aagcgacccc tgaactgctg 900

gcgctggttg gcgaagaagg tgccgcagaa ccgtatcgct atctgatgaa aaacctgcgt 960

tctcgcctga tggcgacaca ggcatggctg gaagcgcgcc tgaaaggcga agaactgcca 1020

aaaccagaag gcctgctgac acaaaacgaa gaactgtggg aaccgctcta cgcttgctac 1080

cagtcacttc aggcgtgtgg catgggtatt atcgccaacg gcgatctgct cgacaccctg 1140

cgccgcgtga aatgtttcgg cgtaccgctg gtccgtattg atatccgtca ggagagcacg 1200

cgtcataccg aagcgctggg cgagctgacc cgctacctcg gtatcggcga ctacgaaagc 1260

tggtcagagg ccgacaaaca ggcgttcctg atccgcgaac tgaactccaa acgtccgctt 1320

ctgccgcgca actggcaacc aagcgccgaa acgcgcgaag tgctcgatac ctgccaggtg 1380

attgccgaag caccgcaagg ctccattgcc gcctacgtga tctcgatggc gaaaacgccg 1440

tccgacgtac tggctgtcca cctgctgctg aaagaagcgg gtatcgggtt tgcgatgccg 1500

gttgctccgc tgtttgaaac cctcgatgat ctgaacaacg ccaacgatgt catgacccag 1560

ctgctcaata ttgactggta tcgtggcctg attcagggca aacagatggt gatgattggc 1620

tattccgact cagcaaaaga tgcgggagtg atggcagctt cctgggcgca atatcaggca 1680

caggatgcat taatcaaaac ctgcgaaaaa gcgggtattg agctgacgtt gttccacggt 1740

cgcggcggtt ccattggtcg cggcggcgca cctgctcatg cggcgctgct gtcacaaccg 1800

ccaggaagcc tgaaaggcgg cctgcgcgta accgaacagg gcgagatgat ccgctttaaa 1860

tatggtctgc cagaaatcac cgtcagcagc ctgtcgcttt ataccggggc gattctggaa 1920

gccaacctgc tgccaccgcc ggagccgaaa gagagctggc gtcgcattat ggatgaactg 1980

tcagtcatct cctgcgatgt ctaccgcggc tacgtacgtg aaaacaaaga ttttgtgcct 2040

tacttccgct ccgctacgcc ggaacaagaa ctgggcaaac tgccgttggg ttcacgtccg 2100

gcgaaacgtc gcccaaccgg cggcgtcgag tcactacgcg ccattccgtg gatcttcgcc 2160

tggacgcaaa accgtctgat gctccccgcc tggctgggtg caggtacggc gctgcaaaaa 2220

gtggtcgaag acggcaaaca gagcgagctg gaggctatgt gccgcgattg gccattcttc 2280

tcgacgcgtc tcggcatgct ggagatggtc ttcgccaaag cagacctgtg gctggcggaa 2340

tactatgacc aacgcctggt agacaaagca ctgtggccgt taggtaaaga gttacgcaac 2400

ctgcaagaag aagacatcaa agtggtgctg gcgattgcca acgattccca tctgatggcc 2460

gatctgccgt ggattgcaga gtctattcag ctacggaata tttacaccga cccgctgaac 2520

gtattgcagg ccgagttgct gcaccgctcc cgccaggcag aaaaagaagg ccaggaaccg 2580

gatcctcgcg tcgaacaagc gttaatggtc actattgccg ggattgcggc aggtatgcgt 2640

aataccggct aa 2652

<210> 10

<211> 1191

<212> DNA

<213> Escherichia coli BL21(DE3) (Escherichia coli BL21(DE3))

<400> 10

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> 11

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

aacagacccc atgggcatga acgaacaata ttccgcatt 39

<210> 12

<211> 49

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gccggatgat taattgtcaa gaattcttag ccggtattac gcatacctg 49

<210> 13

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cacacaggaa acagaccatg tttgagaaca ttaccgccg 39

<210> 14

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gccgcaagct tgtcgactta cagcactgcc acaatcgc 38

<210> 15

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gtcgacaagc ttgcggcc 18

<210> 16

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

atttcctaat gcaggagtcg cat 23

<210> 17

<211> 3423

<212> DNA

<213> Corynebacterium glutamicum (Corynebacterium glutamicum)

<400> 17

gtgtcgactc acacatcttc aacgcttcca gcattcaaaa agatcttggt agcaaaccgc 60

ggcgaaatcg cggtccgtgc tttccgtgca gcactcgaaa ccggtgcagc cacggtagct 120

atttaccccc gtgaagatcg gggatcattc caccgctctt ttgcttctga agctgtccgc 180

attggtaccg aaggctcacc agtcaaggcg tacctggaca tcgatgaaat tatcggtgca 240

gctaaaaaag ttaaagcaga tgccatttac ccgggatacg gcttcctgtc tgaaaatgcc 300

cagcttgccc gcgagtgtgc ggaaaacggc attactttta ttggcccaac cccagaggtt 360

cttgatctca ccggtgataa gtctcgcgcg gtaaccgccg cgaagaaggc tggtctgcca 420

gttttggcgg aatccacccc gagcaaaaac atcgatgaga tcgttaaaag cgctgaaggc 480

cagacttacc ccatctttgt gaaggcagtt gccggtggtg gcggacgcgg tatgcgtttt 540

gttgcttcac ctgatgagct tcgcaaatta gcaacagaag catctcgtga agctgaagcg 600

gctttcggcg atggcgcggt atatgtcgaa cgtgctgtga ttaaccctca gcatattgaa 660

gtgcagatcc ttggcgatca cactggagaa gttgtacacc tttatgaacg tgactgctca 720

ctgcagcgtc gtcaccaaaa agttgtcgaa attgcgccag cacagcattt ggatccagaa 780

ctgcgtgatc gcatttgtgc ggatgcagta aagttctgcc gctccattgg ttaccagggc 840

gcgggaaccg tggaattctt ggtcgatgaa aagggcaacc acgtcttcat cgaaatgaac 900

ccacgtatcc aggttgagca caccgtgact gaagaagtca ccgaggtgga cctggtgaag 960

gcgcagatgc gcttggctgc tggtgcaacc ttgaaggaat tgggtctgac ccaagataag 1020

atcaagaccc acggtgcagc actgcagtgc cgcatcacca cggaagatcc aaacaacggc 1080

ttccgcccag ataccggaac tatcaccgcg taccgctcac caggcggagc tggcgttcgt 1140

cttgacggtg cagctcagct cggtggcgaa atcaccgcac actttgactc catgctggtg 1200

aaaatgacct gccgtggttc cgactttgaa actgctgttg ctcgtgcaca gcgcgcgttg 1260

gctgagttca ccgtgtctgg tgttgcaacc aacattggtt tcttgcgtgc gttgctgcgg 1320

gaagaggact tcacttccaa gcgcatcgcc accggattca ttgccgatca cccgcacctc 1380

cttcaggctc cacctgctga tgatgagcag ggacgcatcc tggattactt ggcagatgtc 1440

accgtgaaca agcctcatgg tgtgcgtcca aaggatgttg cagctcctat cgataagctg 1500

cctaacatca aggatctgcc actgccacgc ggttcccgtg accgcctgaa gcagcttggc 1560

ccagccgcgt ttgctcgtga tctccgtgag caggacgcac tggcagttac tgataccacc 1620

ttccgcgatg cacaccagtc tttgcttgcg acccgagtcc gctcattcgc actgaagcct 1680

gcggcagagg ccgtcgcaaa gctgactcct gagcttttgt ccgtggaggc ctggggcggc 1740

gcgacctacg atgtggcgat gcgtttcctc tttgaggatc cgtgggacag gctcgacgag 1800

ctgcgcgagg cgatgccgaa tgtaaacatt cagatgctgc ttcgcggccg caacaccgtg 1860

ggatacaccc cgtacccaga ctccgtctgc cgcgcgtttg ttaaggaagc tgccagctcc 1920

ggcgtggaca tcttccgcat cttcgacgcg cttaacgacg tctcccagat gcgtccagca 1980

atcgacgcag tcctggagac caacaccgcg gtagccgagg tggctatggc ttattctggt 2040

gatctctctg atccaaatga aaagctctac accctggatt actacctaaa gatggcagag 2100

gagatcgtca agtctggcgc tcacatcttg gccattaagg atatggctgg tctgcttcgc 2160

ccagctgcgg taaccaagct ggtcaccgca ctgcgccgtg aattcgatct gccagtgcac 2220

gtgcacaccc acgacactgc gggtggccag ctggcaacct actttgctgc agctcaagct 2280

ggtgcagatg ctgttgacgg tgcttccgca ccactgtctg gcaccacctc ccagccatcc 2340

ctgtctgcca ttgttgctgc attcgcgcac acccgtcgcg ataccggttt gagcctcgag 2400

gctgtttctg acctcgagcc gtactgggaa gcagtgcgcg gactgtacct gccatttgag 2460

tctggaaccc caggcccaac cggtcgcgtc taccgccacg aaatcccagg cggacagttg 2520

tccaacctgc gtgcacaggc caccgcactg ggccttgcgg atcgtttcga actcatcgaa 2580

gacaactacg cagccgttaa tgagatgctg ggacgcccaa ccaaggtcac cccatcctcc 2640

aaggttgttg gcgacctcgc actccacctc gttggtgcgg gtgtggatcc agcagacttt 2700

gctgccgatc cacaaaagta cgacatccca gactctgtca tcgcgttcct gcgcggcgag 2760

cttggtaacc ctccaggtgg ctggccagag ccactgcgca cccgcgcact ggaaggccgc 2820

tccgaaggca aggcacctct gacggaagtt cctgaggaag agcaggcgca cctcgacgct 2880

gatgattcca aggaacgtcg caatagcctc aaccgcctgc tgttcccgaa gccaaccgaa 2940

gagttcctcg agcaccgtcg ccgcttcggc aacacctctg cgctggatga tcgtgaattc 3000

ttctacggcc tggtcgaagg ccgcgagact ttgatccgcc tgccagatgt gcgcacccca 3060

ctgcttgttc gcctggatgc gatctctgag ccagacgata agggtatgcg caatgttgtg 3120

gccaacgtca acggccagat ccgcccaatg cgtgtgcgtg accgctccgt tgagtctgtc 3180

accgcaaccg cagaaaaggc agattcctcc aacaagggcc atgttgctgc accattcgct 3240

ggtgttgtca ccgtgactgt tgctgaaggt gatgaggtca aggctggaga tgcagtcgca 3300

atcatcgagg ctatgaagat ggaagcaaca atcactgctt ctgttgacgg caaaatcgat 3360

cgcgttgtgg ttcctgctgc aacgaaggtg gaaggtggcg acttgatcgt cgtcgtttcc 3420

taa 3423

<210> 18

<211> 411

<212> DNA

<213> E.coli BL21(DE3) (Escherichia coli BL21(DE3))

<400> 18

atgttgcgta ctatcctggg ctccaaaatt catcgtgcca ccgtcacgca ggcagacttg 60

gattatgtgg gctccgtgac catcgacgcg gacttagtcc acgccgccgg gttgatcgaa 120

ggcgagaaag tggcgattgt agacattacc aacggggctc gcttggaaac ttatgtcatt 180

gtgggtgatg cgggaactgg gaacatctgc attaacgggg ccgcagctca tctgatcaat 240

ccgggcgatt tggtgatcat catgtcatat ttgcaagcga cggatgcaga agctaaagca 300

tatgagccga agatcgtcca tgtcgacgct gataaccgca ttgtggcgct gggaaacgac 360

ctggctgagg ccttgccagg ttcaggcctt ttaaccagtc gctcgatcta g 411

<210> 19

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

cacacaggaa acagaccgtg tcgactcaca catcttcaac g 41

<210> 20

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gccggatgat taattgtcaa gaattcctta ggaaacgacg acgatcaagt c 51

<210> 21

<211> 48

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gaaacagacc ctcgagcaag aggtatatat taatgttgcg tactatcc 48

<210> 22

<211> 42

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

gccaaaacag ccaagcttct agatcgagcg actggttaaa ag 42

<210> 23

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

aagcttggct gttttggcg 19

<210> 24

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

cagctcattt cagaatattt gcca 24

<210> 25

<211> 60

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

tcttcaagct ctggtagctc gtcgtctggg tcttcaagtt ccggcagttc gagctccgga 60

<210> 26

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly Ser

1 5 10 15

Ser Ser Ser Gly

20

<210> 27

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

taataccggc gaattctctt caagctctgg tagctcgtc 39

<210> 28

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

cggcggtaat gttctcaaac attccggagc tcgaactgcc 40

<210> 29

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

atgtttgaga acattaccgc cg 22

<210> 30

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

gaattcgccg gtattacgca 20

<210> 31

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

acggaattgc caagctttct tcaagctctg gtagctcgtc 40

<210> 32

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

ccggagtaat ggtcattccg gagctcgaac tgcc 34

<210> 33

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

atgaccatta ctccggcaac tc 22

<210> 34

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

aagcttggca attccgttca 20

<210> 35

<211> 1299

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 35

Met Asn Glu Gln Tyr Ser Ala Leu Arg Ser Asn Val Ser Met Leu Gly

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Asn Asp Glu Leu Leu Pro Val Ala Arg Ala Phe Ser Gln Phe Leu Asn

65 70 75 80

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

85 90 95

Ala Ala Ser Asn Pro Glu Val Ile Ala Arg Thr Leu Arg Lys Leu Lys

100 105 110

Asn Gln Pro Glu Leu Ser Glu Asp Thr Ile Lys Lys Ala Val Glu Ser

115 120 125

Leu Ser Leu Glu Leu Val Leu Thr Ala His Pro Thr Glu Ile Thr Arg

130 135 140

Arg Thr Leu Ile His Lys Met Val Glu Val Asn Ala Cys Leu Lys Gln

145 150 155 160

Leu Asp Asn Lys Asp Ile Ala Asp Tyr Glu His Asn Gln Leu Met Arg

165 170 175

Arg Leu Arg Gln Leu Ile Ala Gln Ser Trp His Thr Asp Glu Ile Arg

180 185 190

Lys Leu Arg Pro Ser Pro Val Asp Glu Ala Lys Trp Gly Phe Ala Val

195 200 205

Val Glu Asn Ser Leu Trp Gln Gly Val Pro Asn Tyr Leu Arg Glu Leu

210 215 220

Asn Glu Gln Leu Glu Glu Asn Leu Gly Tyr Lys Leu Pro Val Glu Phe

225 230 235 240

Val Pro Val Arg Phe Thr Ser Trp Met Gly Gly Asp Arg Asp Gly Asn

245 250 255

Pro Asn Val Thr Ala Asp Ile Thr Arg His Val Leu Leu Leu Ser Arg

260 265 270

Trp Lys Ala Thr Asp Leu Phe Leu Lys Asp Ile Gln Val Leu Val Ser

275 280 285

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

290 295 300

Glu Glu Gly Ala Ala Glu Pro Tyr Arg Tyr Leu Met Lys Asn Leu Arg

305 310 315 320

Ser Arg Leu Met Ala Thr Gln Ala Trp Leu Glu Ala Arg Leu Lys Gly

325 330 335

Glu Glu Leu Pro Lys Pro Glu Gly Leu Leu Thr Gln Asn Glu Glu Leu

340 345 350

Trp Glu Pro Leu Tyr Ala Cys Tyr Gln Ser Leu Gln Ala Cys Gly Met

355 360 365

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

370 375 380

Cys Phe Gly Val Pro Leu Val Arg Ile Asp Ile Arg Gln Glu Ser Thr

385 390 395 400

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

405 410 415

Asp Tyr Glu Ser Trp Ser Glu Ala Asp Lys Gln Ala Phe Leu Ile Arg

420 425 430

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

435 440 445

Ala Glu Thr Arg Glu Val Leu Asp Thr Cys Gln Val Ile Ala Glu Ala

450 455 460

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

465 470 475 480

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

485 490 495

Phe Ala Met Pro Val Ala Pro Leu Phe Glu Thr Leu Asp Asp Leu Asn

500 505 510

Asn Ala Asn Asp Val Met Thr Gln Leu Leu Asn Ile Asp Trp Tyr Arg

515 520 525

Gly Leu Ile Gln Gly Lys Gln Met Val Met Ile Gly Tyr Ser Asp Ser

530 535 540

Ala Lys Asp Ala Gly Val Met Ala Ala Ser Trp Ala Gln Tyr Gln Ala

545 550 555 560

Gln Asp Ala Leu Ile Lys Thr Cys Glu Lys Ala Gly Ile Glu Leu Thr

565 570 575

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

580 585 590

His Ala Ala Leu Leu Ser Gln Pro Pro Gly Ser Leu Lys Gly Gly Leu

595 600 605

Arg Val Thr Glu Gln Gly Glu Met Ile Arg Phe Lys Tyr Gly Leu Pro

610 615 620

Glu Ile Thr Val Ser Ser Leu Ser Leu Tyr Thr Gly Ala Ile Leu Glu

625 630 635 640

Ala Asn Leu Leu Pro Pro Pro Glu Pro Lys Glu Ser Trp Arg Arg Ile

645 650 655

Met Asp Glu Leu Ser Val Ile Ser Cys Asp Leu Tyr Arg Gly Tyr Val

660 665 670

Arg Glu Asn Lys Asp Phe Val Pro Tyr Phe Arg Ser Ala Thr Pro Glu

675 680 685

Gln Glu Leu Gly Lys Leu Pro Leu Gly Ser Arg Pro Ala Lys Arg Arg

690 695 700

Pro Thr Gly Gly Val Glu Ser Leu Arg Ala Ile Pro Trp Ile Phe Ala

705 710 715 720

Trp Thr Gln Asn Arg Leu Met Leu Pro Ala Trp Leu Gly Ala Gly Thr

725 730 735

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

740 745 750

Met Cys Arg Asp Trp Pro Phe Phe Ser Thr Arg Leu Gly Met Leu Glu

755 760 765

Met Val Phe Ala Lys Ala Asp Leu Trp Leu Ala Glu Tyr Tyr Asp Gln

770 775 780

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

785 790 795 800

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

805 810 815

His Leu Met Ala Asp Leu Pro Trp Ile Ala Glu Ser Ile Gln Leu Arg

820 825 830

Asn Ile Tyr Thr Asp Pro Leu Asn Val Leu Gln Ala Glu Leu Leu His

835 840 845

Arg Ser Arg Gln Ala Glu Lys Glu Gly Gln Glu Pro Asp Pro Arg Val

850 855 860

Glu Gln Ala Leu Met Val Thr Ile Ala Gly Ile Ala Ala Gly Met Arg

865 870 875 880

Asn Thr Gly Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Ser

885 890 895

Ser Gly Ser Ser Ser Ser Gly Met Phe Glu Asn Ile Thr Ala Ala Pro

900 905 910

Ala Asp Pro Ile Leu Gly Leu Ala Asp Leu Phe Arg Ala Asp Glu Arg

915 920 925

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

930 935 940

Lys Thr Pro Val Leu Thr Ser Val Lys Lys Ala Glu Gln Tyr Leu Leu

945 950 955 960

Glu Asn Glu Thr Thr Lys Asn Tyr Leu Gly Ile Asp Gly Ile Pro Glu

965 970 975

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

980 985 990

Ile Asn Asp Lys Arg Ala Arg Thr Ala Gln Thr Pro Gly Gly Thr Gly

995 1000 1005

Ala Leu Arg Val Ala Ala Asp Phe Leu Ala Lys Asn Thr Ser Val Lys

1010 1015 1020

Arg Val Trp Val Ser Asn Pro Ser Trp Pro Asn His Lys Ser Val Phe

1025 1030 1035 1040

Asn Ser Ala Gly Leu Glu Val Arg Glu Tyr Ala Tyr Tyr Asp Ala Glu

1045 1050 1055

Asn His Thr Leu Asp Phe Asp Ala Leu Ile Asn Ser Leu Asn Glu Ala

1060 1065 1070

Gln Ala Gly Asp Val Val Leu Phe His Gly Cys Cys His Asn Pro Thr

1075 1080 1085

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

1090 1095 1100

Val Glu Lys Gly Trp Leu Pro Leu Phe Asp Phe Ala Tyr Gln Gly Phe

1105 1110 1115 1120

Ala Arg Gly Leu Glu Glu Asp Ala Glu Gly Leu Arg Ala Phe Ala Ala

1125 1130 1135

Met His Lys Glu Leu Ile Val Ala Ser Ser Tyr Ser Lys Asn Phe Gly

1140 1145 1150

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

1155 1160 1165

Glu Thr Val Asp Arg Ala Phe Ser Gln Met Lys Ala Ala Ile Arg Ala

1170 1175 1180

Asn Tyr Ser Asn Pro Pro Ala His Gly Ala Ser Val Val Ala Thr Ile

1185 1190 1195 1200

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

1205 1210 1215

Met Arg Gln Arg Ile Gln Arg Met Arg Gln Leu Phe Val Asn Thr Leu

1220 1225 1230

Gln Glu Lys Gly Ala Asn Arg Asp Phe Ser Phe Ile Ile Lys Gln Asn

1235 1240 1245

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

1250 1255 1260

Arg Glu Glu Phe Gly Val Tyr Ala Val Ala Ser Gly Arg Val Asn Val

1265 1270 1275 1280

Ala Gly Met Thr Pro Asp Asn Met Ala Pro Leu Cys Glu Ala Ile Val

1285 1290 1295

Ala Val Leu

<210> 36

<211> 930

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 36

Met Thr Ile Thr Pro Ala Thr His Ala Ile Ser Ile Asn Pro Ala Thr

1 5 10 15

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

20 25 30

Asn Ala Leu Gln Leu Ala Ala Ala Gly Phe Arg Asp Trp Arg Glu Thr

35 40 45

Asn Ile Asp Tyr Arg Ala Glu Lys Leu Arg Asp Ile Gly Lys Ala Leu

50 55 60

Arg Ala Arg Ser Glu Glu Met Ala Gln Met Ile Thr Arg Glu Met Gly

65 70 75 80

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

85 90 95

Cys Asp Trp Tyr Ala Glu His Gly Pro Ala Met Leu Lys Ala Glu Pro

100 105 110

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

115 120 125

Thr Ile Leu Ala Ile Met Pro Trp Asn Phe Pro Leu Trp Gln Val Met

130 135 140

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

145 150 155 160

His Ala Pro Asn Val Met Gly Cys Ala Gln Leu Ile Ala Gln Val Phe

165 170 175

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

180 185 190

Asn Asp Gly Val Ser Gln Met Ile Lys Asp Ser Arg Ile Ala Ala Val

195 200 205

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

210 215 220

Gly Ala Ala Leu Lys Lys Cys Val Leu Glu Leu Gly Gly Ser Asp Pro

225 230 235 240

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

245 250 255

Val Ala Gly Arg Tyr Gln Asn Thr Gly Gln Val Cys Ala Ala Ala Lys

260 265 270

Arg Phe Ile Ile Glu Glu Gly Ile Ala Ser Ala Phe Thr Glu Arg Phe

275 280 285

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

290 295 300

Asn Ala Leu Gly Pro Met Ala Arg Phe Asp Leu Arg Asp Glu Leu His

305 310 315 320

His Gln Val Glu Lys Thr Leu Ala Gln Gly Ala Arg Leu Leu Leu Gly

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

Asp Glu Thr Gln Ala Arg Gln Met Ala Ala Arg Leu Glu Cys Gly Gly

405 410 415

Val Phe Ile Asn Gly Tyr Cys Ala Ser Asp Ala Arg Val Ala Phe Gly

420 425 430

Gly Val Lys Lys Ser Gly Phe Gly Arg Glu Leu Ser His Phe Gly Leu

435 440 445

His Glu Phe Cys Asn Ile Gln Thr Val Trp Lys Asp Arg Ile Ser Ser

450 455 460

Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly Ser Ser Ser

465 470 475 480

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

485 490 495

Leu Asn Leu Arg Ala His Trp Met Pro Phe Ser Ala Asn Arg Asn Phe

500 505 510

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

515 520 525

Asp Asp Lys Gly Arg Lys Val Tyr Asp Ser Leu Ser Gly Leu Trp Thr

530 535 540

Cys Gly Ala Gly His Ser Arg Lys Glu Ile Gln Glu Ala Val Ala Arg

545 550 555 560

Gln Leu Gly Thr Leu Asp Tyr Ser Pro Gly Phe Gln Tyr Gly His Pro

565 570 575

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

580 585 590

Leu Asn His Val Phe Phe Thr Gly Ser Gly Ser Glu Cys Ala Asp Thr

595 600 605

Ser Ile Lys Met Ala Arg Ala Tyr Trp Arg Leu Lys Gly Gln Pro Gln

610 615 620

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

625 630 635 640

Ala Gly Thr Ser Leu Gly Gly Ile Gly Gly Asn Arg Lys Met Phe Gly

645 650 655

Gln Leu Met Asp Val Asp His Leu Pro His Thr Leu Gln Pro Gly Met

660 665 670

Ala Phe Thr Arg Gly Met Ala Gln Thr Gly Gly Val Glu Leu Ala Asn

675 680 685

Glu Leu Leu Lys Leu Ile Glu Leu His Asp Ala Ser Asn Ile Ala Ala

690 695 700

Val Ile Val Glu Pro Met Ser Gly Ser Ala Gly Val Leu Val Pro Pro

705 710 715 720

Val Gly Tyr Leu Gln Arg Leu Arg Glu Ile Cys Asp Gln His Asn Ile

725 730 735

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

740 745 750

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

755 760 765

Ala Lys Gln Val Thr Asn Gly Ala Val Pro Met Gly Ala Val Ile Ala

770 775 780

Ser Ser Glu Ile Tyr Asp Thr Phe Met Asn Gln Ala Leu Pro Glu His

785 790 795 800

Ala Val Glu Phe Ser His Gly Tyr Thr Tyr Ser Ala His Pro Val Ala

805 810 815

Cys Ala Ala Gly Leu Ala Ala Leu Asp Ile Leu Ala Arg Asp Asn Leu

820 825 830

Val Gln Gln Ser Ala Glu Leu Ala Pro His Phe Glu Lys Gly Leu His

835 840 845

Gly Leu Gln Gly Ala Lys Asn Val Ile Asp Ile Arg Asn Cys Gly Leu

850 855 860

Ala Gly Ala Ile Gln Ile Ala Pro Arg Asp Gly Asp Pro Thr Val Arg

865 870 875 880

Pro Phe Glu Ala Gly Met Lys Leu Trp Gln Gln Gly Phe Tyr Val Arg

885 890 895

Phe Gly Gly Asp Thr Leu Gln Phe Gly Pro Thr Phe Asn Ala Arg Pro

900 905 910

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

915 920 925

Ile Ala

930

<210> 37

<211> 1140

<212> PRT

<213> Corynebacterium glutamicum (Corynebacterium glutamicum)

<400> 37

Met Ser Thr His Thr Ser Ser Thr Leu Pro Ala Phe Lys Lys Ile Leu

1 5 10 15

Val Ala Asn Arg Gly Glu Ile Ala Val Arg Ala Phe Arg Ala Ala Leu

20 25 30

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

35 40 45

Ser Phe His Arg Ser Phe Ala Ser Glu Ala Val Arg Ile Gly Thr Glu

50 55 60

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

65 70 75 80

Ala Lys Lys Val Lys Ala Asp Ala Ile Tyr Pro Gly Tyr Gly Phe Leu

85 90 95

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

100 105 110

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

115 120 125

Arg Ala Val Thr Ala Ala Lys Lys Ala Gly Leu Pro Val Leu Ala Glu

130 135 140

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

145 150 155 160

Gln Thr Tyr Pro Ile Phe Val Lys Ala Val Ala Gly Gly Gly Gly Arg

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

Leu Asp Pro Glu Leu Arg Asp Arg Ile Cys Ala Asp Ala Val Lys Phe

260 265 270

Cys Arg Ser Ile Gly Tyr Gln Gly Ala Gly Thr Val Glu Phe Leu Val

275 280 285

Asp Glu Lys Gly Asn His Val Phe Ile Glu Met Asn Pro Arg Ile Gln

290 295 300

Val Glu His Thr Val Thr Glu Glu Val Thr Glu Val Asp Leu Val Lys

305 310 315 320

Ala Gln Met Arg Leu Ala Ala Gly Ala Thr Leu Lys Glu Leu Gly Leu

325 330 335

Thr Gln Asp Lys Ile Lys Thr His Gly Ala Ala Leu Gln Cys Arg Ile

340 345 350

Thr Thr Glu Asp Pro Asn Asn Gly Phe Arg Pro Asp Thr Gly Thr Ile

355 360 365

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

370 375 380

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

385 390 395 400

Lys Met Thr Cys Arg Gly Ser Asp Phe Glu Thr Ala Val Ala Arg Ala

405 410 415

Gln Arg Ala Leu Ala Glu Phe Thr Val Ser Gly Val Ala Thr Asn Ile

420 425 430

Gly Phe Leu Arg Ala Leu Leu Arg Glu Glu Asp Phe Thr Ser Lys Arg

435 440 445

Ile Ala Thr Gly Phe Ile Ala Asp His Pro His Leu Leu Gln Ala Pro

450 455 460

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

465 470 475 480

Thr Val Asn Lys Pro His Gly Val Arg Pro Lys Asp Val Ala Ala Pro

485 490 495

Ile Asp Lys Leu Pro Asn Ile Lys Asp Leu Pro Leu Pro Arg Gly Ser

500 505 510

Arg Asp Arg Leu Lys Gln Leu Gly Pro Ala Ala Phe Ala Arg Asp Leu

515 520 525

Arg Glu Gln Asp Ala Leu Ala Val Thr Asp Thr Thr Phe Arg Asp Ala

530 535 540

His Gln Ser Leu Leu Ala Thr Arg Val Arg Ser Phe Ala Leu Lys Pro

545 550 555 560

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

565 570 575

Ala Trp Gly Gly Ala Thr Tyr Asp Val Ala Met Arg Phe Leu Phe Glu

580 585 590

Asp Pro Trp Asp Arg Leu Asp Glu Leu Arg Glu Ala Met Pro Asn Val

595 600 605

Asn Ile Gln Met Leu Leu Arg Gly Arg Asn Thr Val Gly Tyr Thr Pro

610 615 620

Tyr Pro Asp Ser Val Cys Arg Ala Phe Val Lys Glu Ala Ala Ser Ser

625 630 635 640

Gly Val Asp Ile Phe Arg Ile Phe Asp Ala Leu Asn Asp Val Ser Gln

645 650 655

Met Arg Pro Ala Ile Asp Ala Val Leu Glu Thr Asn Thr Ala Val Ala

660 665 670

Glu Val Ala Met Ala Tyr Ser Gly Asp Leu Ser Asp Pro Asn Glu Lys

675 680 685

Leu Tyr Thr Leu Asp Tyr Tyr Leu Lys Met Ala Glu Glu Ile Val Lys

690 695 700

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

705 710 715 720

Pro Ala Ala Val Thr Lys Leu Val Thr Ala Leu Arg Arg Glu Phe Asp

725 730 735

Leu Pro Val His Val His Thr His Asp Thr Ala Gly Gly Gln Leu Ala

740 745 750

Thr Tyr Phe Ala Ala Ala Gln Ala Gly Ala Asp Ala Val Asp Gly Ala

755 760 765

Ser Ala Pro Leu Ser Gly Thr Thr Ser Gln Pro Ser Leu Ser Ala Ile

770 775 780

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

785 790 795 800

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

805 810 815

Leu Pro Phe Glu Ser Gly Thr Pro Gly Pro Thr Gly Arg Val Tyr Arg

820 825 830

His Glu Ile Pro Gly Gly Gln Leu Ser Asn Leu Arg Ala Gln Ala Thr

835 840 845

Ala Leu Gly Leu Ala Asp Arg Phe Glu Leu Ile Glu Asp Asn Tyr Ala

850 855 860

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

865 870 875 880

Lys Val Val Gly Asp Leu Ala Leu His Leu Val Gly Ala Gly Val Asp

885 890 895

Pro Ala Asp Phe Ala Ala Asp Pro Gln Lys Tyr Asp Ile Pro Asp Ser

900 905 910

Val Ile Ala Phe Leu Arg Gly Glu Leu Gly Asn Pro Pro Gly Gly Trp

915 920 925

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

930 935 940

Ala Pro Leu Thr Glu Val Pro Glu Glu Glu Gln Ala His Leu Asp Ala

945 950 955 960

Asp Asp Ser Lys Glu Arg Arg Asn Ser Leu Asn Arg Leu Leu Phe Pro

965 970 975

Lys Pro Thr Glu Glu Phe Leu Glu His Arg Arg Arg Phe Gly Asn Thr

980 985 990

Ser Ala Leu Asp Asp Arg Glu Phe Phe Tyr Gly Leu Val Glu Gly Arg

995 1000 1005

Glu Thr Leu Ile Arg Leu Pro Asp Val Arg Thr Pro Leu Leu Val Arg

1010 1015 1020

Leu Asp Ala Ile Ser Glu Pro Asp Asp Lys Gly Met Arg Asn Val Val

1025 1030 1035 1040

Ala Asn Val Asn Gly Gln Ile Arg Pro Met Arg Val Arg Asp Arg Ser

1045 1050 1055

Val Glu Ser Val Thr Ala Thr Ala Glu Lys Ala Asp Ser Ser Asn Lys

1060 1065 1070

Gly His Val Ala Ala Pro Phe Ala Gly Val Val Thr Val Thr Val Ala

1075 1080 1085

Glu Gly Asp Glu Val Lys Ala Gly Asp Ala Val Ala Ile Ile Glu Ala

1090 1095 1100

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

1105 1110 1115 1120

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

1125 1130 1135

Val Val Val Ser

1140

<210> 38

<211> 136

<212> PRT

<213> E.coli BL21(DE3) (Escherichia coli BL21(DE3))

<400> 38

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Gly Thr Gly Asn Ile Cys Ile Asn Gly Ala Ala Ala His Leu Ile Asn

65 70 75 80

Pro Gly Asp Leu Val Ile Ile Met Ser Tyr Leu Gln Ala Thr Asp Ala

85 90 95

Glu Ala Lys Ala Tyr Glu Pro Lys Ile Val His Val Asp Ala Asp Asn

100 105 110

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

115 120 125

Gly Leu Leu Thr Ser Arg Ser Ile

130 135

<210> 39

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

ttgacaatta atcatccggc tcgtataatg tgtggaattt tgggcagata acaatttcac 60

acaggaaaca gacc 74

Claims (10)

1. A recombinant Escherichia coli (Escherichia coli) capable of synthesizing malonic acid from oxaloacetate and aspartate by using glucose as a raw material is characterized in that the recombinant Escherichia coli is composed of an over-expression Escherichia coli-derived phosphoenolpyruvate carboxylase ppc, an aspartate aminotransferase aspC and an aspartate-alpha-decarboxylase panD, and Pseudomonas aeruginosa-derived beta-alanine pyruvate aminotransferase pa0132, Escherichia coli-derived succinic semialdehyde dehydrogenase yneI and Corynebacterium glutamicum-derived pyruvate carboxylase pyc.

2. The recombinant Escherichia coli of claim 1, wherein said modular constitutive overexpression is fusion expression of said phosphoenolpyruvate carboxylase ppc and said aspartate aminotransferase aspC, separate expression of said aspartate- α -decarboxylase panD and said pyruvate carboxylase pyc, and fusion expression of said succinate semialdehyde dehydrogenase yneI and said β -alanine pyruvate aminotransferase pa 0132.

3. The recombinant E.coli of claim 1 or 2, wherein the promoter used for modular constitutive overexpression is the constitutive promoter ML 2719;

taking escherichia coli BL21(DE3) as a host;

the phosphoenolpyruvate carboxylase ppc, the aspartate aminotransferase aspC and the aspartate- α -decarboxylase panD are from escherichia coli BL21(DE 3); and/or

The succinic semialdehyde dehydrogenase yneI is derived from Escherichia coli K12.

4. The recombinant Escherichia coli of any one of claims 1 to 3, wherein the phosphoenolpyruvate carboxylase ppc has a protein sequence shown in positions 1 to 883 in SEQ ID NO 35;

the protein sequence of the aspartate aminotransferase aspC is shown as position 904-1299 in SEQ ID NO 35;

the protein sequence of the aspartate-alpha-decarboxylase panD is shown as SEQ ID NO 38;

the protein sequence of the beta-alanine pyruvate transaminase pa0132 is shown as position 483-930 in SEQ ID NO 36;

the protein sequence of the succinic semialdehyde dehydrogenase yneI is shown as 1-462 in SEQ ID NO: 36; and/or

The protein sequence of the pyruvate carboxylase pyc is shown as SEQ ID NO: 37.

5. A method for constructing the recombinant Escherichia coli as claimed in any one of claims 1 to 4, which comprises transferring one or more plasmids containing the gene ppc encoding phosphoenolpyruvate carboxylase derived from Escherichia coli, the gene aspC encoding aspartate aminotransferase, the gene panD encoding aspartate- α -decarboxylase, and the gene pa0132 encoding β -alanine pyruvate aminotransferase derived from Pseudomonas aeruginosa, the gene yneI encoding succinate semialdehyde dehydrogenase derived from Escherichia coli, and the gene pyc encoding pyruvate carboxylase derived from Corynebacterium glutamicum into an Escherichia coli host for modular constitutive overexpression.

6. The method of claim 5, wherein said modular constitutive overexpression comprises the step of modular constitutive overexpression of a plasmid fusion expressing phosphoenolpyruvate carboxylase gene ppc and aspartate aminotransferase gene aspC, a plasmid separately expressing said aspartate- α -dehydrogenase gene panD and said pyruvate carboxylase gene pyc, and a plasmid fusion expressing said succinate semialdehyde dehydrogenase gene ynel and said β -alanine pyruvate aminotransferase gene pa0132 into an E.coli host.

7. The method according to claim 5 or 6, characterized in that the promoter used in the modular constitutive overexpression is the constitutive promoter ML 2719;

the Escherichia coli host is Escherichia coli BL21(DE 3);

the phosphoenolpyruvate carboxylase gene ppc, the aspartate aminotransferase gene aspC and the aspartate-alpha-dehydrogenase gene panD are from escherichia coli BL21(DE 3); and/or

The succinic semialdehyde dehydrogenase gene yneI is derived from Escherichia coli K12.

8. The method according to any one of claims 5 to 7, wherein the phosphoenolpyruvate carboxylase gene ppc has the sequence shown in SEQ ID NO 9;

the sequence of the aspartate aminotransferase gene aspC is shown as SEQ ID NO. 10;

the sequence of the aspartate-alpha-dehydrogenase gene panD is shown as SEQ ID NO 18;

the sequence of the beta-alanine pyruvate transaminase gene pa0132 is shown as SEQ ID NO 2;

the sequence of the succinic semialdehyde dehydrogenase gene yneI is shown in SEQ ID NO 1; and/or

The sequence of the pyruvate carboxylase gene pyc is shown in SEQ ID NO. 17.

9. A method for producing malonic acid by fermentation, comprising inoculating the recombinant Escherichia coli of any one of claims 1 to 4 into an SOB medium containing 4 to 8g/L glucose, and fermenting at 30 to 37 ℃ and 300-.

10. Use of the recombinant E.coli of any one of claims 1 to 4 or the method of claim 9 for the preparation of malonic acid and its derivatives.

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