CN113122489A

CN113122489A - Recombinant escherichia coli for producing glycolic acid and construction method and application thereof

Info

Publication number: CN113122489A
Application number: CN202010041479.9A
Authority: CN
Inventors: 刘伟丰; 杨梦杰; 朴晓宇; 陶勇
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-01-15
Filing date: 2020-01-15
Publication date: 2021-07-16
Anticipated expiration: 2040-01-15
Also published as: CN113122489B

Abstract

The invention discloses a recombinant escherichia coli for producing glycollic acid and a construction method and application thereof. The invention firstly discloses recombinant escherichia coli, compared with acceptor escherichia coli, the expression level and/or content and/or activity of the gene of protein A and/or the expression level and/or content and/or activity of the gene of protein B are reduced; the protein A is at least one of the following: malic acid thiokinase; malyl-coa lyase; glyoxylate reductase; isocitrate lyase; isocitrate dehydrogenase kinase/phosphatase; the protein B is at least one of the following: NAD-dependent-malic enzyme; NADP-dependent-malic enzyme; a malate synthase A; glyoxylate pathway transcription repressing factor; glycolate oxidase. Further provided are methods of preparing glycolic acid. The invention takes glucose as raw material to construct recombinant escherichia coli, so that the glucose has higher conversion rate, and the yield of glycolic acid is greatly improved.

Description

Recombinant escherichia coli for producing glycolic acid and construction method and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to recombinant escherichia coli for producing glycolic acid and a construction method and application thereof.

Background

Glycolic acid (english name is Glycolic acid), also known as Glycolic acid, is the simplest alpha-hydroxy acid and is widely used in industrial processes, the cosmetic industry and in the synthesis of polymers (PGA, PLGA). The price of the high-purity glycolic acid is 30000-40000 yuan/ton.

At present, the synthesis of glycolic acid mainly comprises a chemical synthesis method and a biological synthesis method. The chemical synthesis mainly comprises a chloroacetic acid method, a formaldehyde carbonylation method, a cyanidation method, an ester exchange method and the like, but the chemical reaction has the defects of severe reaction conditions, difficult recycling of a catalyst, environmental pollution and the like. The biosynthesis method can be further divided into a biological enzyme method synthesis and a total biological synthesis, wherein the biological enzyme method mainly utilizes enzymes such as nitrilase producing enzyme and glycerol oxidase producing enzyme to respectively hydrolyze glycolonitrile and oxidize ethylene glycol to obtain glycolic acid. The methods are not widely used at present because the precursor substances required by the biological enzyme method are expensive and the precursor substances themselves or their degradation products have toxic effects. In order to solve the above problems, researchers have attempted to produce glycolic acid using microorganisms as cell factories and compounds such as glucose, xylose, ethanol, ethylene glycol, etc. as carbon sources. At present, 56.44g/L of glycolic acid is obtained by taking glucose as a carbon source and utilizing the glyoxylate pathway in escherichia coli at the highest energy; d-xylose and ethanol are used as carbon sources, and 15g/L glycolic acid can be obtained in the yeast engineering bacteria; ethylene glycol is used as a carbon source, and 105g/L and 110g/L of glycolic acid can be obtained by using pichia pastoris and rhodotorula pastoris respectively. In addition, Corynebacterium glutamicum, chemoautotrophic iron-sulfur bacteria, and Burkholderia were reported to be useful for glycolic acid production.

The main technical limitation of the biological method for synthesizing the glycollic acid is still the low theoretical conversion rate of the adopted route, so a new high-conversion rate route needs to be developed.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a glycolic acid strain with high yield so as to realize the high-efficiency synthesis of glycolic acid.

In order to solve the above technical problems, the present invention provides recombinant Escherichia coli.

Compared with a receptor Escherichia coli, the recombinant Escherichia coli has the advantages that the expression quantity of the gene of the protein A in the recombinant Escherichia coli is increased, the content of the protein A is increased, the activity of the protein A is increased, the expression quantity of the gene of the protein B in the recombinant Escherichia coli is reduced, the content of the protein B is reduced, and the activity of the protein B is reduced;

the protein A is selected from at least one of the following:

A1) malic acid thiokinase;

A2) malyl-coa lyase;

A3) glyoxylate reductase;

A4) isocitrate dehydrogenase kinase/phosphatase;

A5) isocitrate lyase;

the protein B is selected from at least one of the following:

B1) NAD-dependent-malic enzyme;

B2) NADP-dependent-malic enzyme;

B3) a malate synthase A;

B4) glyoxylate pathway transcription repressing factor;

B5) glycolate oxidase.

In the above recombinant Escherichia coli, the recipient Escherichia coli may be Escherichia coli K12, more specifically Escherichia coli BW 25113.

In the recombinant escherichia coli, the gene of the malyl thiokinase is derived from Methylococcus capsulatus (Methylococcus capsulatus), and the gene of the malyl-CoA lyase is derived from Rhodobacter sphaeroides (Rhodobacter sphaeroides).

In the recombinant Escherichia coli, the glyoxylate reductase can be represented by C1) or C2):

C1) protein coded by DNA molecule shown in SEQ ID No. 2;

C2) a protein having 90% or more identity and function identity to the protein represented by C1) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein represented by C1);

the isocitrate lyase can be C3) or C4):

C3) a protein encoded by the DNA molecule shown in SEQ ID No.3, positions 1-1305;

C4) a protein having 90% or more identity and function identity to the protein represented by C3) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein represented by C3);

the isocitrate dehydrogenase kinase/phosphatase may be represented by C5) or C6):

C5) a protein encoded by the DNA molecule as shown in position 1488-3224 of SEQ ID No. 3;

C6) a protein having 90% or more identity and function identity to the protein represented by C5) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein represented by C5);

the malate thiokinase can be C7) or C8):

C7) a protein encoded by the DNA molecule shown in SEQ ID No.4 at positions 1-2088;

C8) a protein having 90% or more identity and function identity to the protein represented by C7) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein represented by C7);

the malyl-coa lyase may be C9) or C10):

C9) a protein encoded by the DNA molecule as shown in positions 2105-3061 of SEQ ID No. 4;

C10) a protein having 90% or more identity and function identity to the protein represented by C9) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein represented by C9);

the NAD-dependent malic enzyme can be C11) or C12):

C11) protein coded by DNA molecule shown in SEQ ID No. 5;

C12) a protein having 90% or more identity and function identity to the protein represented by C11) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein represented by C11);

the NADP-dependent malic enzyme can be C13) or C14):

C13) protein coded by DNA molecule shown in SEQ ID No. 6;

C14) a protein having 90% or more identity and function identity to the protein represented by C13) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein represented by C13);

the malate synthase A can be represented by C15) or C16):

C15) protein coded by DNA molecule shown in SEQ ID No. 7;

C16) a protein having 90% or more identity and function identity to the protein represented by C15) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein represented by C15);

the glyoxylate pathway transcription inhibitor can be C17) or C18):

C17) protein coded by DNA molecule shown in SEQ ID No. 8;

C18) a protein having 90% or more identity and function identity to the protein represented by C17) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein represented by C17);

the glycolate oxidase can be represented by C19) or C20):

C19) protein coded by DNA molecule shown in SEQ ID No. 9;

C20) a protein having 90% or more identity and function identity to the protein represented by C19) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein represented by C19).

Wherein, SEQ ID No.2 consists of 939 nucleotides, and the 1 st to 939 th positions are coding sequences which code glyoxylate reductase.

The 1 st to 1305 th positions of SEQ ID No.3 consist of 1305 nucleotides, and the 1 st to 1305 th positions of SEQ ID No.3 are coding sequences which code for isocitrate lyase.

The position 1488-3224 of SEQ ID No.3 consists of 1737 nucleotides, the position 1488-3224 is a coding sequence, encoding isocitrate dehydrogenase kinase/phosphatase.

The 1 st to 2088 th sites of SEQ ID No.4 consist of 2088 nucleotides, and the 1 st to 2088 th sites of SEQ ID No.4 are coding sequences and code for malate thiokinase.

The 2105-3061 position of SEQ ID No.4 consists of 957 nucleotides, the 2105-3061 position is a coding sequence, and the coding sequence codes malyl-CoA lyase.

SEQ ID No.5 consists of 1695 nucleotides, with coding sequences at positions 1-1695, encoding an NAD-dependent malic enzyme.

SEQ ID No.6 consists of 2217 nucleotides, and positions 1-2217 are coding sequences which encode NADP dependent-malic enzyme.

SEQ ID No.7 consists of 1599 nucleotides, positions 1-1599 of which are coding sequences, encoding malate synthase A.

SEQ ID No.8 consists of 822 nucleotides, with coding sequences at positions 1-822, encoding glyoxylate pathway transcription repressing factors.

SEQ ID No.9 consists of 1497 nucleotides, and positions 1-1497 are coding sequences, which code for glycolate oxidase.

In the above recombinant Escherichia coli, identity means identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above recombinant E.coli, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the recombinant Escherichia coli, the gene (ghrA) of glyoxylate reductase may be represented by D1) or D2) as follows:

D1) the coding sequence is DNA molecule shown in SEQ ID No. 2;

D2) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.2 and has the same function as SEQ ID No. 2;

the isocitrate lyase gene (aceA) may be D3) or D4) as follows:

D3) a DNA molecule with the coding sequence shown as SEQ ID No3 1-1305;

D4) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides from the 1 st to the 1305 th positions of SEQ ID No.3 and has the same function with the 1 st to the 1305 th positions of SEQ ID No. 3;

the isocitrate dehydrogenase kinase/phosphatase gene (aceK) may be as shown in D5) or D6):

D5) a DNA molecule with the coding sequence shown in the 1488-3224 position of SEQ ID No. 3;

D6) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides at the position 1488-3224 of the SEQ ID No.3 and has the same function with the position 1488-3224 of the SEQ ID No. 3;

the gene (mtk) of the malate thiokinase can be D7) or D8) as follows:

D7) the coding sequence is DNA molecule shown in 1 st to 2088 th sites of SEQ ID No. 4;

D8) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides from the 1 st to the 2088 th positions of SEQ ID No.4 and has the same function with the 1 st to the 2088 th positions of SEQ ID No. 4;

the gene (mcl) of the malyl-CoA lyase may be D9) or D10) as follows:

D9) a DNA molecule with the coding sequence shown as the 2105-3061 site of SEQ ID No. 4;

D10) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides at the 2105-3061 site of the SEQ ID No.4 and has the same function as the 2105-3061 site of the SEQ ID No. 4;

the gene (maeA) of the NAD dependent-malic enzyme can be D11) or D12):

D11) the coding sequence is DNA molecule shown in SEQ ID No. 5;

D12) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.5 and has the same function as SEQ ID No. 5;

the NADP dependent-malic enzyme gene (maeB) can be D13) or D14):

D13) the coding sequence is DNA molecule shown in SEQ ID No. 6;

D14) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.6 and has the same function as SEQ ID No. 3;

the gene (aceB) of the malate synthase A can be D15) or D16):

D15) the coding sequence is DNA molecule shown in SEQ ID No. 7;

D16) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.7 and has the same function as SEQ ID No. 7;

the gene (iclR) of the glyoxylate pathway transcription repressing factor can be D17) or D18):

D17) the coding sequence is DNA molecule shown in SEQ ID No. 8;

D18) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.8 and has the same function as SEQ ID No. 8;

the gene (glcD) of the glycolate oxidase can be D19) or D20):

D19) the coding sequence is DNA molecule shown in SEQ ID No. 9;

D20) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in SEQ ID No.9 and has the same function as SEQ ID No. 9.

The invention further provides a construction method of the recombinant Escherichia coli.

The construction method of the recombinant escherichia coli comprises the following steps: increasing the expression level of the gene of the protein A and/or the content of the protein A and/or the activity of the protein A in the recipient Escherichia coli, and/or decreasing the expression level of the gene of the protein B and/or the content of the protein B and/or the activity of the protein B in the recipient Escherichia coli:

the protein A is selected from at least one of the following:

A1) glyoxylate reductase;

A2) isocitrate lyase;

A3) isocitrate dehydrogenase kinase/phosphatase;

A4) malic acid thiokinase;

A5) malyl-coa lyase;

the protein B is selected from at least one of the following:

B1) NAD-dependent-malic enzyme;

B2) NADP-dependent-malic enzyme;

B3) a malate synthase A;

B4) glyoxylate pathway transcription repressing factor;

B5) glycolate oxidase.

In a specific embodiment of the present invention, the recombinant escherichia coli may be constructed by any one of the following methods P1) -P3):

p1) increasing the expression level of the genes of glyoxylate reductase, isocitrate lyase, isocitrate dehydrogenase kinase/phosphatase, malate thiokinase and maloyl-CoA lyase and/or the activity of glyoxylate reductase, isocitrate lyase, isocitrate dehydrogenase kinase/phosphatase, malate thiokinase and maloyl-CoA lyase in recipient E.coli and reducing the expression level of the genes of NAD-dependent-malic enzyme, NADP-dependent-malic enzyme, malate synthase A, glyoxylate pathway transcription inhibitor and glycolate oxidase and/or NAD-dependent-malic enzyme, malate synthase A, malate synthase and malate synthase, (ii) the content of NADP-dependent-malic enzyme, malate synthase a, glyoxylate pathway transcriptional inhibitor and glycolate oxidase and/or the activity of NAD-dependent-malic enzyme, NADP-dependent-malic enzyme, malate synthase a, glyoxylate pathway transcriptional inhibitor and glycolate oxidase;

p2) increasing the expression level of the genes glyoxylate reductase, isocitrate lyase, isocitrate dehydrogenase kinase/phosphatase, malate thiokinase and maloyl-CoA lyase and/or the activity of the genes glyoxylate reductase, isocitrate lyase, isocitrate dehydrogenase kinase/phosphatase, malate thiokinase and maloyl-CoA lyase in recipient E.coli and/or the expression level of the genes of glyoxylate reductase, isocitrate lyase, isocitrate dehydrogenase kinase/phosphatase, malate thiokinase and maloyl-CoA lyase in said recipient E.coli and reducing the expression level of the genes of NAD-dependent-malic enzyme, NADP-dependent-malic enzyme and malate synthase A and/or NAD-dependent-malic enzyme, NADP-dependent-malic enzyme, malate synthase A, The content of glyoxylate pathway transcription inhibitor and glycolate oxidase and/or the activity of NAD-dependent-malic enzyme, NADP-dependent-malic enzyme, malate synthase a, glyoxylate pathway transcription inhibitor and glycolate oxidase;

p3) increasing the expression level of the genes for glyoxylate reductase, isocitrate lyase, isocitrate dehydrogenase kinase/phosphatase, malate thiokinase and malyl-coa lyase and/or the content of glyoxylate reductase, isocitrate lyase, isocitrate dehydrogenase kinase/phosphatase, malate thiokinase and malyl-coa lyase and/or the activity of glyoxylate reductase, isocitrate lyase, isocitrate dehydrogenase kinase/phosphatase, malate thiokinase and malyl-coa lyase in the recipient escherichia coli.

In the method, the construction method is realized by introducing the gene of the protein A into recipient Escherichia coli; the acceptor escherichia coli is an escherichia coli mutant or wild type escherichia coli;

the protein A is selected from at least one of the following:

A1) glyoxylate reductase;

A2) isocitrate lyase;

A3) isocitrate dehydrogenase kinase/phosphatase;

A4) malic acid thiokinase;

A5) malyl-coa lyase.

In the method, the escherichia coli mutant is obtained by modifying the wild type escherichia coli genome with all, any four, any three, any two or any one of the following m1) -m 5):

m1) knocking out the gene of NAD-dependent-malic enzyme (maeA);

m2) knocking out the gene (maeB) of NADP dependent-malic enzyme;

m3) knocking out the gene (aceB) of malate synthase A;

m4) knocking out the gene (iclR) of the transcription repressing factor of the glyoxylate pathway;

m5) knock out the gene (glcD) of glycolate oxidase.

In a specific embodiment of the present invention, the escherichia coli mutant is any one of:

m1) the Escherichia coli mutant is obtained by modifying the wild Escherichia coli with M1) -M5);

m2) the Escherichia coli mutant is obtained by modifying the wild Escherichia coli with M1) -M4);

m3) the Escherichia coli mutant is obtained by modifying the wild Escherichia coli with M1) -M3);

m4) the Escherichia coli mutant is obtained by modifying the wild Escherichia coli with M1) and M2);

m5) the Escherichia coli mutant is obtained by modifying the wild type Escherichia coli with the above M1).

In the above method, the wild type Escherichia coli is Escherichia coli K12, more specifically Escherichia coli BW 25113.

In the method, the knockout is performed by using a P1 phage transduction knockout gene technology.

In a specific embodiment of the present invention, the glyoxylate reductase gene (ghrA), the isocitrate lyase gene (aceA), and the isocitrate dehydrogenase kinase/phosphatase gene (aceK) are introduced into recipient escherichia coli through a recombinant vector a, which is a recombinant expression vector pYB1s-ghrA obtained by replacing a fragment between NcoI and EcoRI sites of the YB1s vector with a ghrA gene and maintaining the other sequences of the YB1s vector, and a recombinant expression vector pYB 1-1 s-ghrA-aceAK obtained by replacing a fragment between EcoRI and PstI sites of pYB1s-ghrA recombinant expression vector with a fragment comprising aceA and aceK genes and maintaining the other sequences of pYB1s-ghrA vector.

The gene (mtk) of the malate thiokinase and the gene (mcl) of the malyl-CoA lyase are introduced into a receptor escherichia coli through a recombinant vector B, and the recombinant vector B is a recombinant expression vector pSB1a-mtk-mcl obtained by replacing a fragment between NcoI and EcoRI sites of a pSB1a vector with a fragment containing mtk and mcl genes and keeping other sequences of the pSB1a vector unchanged.

In the above method, the gene (mtk) of malate thiokinase is derived from Methylococcus capsulatus, and the gene (mcl) of malyl-CoA lyase is derived from Rhodobacter sphaeroides.

In the method, the glyoxylate reductase can be represented by C1) or C2):

C1) protein coded by DNA molecule shown in SEQ ID No. 2;

the isocitrate lyase can be C3) or C4):

the malate thiokinase can be C7) or C8):

the malyl-coa lyase may be C9) or C10):

the NAD-dependent malic enzyme can be C11) or C12):

C11) protein coded by DNA molecule shown in SEQ ID No. 5;

the NADP-dependent malic enzyme can be C13) or C14):

C13) protein coded by DNA molecule shown in SEQ ID No. 6;

the malate synthase A can be represented by C15) or C16):

C15) protein coded by DNA molecule shown in SEQ ID No. 7;

the glyoxylate pathway transcription inhibitor can be C17) or C18):

C17) protein coded by DNA molecule shown in SEQ ID No. 8;

the glycolate oxidase can be represented by C19) or C20):

C19) protein coded by DNA molecule shown in SEQ ID No. 9;

SEQ ID No.9 consists of 1497 nucleotides, with coding sequences at positions 1-1497, which encode malyl-CoA lyase.

In the above methods, identity refers to the identity of amino acid sequences. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above method, the 90% or greater identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

In the above method, the glyoxylate reductase gene (ghrA) may be represented by D1) or D2) as follows:

D1) the coding sequence is DNA molecule shown in SEQ ID No. 2;

the isocitrate lyase gene (aceA) may be D3) or D4) as follows:

D3) the coding sequence is DNA molecule shown in 1 st to 1305 th of SEQ ID No. 3;

the gene (mtk) of the malate thiokinase can be D7) or D8) as follows:

the gene (mcl) of the malyl-CoA lyase may be D9) or D10) as follows:

the gene (maeA) of the NAD dependent-malic enzyme can be D11) or D12):

D11) the coding sequence is DNA molecule shown in SEQ ID No. 5;

the NADP dependent-malic enzyme gene (maeB) can be D13) or D14):

D13) the coding sequence is DNA molecule shown in SEQ ID No. 6;

D14) a DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides to SEQ ID No.6 and has the same function as SEQ ID No. 6;

the gene (aceB) of the malate synthase A can be D15) or D16):

D15) the coding sequence is DNA molecule shown in SEQ ID No. 7;

D17) the coding sequence is DNA molecule shown in SEQ ID No. 8;

the gene (glcD) of the glycolate oxidase can be D19) or D20):

D19) the coding sequence is DNA molecule shown in SEQ ID No. 9;

The recombinant Escherichia coli obtained by the construction method and the application thereof in the preparation of glycolic acid are also within the protection scope of the invention.

The invention further discloses a method for preparing glycolic acid.

The method for preparing glycolic acid comprises the following steps: the recombinant escherichia coli is used for catalyzing glucose reaction to obtain glycolic acid.

Specifically, the recombinant escherichia coli is subjected to arabinose induction culture to obtain induced recombinant escherichia coli, and the induced recombinant escherichia coli is used for catalyzing glucose reaction to obtain glycolic acid.

In the method, the arabinose induction culture is carried out in a culture medium containing arabinose, and the temperature of the induction culture is 30 ℃ and the time is 16 h.

In the above method, the arabinose is L-arabinose.

The invention constructs recombinant escherichia coli, and a large amount of phosphoenolpyruvate is accumulated by changing a glucose uptake pathway and a glycolysis pathway, so that a carbon fixation pathway is enhanced to obtain a large amount of precursor oxaloacetate; the synthesis of intermediate product glyoxylate is enhanced and the loss of carbon and sulfur is reduced by weakening the oxidation chain of the tricarboxylic acid cycle and enhancing the first step reaction of the glyoxylate path. And a reverse glyoxylate pathway is introduced, so that acetyl coenzyme A entering a tricarboxylic acid cycle and acetyl coenzyme A released by the reverse glyoxylate pathway form a cycle, and the synthesis efficiency of glyoxylate is further improved under the propulsion of a large amount of oxaloacetate; finally, the expression of glyoxylate reductase gene required by a glycollic acid synthesis path is enhanced, thereby obtaining the capability of efficiently synthesizing glycollic acid.

The invention takes glucose as raw material to ferment and convert, and simultaneously introduces the carbon fixation way and the recombinant escherichia coli constructed by the reverse glyoxylate way to lead the glucose to have higher conversion rate, greatly improves the yield of the glycollic acid and leads the glucose to have great potential for synthesizing the glycollic acid.

Drawings

FIG. 1 shows glycolic acid production of a recombinant E.coli engineered strain using glucose as a raw material.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. 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.

In the following examples, E.coli BW25113(Datsenko KA, Wanner BL. one-step inactivation of chromosomal genes in Escherichia coli K-12using PCR products. Proc. Natl. Acad. Sci. U.S.A.2000; 97(12):6640-6645.) is a non-pathogenic bacterium with clear genetic background, short generation time, easy culture and low cost of culture medium raw materials. Coli BW25113 is publicly available from the institute of microbiology, academy of sciences, and this biomaterial is only used for repeating the relevant experiments of the present invention, and is not used for other purposes.

Example 1 construction of recombinant engineered Escherichia coli Strain GA00-GA07

1. A recombinant plasmid expressing glyoxylate reductase derived from the large intestine (pYB1s-ghRA) was constructed.

(1) Extraction of Escherichia coli genome DNA and PCR amplification of ghrA gene.

The genomic DNA of E.coli BW25113 was extracted using a bacterial genome extraction kit (Tiangen Biochemical technology Co., Ltd., product catalog DP 302). Extracting total DNA of Escherichia coli genome as template, using ghrA-F and ghrA-R as primer (Table 1), using high fidelity TransStart FastPfu DNA polymerase (Beijing Quanyujin Biotechnology Co., Ltd., product catalog is AP221) to perform PCR amplification, obtaining PCR product, performing agarose gel electrophoresis to the PCR product, recovering target segment, and obtaining gene segment ghrA-NX containing gene ghrA (nucleotide sequence shown as SEQ ID No. 2).

(2) Constructing a recombinant expression vector containing the ghrA gene.

The vector pYB1s (vector pYB1s has the nucleotide sequence shown in SEQ ID No.1, wherein the 86 th to 964 th sites are araC gene coding sequences, and the 1238 th and 1266 th sites are P-beta-amino acids) is digested with NcoI and XhoI_BADA promoter sequence, wherein the 1295-position 1299 is an RBS sequence, the 1307-position 1312 is an NcoI site, the 1366-position 1371 is an XhoI site, the 1393-position 1398 is an EcoRI site, the 1414-position 1419 is a PstI site, and the 1501-position 1658 is a T site_rrnBTerminator sequence, ORI gene coding sequence of P15A replication initiation site at position 1667-2457 and streptomycin resistance gene coding sequence at position 2561-3349), recovering large fragment pYB1s-NX of the vector. The gene fragment ghrA-NX and the vector large fragment pYB1s-NX are ligated by a Gibson assembly method (Gibson DG, Young L, et al. enzymatic assembly of DNA molecules up to recombinant human cloned nucleic acids. Nat. methods. 2009; 6(5):343-345) to obtain a ligation product. With CaCl₂Method for transforming the ligation product into E.coli DH5 alpha competent cells (purchased from Beijing Quanjin Biotechnology Ltd., catalog CD201) and uniformly spreading the cells on an LB plate containing streptomycinThen, the mixture was cultured overnight at 37 ℃ to select clones, and the clones capable of amplifying the target fragment were identified by using primers 105-F and ghrA-R (Table 1) and sequenced, and the positive clones were selected to extract plasmids, and the obtained positive plasmids were named pYB1 s-ghrA.

2. Recombinant plasmids (pYB1s-ghrA-aceAK) that synergistically expressed large intestine-derived glyoxylate reductase, isocitrate lyase, and isocitrate dehydrogenase kinase/phosphatase were constructed.

The genomic DNA of E.coli was extracted, and the gene fragment aceAK-NX comprising the gene aceAK (nucleotide sequence shown in SEQ ID No.3) was obtained by amplification with primers aceA-F and aceK-R, while the RBS sequence was introduced into the primers. The vector pYB1s-ghrA was digested with EcoRI and PstI to obtain the vector large fragment YB1 s-ghrA-EP. The gene fragment aceAK-NX and the carrier large fragment YB1s-ghrA-EP are subjected to ligation reaction, and the obtained ligation product is CaCl₂Coli DH 5. alpha. competent cells were transformed, spread evenly on LB plates containing streptomycin, and cultured overnight at 37 ℃. Clones were selected, clones capable of amplifying the desired fragment were identified with primers aceA-F and 124-R (Table 1) and sequenced, positive clones were screened and the positive plasmid obtained was named pYB1 s-ghrA-aceAK.

3. Constructing a recombinant plasmid pSB1a-mtk (Mca) -mcl (R) which synergistically expresses Methycoccus capsulatus-derived malate thiokinase and Rhodobacter sphaeroides-derived malyl-CoA lyase.

Escherichia coli codon-optimized malate thiokinase gene mtk derived from Methycoccus capsulatus and Rhodobacter sphaeroides-derived malyl-CoA lyase gene mcl were artificially synthesized, and RBS sequence was added before gene mcl, and the nucleotide sequence of mtk (mca) -mcl (Rsp) -NX fragment (mtk containing RBS (mca) -mcl (Rsp)) was shown in SEQ ID No.3 by using primers mtk (mca) -F and mcl (Rsp) -R (Table 1) according to the above method for amplifying ghrA. The vector pSB1a (the nucleotide sequence of the vector YB1s is shown in SEQ ID No.10, wherein, the 86 th to 964 th sites are araC gene coding sequences, and the 1238 th and 1266 th sites are P) is digested by NcoI and XhoI_BADA promoter sequence, wherein the 1295-position 1299 is an RBS sequence, the 1307-position 1312 is an NcoI site, the 1366-position 1371 is an XhoI site, and the 1501-position 1658 is a T site_rrnBTerminator sequence, 2 ndThe RepA gene coding sequence of the P15A replication initiation site at position 260-3210 and the ampicillin resistance gene coding sequence at position 3832-4692), and recovering the large vector fragment SB1 a-NX. The mtk (mca) -mcl (rsp) -NX fragment and the vector fragment SB1a-NX were Gibson assembled, the ligation product obtained was transformed into E.coli DH5 competent cells, which were then spread evenly on LB plate containing ampicillin overnight at 37 ℃, clones were selected, clones capable of amplifying the desired fragment were identified with primers 105-F and 124-R (Table 1) and sequenced, positive clones were selected, and the positive plasmid obtained was named pSB1a-mtk (mca) -mcl (rsp).

4. Construction of host bacteria

(1) Knock-out of NAD-dependent-malic enzyme gene maeA

The coding sequence of the NAD-dependent-malic enzyme gene maeA is shown in SEQ ID No.5, positions 1-1695.

(1-a) preparation of P1 phage containing E.coli Gene fragment with maeA knockout trait, i.e., phage P1 vir. DELTA.maeA

Coli gene fragments containing a maeA knockout trait were derived from Escherichia coli strain JW5238, a W3110 series strain containing a maeA knockout trait, purchased from Japan national institute of genetics (NIG, Japan), in which the NAD-dependent-malate gene maeA was replaced with a kanamycin-resistant gene (about 1300bp) having FRT sites at both ends to knock out the maeA gene (Baba T, Ara T, et al.

The preparation process of the P1 phage is specifically as follows: after culturing the strain JW5238 overnight at 37 ℃, the strain was transferred to a medium containing 5mmol/LCaCl₂And 0.1% glucose in LB culture medium, culturing at 37 deg.C for 1h, adding wild type P1 bacteriophage, culturing for 1-3h, adding several drops of chloroform, culturing for several minutes, centrifuging, and collecting supernatant to obtain bacteriophage P1vir Δ maeA containing Escherichia coli gene fragment with maeA knockout character.

(1-b) constructing an escherichia coli strain GA01-Kan by utilizing a P1 phage transduction technology, and specifically comprising the following steps: XY24 (recipient bacterium) cultured overnight (non-patent document describing the bacterium as "MetaThe bacillus engineering of Escherichia coli for production of L-aspartic and said microorganisms with high biological yield. biological engineering.54:244-254. "), 1.5mL of the bacterial suspension was centrifuged at 8000rpm for 3 minutes, followed by 0.75mL of P1 salt solution (water as solvent, 10mM CaCl as solute)₂And 5mM MgSO₄) Re-suspending XY24 thallus cells to obtain XY24 thallus cell suspension; mu.L of phage P1 vir. DELTA.maeA was mixed with 100. mu.L of XY24 bacterial cell suspension, and incubated at 37 ℃ for 30 min. Then, 1mL of LB medium and 200. mu.L of 1mol/L sodium citrate were added, and the culture was continued at 37 ℃ for 1 hour, the cells were collected by centrifugation, resuspended in 200. mu.L of LB medium, and then plated on an LB plate containing kanamycin (kanamycin concentration: 50. mu.g/mL), cultured overnight at 37 ℃, followed by selection of clones, PCR amplification and identification (amplification of 1900bp band was positive) using the primers maeA-F/maeA-R (Table 1), and the selected positive clones were named GA 01-Kan.

(1-c) Elimination of resistance: the pCP20 plasmid (purchased from Clontech) was transformed into GA01-Kan by calcium chloride transformation, and after overnight culture at 30 ℃ on LB plates containing ampicillin, clones were selected to obtain recombinant Escherichia coli GA01-Kan/pCP20 containing plasmid pCP 20. After culturing in LB medium containing ampicillin resistance at 30 ℃, spreading on an LB plate without resistance for overnight culture at 42 ℃, selecting clones, identifying by PCR amplification with primers maeA-F/maeA-R (Table 1) (600 bp of amplified band is positive), selecting positive clones and naming as Escherichia coli mutant GA 01.

(2) Knock-out of NADP dependent-malic enzyme gene maeB.

The coding sequence of the NADP dependent-malic enzyme gene maeB is shown in SEQ ID No.6, positions 1-2217.

Starting from the E.coli mutant GA01, the NADP dependent-malate gene maeB was knocked out using the same method as in step (1), to obtain the E.coli mutant GA 02. Wherein the difference from the step (1) is that: the strains and the primer names are different, namely the 'Escherichia coli gene fragment containing the maeA knockout character is from Escherichia coli strain JW 5238' is replaced by 'Escherichia coli gene fragment containing the maeB knockout character is from Escherichia coli strain JW2447 (purchased from the national institute of genetics (NIG, Japan))'; the name of the corresponding primer is changed from maeA to maeB, as shown in Table 1.

(3) Knockout of malate synthase a gene aceB.

The coding sequence of the malate synthase A gene aceB is shown in SEQ ID No.7, positions 1-1599.

Starting from the E.coli mutant GA02, the malate synthase gene aceB was knocked out using the same method as in step (1), to obtain E.coli GA 03. Wherein the difference from the step (1) is that: strains and primer names are different, namely, the 'Escherichia coli gene fragment containing maeA knockout character is from Escherichia coli strain JW 5238' is replaced by the 'Escherichia coli gene fragment containing aceB knockout character is from Escherichia coli strain JW3974 (purchased from the national institute of genetics (NIG, Japan))'; the name of the corresponding primer is changed from maeA to aceB, which is shown in Table 1.

(4) Knock-out of the glyoxylate pathway transcription repressor gene iclR.

The coding sequence of the glyoxylate pathway transcription repression factor gene iclR is shown as 1-822 sites of SEQ ID No. 8.

Starting from the E.coli mutant GA03, the glyoxylate pathway transcription repressing factor gene iclR was knocked out by the same method as in step (1). Recombinant E.coli GA04 was obtained. Wherein the difference from the step (1) is that: strains and primer names are different, namely, the 'Escherichia coli gene fragment containing maeA knockout character is from Escherichia coli strain JW 5238' is replaced by the 'Escherichia coli gene fragment containing iclR knockout character is from Escherichia coli strain JW3978 (purchased from the national institute of genetics (NIG, Japan))'; the name of the corresponding primer is changed from maeA to icl, which is shown in Table 1.

(5) Knock-out of glycolate oxidase gene glcD.

The coding sequence of the glycollic oxidase gene glcD is shown in SEQ ID No.9, positions 1-1497.

Escherichia coli GA05 was obtained by knocking out the glcD gene of glycolate oxidase in the same manner as in step (1) from the Escherichia coli mutant GA 04. Wherein the difference from the step (1) is that: strains and primer names are different, namely, the 'Escherichia coli gene fragment containing the maeA knockout character is from Escherichia coli strain JW 5238' is replaced by the 'Escherichia coli gene fragment containing the glcD knockout character is from Escherichia coli strain JW2946 (purchased from the national institute of genetics (NIG, Japan))'; the name of the corresponding primer is changed from maeA to glcD, as shown in Table 1.

5. Construction of recombinant Escherichia coli

(1) Construction of recombinant E.coli GA06

Preparing competent cells from Escherichia coli mutant GA05, and introducing plasmid pYB1s-ghrA-aceAK into CaCl₂GA05 is transformed by the method, the transformed GA05 is coated on an LB plate containing streptomycin, the LB plate is cultured overnight at 37 ℃, clones are selected, the corresponding target fragments are identified by the primers in the steps 1 to 4, and the correctly identified clone is named as recombinant escherichia coli GA 06.

(2) Construction of recombinant E.coli GA07

Preparing competent cells from Escherichia coli mutant GA06, and introducing plasmid pSB1a-mtk-mcl into CaCl₂GA06 is transformed by the method, the transformed GA06 is coated on an LB plate containing streptomycin and ampicillin, the LB plate is cultured overnight at 37 ℃, clones are selected, the corresponding target fragments are identified by the primers in the steps 1-4, and the correctly identified clone is named as recombinant Escherichia coli GA 07.

(3) Construction of recombinant E.coli GA 00.

Preparing competent cells from Escherichia coli mutant GA05, and extracting plasmids pYB1s and pSB1a with CaCl₂GA05 is transformed by the method, the transformed GA05 is coated on an LB plate containing streptomycin and ampicillin, the LB plate is cultured overnight at 37 ℃, clones are selected, the corresponding target fragments are identified by the primers in the steps 1-4, and the correctly identified clone is named as recombinant Escherichia coli GA 00.

TABLE 1 primer sequence List

Example 2 production of glycolic acid Using recombinant Escherichia coli strains GA00, GA06, and GA07 with glucose as the raw Material

1. Formula of culture medium

(11) The LB medium had the following composition:

yeast powder: 5g/L

Peptone: 10g/L

NaCl：10g/L

(12) The formula of the self-induction culture medium ZYM is as follows:

100mL A +2mL B +2mL C + 200. mu. L D + 100. mu. L E (in the following, the concentrations are in mass percent);

a, ZY: 1% tryptone, 0.5% yeast powder;

B.50×M：1.25M Na₂HPO₄，1.25M KH₂PO₄，2.5M NH₄cl and 0.25M Na₂SO₄；

C.50 × 5052: 25% glycerol, 2.5% glucose, 10% L-arabinose;

D.500×MgSO₄：1M MgSO₄

e.1000 × microelements: 50mM FeCl₃，20mM CaCl₂，10mM MnCl₂，10mM ZnSO₄，CoCl₂、NiCl₂、Na₂Mo₄、Na₂SeO₃And H₃BO₃2mM each.

(13) The components of the conversion solution are as follows:

glucose 10g/L, NaHCO₃ 100mM/L、100mM KH₂PO₄/K₂HPO₄ buffer。

2. Culture of cells and Induction of enzymes

The relevant engineered strains GA00, GA06, GA07 preserved in Glycerol tube were grown as single clones on LB plates containing the corresponding antibiotics (described in example 1 (5)) at 37 ℃. Selecting a single clone, inoculating the single clone into a liquid LB culture medium containing corresponding antibiotics, carrying out shake culture (the rotation speed is 200rpm) for 16h at 37 ℃, inoculating the single clone into a shake flask containing 50mL of ZYM self-induced culture medium according to the inoculation amount of 1%, adding arabinose with the final concentration of 0.2% and streptomycin and ampicillin with the final concentration of one thousandth, carrying out induction culture for 16h at 30 ℃, and collecting thalli.

3. Whole cell catalyzed transformation

The collected 150OD thalli are centrifuged for 10min at 4 ℃ and 5000rpm, washed twice by using 0.85% physiological saline, the supernatant is discarded, the thalli are resuspended by using 5ml of transformation solution, the thalli are transferred to a test tube, the final OD value is 30, the cells are transformed at 37 ℃ and 200rpm, samples are respectively sampled after 4h, 6h and 8h of transformation, the samples are centrifuged for 10min at 4 ℃ and 13000rpm, the supernatant is taken, the samples are filtered by using a 0.22 mu m filter membrane, the amounts of organic acid and glucose are detected by using High Performance Liquid Chromatography (HPLC), the experiment is set up for three times of repetition, and the results are averaged.

The high performance liquid chromatography detection instrument and the detection conditions are as follows:

a detection instrument: shimadzu LC-20AT 220V

Detection conditions are as follows: the mobile phase was 5mM H₂SO₄The flow rate is 0.6mL/min, the type of the detected chromatographic column is Aminex HPX-87H (300mm multiplied by 7.8mm), the column temperature is 55 ℃, the differential detection wavelength is 210nm, the sample injection amount is 10 mu L, and the detection and analysis time of each sample is 30 min.

A standard curve is made by referring to the peak emergence time and the peak area of a standard product (purchased from sigma-aldrich company, product number 420581), and the content of glycolic acid in the sample is calculated according to the peak area of the sample.

The results are shown in FIG. 1, from which it can be seen that: the glycolic acid yield of GA07 was 4.13g/L, that of GA06 was 1.94g/L, and that of GA00 was 0, indicating that the properties of the constructed strain are important for glycolic acid yield.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

SEQUENCE LISTING

<110> institute of microbiology of Chinese academy of sciences

<120> recombinant escherichia coli for producing glycolic acid and construction method and application thereof

<130> GNCFY200201

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 3528

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac tccgtcaagc 60

cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca ttcacttttt 120

cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta aatacccgcg 180

agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata ggcatccggg 240

tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag cttaagacgc 300

taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag caaacatgct 360

gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg tactgacaag 420

cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct tccatgcgcc 480

gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc ccttcccctt 540

gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc gcttcatccg 600

ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca tgccagtagg 660

cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga tgacgaccgt 720

agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa acaaattctc 780

gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata taacctttca 840

ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc ggcgttaaac 900

ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt tgcgcttcag 960

ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat tgcatcagac 1020

attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta accccgctta 1080

ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt aacaaaagtg 1140

tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca ctttgctatg 1200

ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta tcgcaactct 1260

ctactgtttc tccatacccg ttttttgggc taacaggagg aattaaccat gggtacctct 1320

catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagcctcga gggtagatct 1380

ggtactagtg gtgaattcgg tgagctcggt ctgcagctgg tgccgcgcgg cagccaccac 1440

caccaccacc actaatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 1500

tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 1560

gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat 1620

caaataaaac gaaaggctca gtcgaaagac tgggcctttc gtcgacgata acgcaggaaa 1680

gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc 1740

gttccatgaa aacgtcctag aagatgccag gaggatactt agcagagaga caataaggcc 1800

ggagcgaagc cgtttttcca taggctccgc ccccctgacg aacatcacga aatctgacgc 1860

tcaaatcagt ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgat 1920

ggctccctct tgcgctctcc tgttcccgtc ctgcggcgtc cgtgttgtgg tggaggcttt 1980

acccaaatca ccacgtcccg ttccgtgtag acagttcgct ccaagctggg ctgtgtgcaa 2040

gaaccccccg ttcagcccga ctgctgcgcc ttatccggta actatcatct tgagtccaac 2100

ccggaaagac acgacaaaac gccactggca gcagccattg gtaactgaga attagtggat 2160

ttagatatcg agagtcttga agtggtggcc taacagaggc tacactgaaa ggacagtatt 2220

tggtatctgc gctccactaa agccagttac caggttaagc agttccccaa ctgacttaac 2280

cttcgatcaa accgcctccc caggcggttt tttcgtttac agagcaggag attacgacga 2340

tcgtaaaagg atctcaagaa gatcctttac ggattcccga caccaggatc tagaagatcc 2400

tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatgcg 2460

gccgcctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 2520

accctgataa atgcttcaat aatattgaaa aaggaagagt atgagggaag cggtgatcgc 2580

cgaagtatcg actcaactat cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac 2640

gttgctggcc gtacatttgt acggctccgc agtggatggc ggcctgaagc cacacagtga 2700

tattgatttg ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc gagctttgat 2760

caacgacctt ttggaaactt cggcttcccc tggagagagc gagattctcc gcgctgtaga 2820

agtcaccatt gttgtgcacg acgacatcat tccgtggcgt tatccagcta agcgcgaact 2880

gcaatttgga gaatggcagc gcaatgacat tcttgcaggt atcttcgagc cagccacgat 2940

cgacattgat ctggctatct tgctgacaaa agcaagagaa catagcgttg ccttggtagg 3000

tccagcggcg gaggaactct ttgatccggt tcctgaacag gatctatttg aggcgctaaa 3060

tgaaacctta acgctatgga actcgccgcc cgactgggct ggcgatgagc gaaatgtagt 3120

gcttacgttg tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt 3180

cgctgccgac tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca tacttgaagc 3240

tagacaggct tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag atcagttgga 3300

agaatttgtc cactacgtga aaggcgagat caccaaggta gtcggcaaat aatgtctaac 3360

aattcgttca agccgagggg ccgcaagatc cggccacgat gacccggtcg tcggttcagg 3420

gcagggtcgt taaatagccg cttatgtcta ttgctggttt accggtttat tgactaccgg 3480

aagcagtgtg accgtgtgct tctcaaatgc ctgaggtttc aggcatgc 3528

<210> 2

<211> 939

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggatatca tcttttatca cccaacgttc gatacccaat ggtggattga ggcactgcgc 60

aaagctattc ctcaggcaag agtcagagca tggaaaagcg gagataatga ctctgctgat 120

tatgctttag tctggcatcc tcctgttgaa atgctggcag ggcgcgatct taaagcggtg 180

ttcgcactcg gggccggtgt tgattctatt ttgagcaagc tacaggcaca ccctgaaatg 240

ctgaaccctt ctgttccact ttttcgcctg gaagataccg gtatgggcga gcaaatgcag 300

gaatatgctg tcagtcaggt gctgcattgg tttcgacgtt ttgacgatta tcgcatccag 360

caaaatagtt cgcattggca accgctgcct gaatatcatc gggaagattt taccatcggc 420

attttgggcg caggcgtact gggcagtaaa gttgctcaga gtctgcaaac ctggcgcttt 480

ccgctgcgtt gctggagtcg aacccgtaaa tcgtggcctg gcgtgcaaag ctttgccgga 540

cgggaagaac tgtctgcatt tctgagccaa tgtcgggtat tgattaattt gttaccgaat 600

acccctgaaa ccgtcggcat tattaatcaa caattactcg aaaaattacc ggatggcgcg 660

tatctcctca acctggcgcg tggtgttcat gttgtggaag atgacctgct cgcggcgctg 720

gatagcggca aagttaaagg cgcaatgttg gatgttttta atcgtgaacc cttaccgcct 780

gaaagtccgc tctggcaaca tccacgcgtg acgataacac cacatgtcgc cgcgattacc 840

cgtcccgctg aagctgtgga gtacatttct cgcaccattg cccagctcga aaaaggggag 900

agggtctgcg ggcaagtcga ccgcgcacgc ggctactaa 939

<210> 3

<211> 3224

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgaaaaccc gtacacaaca aattgaagaa ttacagaaag agtggactca accgcgttgg 60

gaaggcatta ctcgcccata cagtgcggaa gatgtggtga aattacgcgg ttcagtcaat 120

cctgaatgca cgctggcgca actgggcgca gcgaaaatgt ggcgtctgct gcacggtgag 180

tcgaaaaaag gctacatcaa cagcctcggc gcactgactg gcggtcaggc gctgcaacag 240

gcgaaagcgg gtattgaagc agtctatctg tcgggatggc aggtagcggc ggacgctaac 300

ctggcggcca gcatgtatcc ggatcagtcg ctctatccgg caaactcggt gccagctgtg 360

gtggagcgga tcaacaacac cttccgtcgt gccgatcaga tccaatggtc cgcgggcatt 420

gagccgggcg atccgcgcta tgtcgattac ttcctgccga tcgttgccga tgcggaagcc 480

ggttttggcg gtgtcctgaa tgcctttgaa ctgatgaaag cgatgattga agccggtgca 540

gcggcagttc acttcgaaga tcagctggcg tcagtgaaga aatgcggtca catgggcggc 600

aaagttttag tgccaactca ggaagctatt cagaaactgg tcgcggcgcg tctggcagct 660

gacgtgacgg gcgttccaac cctgctggtt gcccgtaccg atgctgatgc ggcggatctg 720

atcacctccg attgcgaccc gtatgacagc gaatttatta ccggcgagcg taccagtgaa 780

ggcttcttcc gtactcatgc gggcattgag caagcgatca gccgtggcct ggcgtatgcg 840

ccatatgctg acctggtctg gtgtgaaacc tccacgccgg atctggaact ggcgcgtcgc 900

tttgcacaag ctatccacgc gaaatatccg ggcaaactgc tggcttataa ctgctcgccg 960

tcgttcaact ggcagaaaaa cctcgacgac aaaactattg ccagcttcca gcagcagctg 1020

tcggatatgg gctacaagtt ccagttcatc accctggcag gtatccacag catgtggttc 1080

aacatgtttg acctggcaaa cgcctatgcc cagggcgagg gtatgaagca ctacgttgag 1140

aaagtgcagc agccggaatt tgccgccgcg aaagatggct ataccttcgt atctcaccag 1200

caggaagtgg gtacaggtta cttcgataaa gtgacgacta ttattcaggg cggcacgtct 1260

tcagtcaccg cgctgaccgg ctccactgaa gaatcgcagt tctaagcaac aacaaccgtt 1320

gctgactgta ggccggataa ggcgttcacg ccgcatccgg caatcggtgc acgatgcctg 1380

atgcgacgct tgcgcgtctt atcatgccta cagccgttgc cgaacgtagg ctggataagg 1440

cgtttacgcc gcatccggca attctctgct cctgatgagg gcgctaaatg ccgcgtggcc 1500

tggaattatt gattgctcaa accattttgc aaggcttcga tgctcagtat ggtcgattcc 1560

tcgaagtgac ctccggtgcg cagcagcgtt tcgaacaggc cgactggcat gctgtccagc 1620

aggcgatgaa aaaccgtatc catctttacg atcatcacgt tggtctggtc gtggagcaac 1680

tgcgctgcat tactaacggc caaagtacgg acgcggcatt tttactacgt gttaaagagc 1740

attacacccg gctgttgccg gattacccgc gcttcgagat tgcggagagc ttttttaact 1800

ccgtgtactg tcggttattt gaccaccgct cgcttactcc cgagcggctt tttatcttta 1860

gctctcagcc agagcgccgc tttcgtacca ttccccgccc gctggcgaaa gactttcacc 1920

ccgatcacgg ctgggaatct ctactgatgc gcgttatcag cgacctaccg ctgcgcctgc 1980

gctggcagaa taaaagccgt gacatccatt acattattcg ccatctgacg gaaacgctgg 2040

ggacagacaa cctcgcggaa agtcatttac aggtggcgaa cgaactgttt taccgcaata 2100

aagccgcctg gctggtaggc aaactgatca caccttccgg cacattgcca tttttgctgc 2160

cgatccacca gacggacgac ggcgagttat ttattgatac ctgcctgacg acgaccgccg 2220

aagcgagcat tgtttttggc tttgcgcgtt cttattttat ggtttatgcg ccgctgcccg 2280

cagcgctggt cgagtggcta cgggaaattc tgccaggtaa aaccaccgct gaattgtata 2340

tggctatcgg ctgccagaag cacgccaaaa ccgaaagcta ccgcgaatat ctcgtttatc 2400

tacagggctg taatgagcag ttcattgaag cgccgggtat tcgtggaatg gtgatgttgg 2460

tgtttacgct gccgggcttt gatcgggtat tcaaagtcat caaagacagg ttcgcgccgc 2520

agaaagagat gtctgccgct cacgttcgtg cctgctatca actggtgaaa gagcacgatc 2580

gcgtgggccg aatggcggac acccaggagt ttgaaaactt tgtgctggag aagcggcata 2640

tttccccggc attaatggaa ttactgcttc aggaagcagc ggaaaaaatc accgatctcg 2700

gcgaacaaat tgtgattcgc catctttata ttgagcggcg gatggtgccg ctcaatatct 2760

ggctggaaca agtggaaggt cagcagttgc gcgacgccat tgaagaatac ggtaacgcta 2820

ttcgccagct tgccgctgct aacattttcc ctggcgacat gctgtttaaa aacttcggtg 2880

tcacccgtca cgggcgtgtg gttttttatg attacgatga aatttgctac atgacggaag 2940

tgaatttccg cgacatcccg ccgccgcgct atccggaaga cgaacttgcc agcgaaccgt 3000

ggtacagcgt ctcgccgggc gatgttttcc cggaagagtt tcgccactgg ctatgcgccg 3060

acccgcgtat tggtccgctg tttgaagaga tgcacgccga cctgttccgc gctgattact 3120

ggcgcgcact acaaaaccgc atacgtgaag ggcatgtgga agatgtttat gcgtatcggc 3180

gcaggcaaag atttagcgta cggtatgggg agatgctttt ttga 3224

<210> 4

<211> 3061

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

atgaacattc acgagtacca ggcgaaggaa ctgctgaaaa cctatggcgt gccggttccg 60

gatggtgcgg tggcgtacag cgatgcgcag gcggcgagcg ttgcggagga aattggtggc 120

agccgttggg tggttaaggc gcaaattcat gcgggtggcc gtggcaaggc gggtggcgtg 180

aaagttgcgc acagcatcga ggaagtgcgt cagtatgcgg acgcgatgct gggcagccac 240

ctggttaccc atcaaaccgg tccgggtggc agcctggtgc agcgtctgtg ggttgagcaa 300

gcgagccaca ttaagaaaga atactatctg ggcttcgtga tcgatcgtgg taaccaacgt 360

atcaccctga ttgcgagcag cgagggtggc atggaaatcg aggaagtggc gaaggagacc 420

ccggaaaaga ttgttaaaga ggtggttgac ccggcgatcg gcctgctgga ttttcagtgc 480

cgtaaagtgg cgaccgcgat tggcctgaag ggtaaactga tgccgcaagc ggttcgtctg 540

atgaaggcga tctaccgttg catgcgtgac aaagatgcgc tgcaagcgga gattaacccg 600

ctggcgatcg tgggcgagag cgacgaaagc ctgatggttc tggatgcgaa gttcaacttt 660

gacgataacg cgctgtaccg tcaacgtacc attaccgaaa tgcgtgacct ggcggaggaa 720

gatccgaaag aggtggaagc gagcggccac ggtctgaact atattgcgct ggacggcaac 780

atcggttgca ttgttaacgg tgcgggtctg gcgatggcga gcctggacgc gatcaccctg 840

cacggtggcc gtccggcgaa cttcctggat gtgggtggcg gtgcgagccc ggagaaggtg 900

accaacgcgt gccgtatcgt tctggaagac ccgaacgtgc gttgcatcct ggttaacatt 960

tttgcgggca tcaaccgttg cgactggatt gcgaagggtc tgatccaggc gtgcgatagc 1020

ctgcaaatta aagtgccgct gatcgttcgt ctggcgggca ccaacgttga tgagggtcgt 1080

aaaattctgg cggaaagcgg cctgagcttc atcaccgcgg aaaacctgga cgatgctgcg 1140

gcgaaggcgg tggcgatcgt taaaggttaa cagtcaggag atataatgag cgtgttcgtt 1200

aacaagcaca gcaaagttat cttccagggt tttaccggcg agcacgcgac ctttcacgcg 1260

aaggacgcga tgcgtatggg cacccgtgtg gttggtggcg tgaccccggg taaaggtggc 1320

acccgtcacc cggacccgga gctggcgcac ctgccggtgt tcgataccgt tgcggaagcg 1380

gtggcggcga ccggtgcgga tgttagcgcg gtgtttgtgc cgccgccgtt taacgcggat 1440

gcgctgatgg aggcgatcga tgcgggtatt cgtgtggcgg ttaccatcgc ggacggcatt 1500

ccggttcacg atatgatccg tctgcaacgt taccgtgttg gcaaggacag catcgtgatt 1560

ggtccgaaca ccccgggcat cattaccccg ggtgaatgca aagtgggcat catgccgagc 1620

cacatttaca agaaaggtaa cgttggcatc gttagccgta gcggcaccct gaactatgag 1680

gcgaccgaac agatggcggc gctgggtctg ggcattacca ccagcgttgg tatcggtggc 1740

gacccgatta acggcaccga tttcgtgacc gttctgcgtg cgtttgaggc ggacccggag 1800

accgaaatcg tggttatgat cggcgagatt ggtggcccgc aagaagtggc tgcggcgcgt 1860

tgggcgaagg aaaacatgac caaaccggtt attggttttg tggcgggtct ggcggcgccg 1920

accggccgtc gtatgggtca cgcgggcgcg atcattagca gcgaggcgga caccgcgggt 1980

gcgaagatgg atgcgatgga agcgctgggc ctgtatgttg cgcgtaaccc ggcgcagatc 2040

ggtcaaaccg tgctgcgtgc ggcgcaggaa catggcattc gtttttaaag aggagaaagg 2100

taccatgagc tttcgtctgc aaccggctcc gccggcgcgt ccgaaccgtt gccaactgtt 2160

tggtccgggc agccgtccgg cgctgtttga gaaaatggcg gcgagcgcgg cggacgtgat 2220

caacctggac ctggaagata gcgttgcgcc ggatgataaa gcgcaggcgc gtgcgaacat 2280

cattgaggcg atcaacggtc tggactgggg ccgtaagtac ctgagcgtgc gtattaacgg 2340

tctggatacc ccgttttggt atcgtgacgt ggttgatctg ctggagcagg cgggtgaccg 2400

tctggatcaa atcatgattc cgaaagtggg ttgcgcggcg gacgtgtatg cggttgatgc 2460

gctggttacc gcgatcgaac gtgcgaaggg tcgtaccaaa ccgctgagct tcgaggtgat 2520

cattgaaagc gcggcgggca tcgcgcacgt tgaggaaatt gcggcgagca gcccgcgtct 2580

gcaagcgatg agcctgggtg cggcggattt tgcggcgagc atgggtatgc agaccaccgg 2640

cattggtggc acccaagaga actactatat gctgcacgac ggccagaaac actggagcga 2700

tccgtggcac tgggcgcaag cggcgattgt ggcggcgtgc cgtacccacg gtattctgcc 2760

ggttgacggt ccgttcggcg attttagcga cgatgaaggt tttcgtgcgc aggcgcgtcg 2820

tagcgcgacc ctgggtatgg tgggcaagtg ggcgatccac ccgaaacaag tggcgctggc 2880

gaacgaggtt tttaccccga gcgaaaccgc ggttaccgag gcgcgtgaaa ttctggcggc 2940

gatggacgcg gcgaaggcgc gtggtgaagg tgcgaccgtg tataaaggtc gtctggttga 3000

tatcgcgagc attaagcagg cggaagtgat cgttcgtcaa gcggaaatga ttagcgcgta 3060

a 3061

<210> 5

<211> 1695

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atggaaccaa aaacaaaaaa acagcgttcg ctttatatcc cttacgctgg ccctgtactg 60

ctggaatttc cgttgttgaa taaaggcagt gccttcagca tggaagaacg ccgtaacttc 120

aacctgctgg ggttactgcc ggaagtggtc gaaaccatcg aagaacaagc ggaacgagca 180

tggatccagt atcagggatt caaaaccgaa atcgacaaac acatctacct gcgtaacatc 240

caggacacta acgaaaccct cttctaccgt ctggtaaaca atcatcttga tgagatgatg 300

cctgttattt ataccccaac cgtcggcgca gcctgtgagc gtttttctga gatctaccgc 360

cgttcacgcg gcgtgtttat ctcttaccag aaccggcaca atatggacga tattctgcaa 420

aacgtgccga accataatat taaagtgatt gtggtgactg acggtgaacg cattctgggg 480

cttggtgacc agggcatcgg cgggatgggc attccgatcg gtaaactgtc gctctatacc 540

gcctgtggcg gcatcagccc ggcgtatacc cttccggtgg tgctggatgt cggaacgaac 600

aaccaacagc tgcttaacga tccgctgtat atgggctggc gtaatccgcg tatcactgac 660

gacgaatact atgaattcgt tgatgaattt atccaggctg tgaaacaacg ctggccagac 720

gtgctgttgc agtttgaaga ctttgctcaa aaaaatgcga tgccgttact taaccgctat 780

cgcaatgaaa tttgttcttt taacgatgac attcagggca ctgcggcggt aacagtcggc 840

acactgatcg cagcaagccg cgcggcaggt ggtcagttaa gcgagaaaaa aatcgtcttc 900

cttggcgcag gttcagcggg atgcggcatt gccgaaatga tcatctccca gacccagcgc 960

gaaggattaa gcgaggaagc ggcgcggcag aaagtcttta tggtcgatcg ctttggcttg 1020

ctgactgaca agatgccgaa cctgctgcct ttccagacca aactggtgca gaagcgcgaa 1080

aacctcagtg actgggatac cgacagcgat gtgctgtcac tgctggatgt ggtgcgcaat 1140

gtaaaaccag atattctgat tggcgtctca ggacagaccg ggctgtttac ggaagagatc 1200

atccgtgaga tgcataaaca ctgtccgcgt ccgatcgtga tgccgctgtc taacccgacg 1260

tcacgcgtgg aagccacacc gcaggacatt atcgcctgga ccgaaggtaa cgcgctggtc 1320

gccacgggca gcccgtttaa tccagtggta tggaaagata aaatctaccc tatcgcccag 1380

tgtaacaacg cctttatttt cccgggcatc ggcctgggtg ttattgcttc cggcgcgtca 1440

cgtatcaccg atgagatgct gatgtcggca agtgaaacgc tggcgcagta ttcaccattg 1500

gtgctgaacg gcgaaggtat ggtactgccg gaactgaaag atattcagaa agtctcccgc 1560

gcaattgcgt ttgcggttgg caaaatggcg cagcagcaag gcgtggcggt gaaaacctct 1620

gccgaagccc tgcaacaggc cattgacgat aatttctggc aagccgaata ccgcgactac 1680

cgccgtacct ccatc 1695

<210> 6

<211> 2217

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atccaggttt ctccaaccaa gcctctggca acacagcgcg atctggcgct ggcctactca 60

ccaggcgttg ccgcaccttg tcttgaaatc gaaaaagacc cgttaaaagc ctacaaatat 120

accgcccgag gtaacctggt ggcggtgatc tctaacggta cggcggtgct ggggttaggc 180

aacattggcg cgctggcagg caaaccggtg atggaaggca agggcgttct gtttaagaaa 240

ttcgccggga ttgatgtatt tgacattgaa gttgacgaac tcgacccgga caaatttatt 300

gaagttgtcg ccgcgctcga accaaccttc ggcggcatca acctcgaaga cattaaagcg 360

ccagaatgtt tctatattga acagaaactg cgcgagcgga tgaatattcc ggtattccac 420

gacgatcagc acggcacggc aattatcagc actgccgcca tcctcaacgg cttgcgcgtg 480

gtggagaaaa acatctccga cgtgcggatg gtggtttccg gcgcgggtgc cgcagcaatc 540

gcctgtatga acctgctggt agcgctgggt ctgcaaaaac ataacatcgt ggtttgcgat 600

tcaaaaggcg ttatctatca gggccgtgag ccaaacatgg cggaaaccaa agccgcatat 660

gcggtggtgg atgacggcaa acgtaccctc gatgatgtga ttgaaggcgc ggatattttc 720

ctgggctgtt ccggcccgaa agtgctgacc caggaaatgg tgaagaaaat ggctcgtgcg 780

ccaatgatcc tggcgctggc gaacccggaa ccggaaattc tgccgccgct ggcgaaagaa 840

gtgcgtccgg atgccatcat ttgcaccggt cgttctgact atccgaacca ggtgaacaac 900

gtcctgtgct tcccgttcat cttccgtggc gcgctggacg ttggcgcaac cgccatcaac 960

gaagagatga aactggcggc ggtacgtgcg attgcagaac tcgcccatgc ggaacagagc 1020

gaagtggtgg cttcagcgta tggcgatcag gatctgagct ttggtccgga atacatcatt 1080

ccaaaaccgt ttgatccgcg cttgatcgtt aagatcgctc ctgcggtcgc taaagccgcg 1140

atggagtcgg gcgtggcgac tcgtccgatt gctgatttcg acgtctacat cgacaagctg 1200

actgagttcg tttacaaaac caacctgttt atgaagccga ttttctccca ggctcgcaaa 1260

gcgccgaagc gcgttgttct gccggaaggg gaagaggcgc gcgttctgca tgccactcag 1320

gaactggtaa cgctgggact ggcgaaaccg atccttatcg gtcgtccgaa cgtgatcgaa 1380

atgcgcattc agaaactggg cttgcagatc aaagcgggcg ttgattttga gatcgtcaat 1440

aacgaatccg atccgcgctt taaagagtac tggaccgaat acttccagat catgaagcgt 1500

cgcggcgtca ctcaggaaca ggcgcagcgg gcgctgatca gtaacccgac agtgatcggc 1560

gcgatcatgg ttcagcgtgg ggaagccgat gcaatgattt gcggtacggt gggtgattat 1620

catgaacatt ttagcgtggt gaaaaatgtc tttggttatc gcgatggcgt tcacaccgca 1680

ggtgccatga acgcgctgct gctgccgagt ggtaacacct ttattgccga tacatatgtt 1740

aatgatgaac cggatgcaga agagctggcg gagatcacct tgatggcggc agaaactgtc 1800

cgtcgttttg gtattgagcc gcgcgttgct ttgttgtcgc actccaactt tggttcttct 1860

gactgcccgt cgtcgagcaa aatgcgtcag gcgctggaac tggtcaggga acgtgcacca 1920

gaactgatga ttgatggtga aatgcacggc gatgcagcgc tggtggaagc gattcgcaac 1980

gaccgtatgc cggacagctc tttgaaaggt tccgccaata ttctggtgat gccgaacatg 2040

gaagctgccc gcattagtta caacttactg cgtgtttcca gctcggaagg tgtgactgtc 2100

ggcccggtgc tgatgggtgt ggcgaaaccg gttcacgtgt taacgccgat cgcatcggtg 2160

cgtcgtatcg tcaacatggt ggcgctggcc gtggtagaag cgcaaaccca accgctg 2217

<210> 7

<211> 1599

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgactgaac aggcaacaac aaccgatgaa ctggctttca caaggccgta tggcgagcag 60

gagaagcaaa ttcttactgc cgaagcggta gaatttctga ctgagctggt gacgcatttt 120

acgccacaac gcaataaact tctggcagcg cgcattcagc agcagcaaga tattgataac 180

ggaacgttgc ctgattttat ttcggaaaca gcttccattc gcgatgctga ttggaaaatt 240

cgcgggattc ctgcggactt agaagaccgc cgcgtagaga taactggccc ggtagagcgc 300

aagatggtga tcaacgcgct caacgccaat gtgaaagtct ttatggccga tttcgaagat 360

tcactggcac cagactggaa caaagtgatc gacgggcaaa ttaacctgcg tgatgcggtt 420

aacggcacca tcagttacac caatgaagca ggcaaaattt accagctcaa gcccaatcca 480

gcggttttga tttgtcgggt acgcggtctg cacttgccgg aaaaacatgt cacctggcgt 540

ggtgaggcaa tccccggcag cctgtttgat tttgcgctct atttcttcca caactatcag 600

gcactgttgg caaagggcag tggtccctat ttctatctgc cgaaaaccca gtcctggcag 660

gaagcggcct ggtggagcga agtcttcagc tatgcagaag atcgctttaa tctgccgcgc 720

ggcaccatca aggcgacgtt gctgattgaa acgctgcccg ccgtgttcca gatggatgaa 780

atccttcacg cgctgcgtga ccatattgtt ggtctgaact gcggtcgttg ggattacatc 840

ttcagctata tcaaaacgtt gaaaaactat cccgatcgcg tcctgccaga cagacaggca 900

gtgacgatgg ataaaccatt cctgaatgct tactcacgcc tgttgattaa aacctgccat 960

aaacgcggtg cttttgcgat gggcggcatg gcggcgttta ttccgagcaa agatgaagag 1020

cacaataacc aggtgctcaa caaagtaaaa gcggataaat cgctggaagc caataacggt 1080

cacgatggca catggatcgc tcacccaggc cttgcggaca cggcaatggc ggtattcaac 1140

gacattctcg gctcccgtaa aaatcagctt gaagtgatgc gcgaacaaga cgcgccgatt 1200

actgccgatc agctgctggc accttgtgat ggtgaacgca ccgaagaagg tatgcgcgcc 1260

aacattcgcg tggctgtgca gtacatcgaa gcgtggatct ctggcaacgg ctgtgtgccg 1320

atttatggcc tgatggaaga tgcggcgacg gctgaaattt cccgtacctc gatctggcag 1380

tggatccatc atcaaaaaac gttgagcaat ggcaaaccgg tgaccaaagc cttgttccgc 1440

cagatgctgg gcgaagagat gaaagtcatt gccagcgaac tgggcgaaga acgtttctcc 1500

caggggcgtt ttgacgatgc cgcacgcttg atggaacaga tcaccacttc cgatgagtta 1560

attgatttcc tgaccctgcc aggctaccgc ctgttagcg 1599

<210> 8

<211> 822

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atggtcgcac ccattcccgc gaaacgcggc agaaaacccg ccgttgccac cgcaccagcg 60

actggacagg ttcagtcttt aacgcgtggc ctgaaattac tggagtggat tgccgaatcc 120

aatggcagtg tggcactcac ggaactggcg caacaagccg ggttacccaa ttccacgacc 180

caccgcctgc taaccacgat gcaacagcag ggtttcgtgc gtcaggttgg cgaactggga 240

cattgggcaa tcggcgcaca tgcctttatg gtcggcagca gctttctcca gagccgtaat 300

ttgttagcga ttgttcaccc tatcctgcgc aatctaatgg aagagtctgg cgaaacggtc 360

aatatggcgg tgcttgatca aagcgatcac gaagcgatta ttatcgacca ggtacagtgt 420

acgcatctga tgcgaatgtc cgcgcctatc ggcggtaaat tgccgatgca cgcttccggt 480

gcgggtaaag cctttttagc ccaactgagc gaagaacagg tgacgaagct gctgcaccgc 540

aaagggttac atgcctatac ccacgcaacg ctggtgtctc ctgtgcattt aaaagaagat 600

ctcgcccaaa cgcgcaaacg gggttattca tttgacgatg aggaacatgc actggggcta 660

cgttgccttg cagcgtgtat tttcgatgag caccgtgaac cgtttgccgc aatttctatt 720

tccggaccga tttcacgtat taccgatgac cgcgtgaccg agtttggcgc gatggtgatt 780

aaagcggcga aggaagtgac gctggcgtac ggtggaatgc gc 822

<210> 9

<211> 1497

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atgagcatct tgtacgaaga gcgtcttgat ggcgctttac ccgatgtcga ccgcacatcg 60

gtactgatgg cactgcgtga gcatgtccct ggacttgaga tcctgcatac cgatgaggag 120

atcattcctt acgagtgtga cgggttgagc gcgtatcgca cgcgtccatt actggttgtt 180

ctgcctaagc aaatggaaca ggtgacagcg attctggctg tctgccatcg cctgcgtgta 240

ccggtggtga cccgtggtgc aggcaccggg ctttctggtg gcgcgctgcc gctggaaaaa 300

ggtgtgttgt tggtgatggc gcgctttaaa gagatcctcg acattaaccc cgttggtcgc 360

cgcgcgcgcg tgcagccagg cgtgcgtaac ctggcgatct cccaggccgt tgcaccgcat 420

aatctctact acgcaccgga cccttcctca caaatcgcct gttccattgg cggcaatgtg 480

gctgaaaatg ccggcggcgt ccactgcctg aaatatggtc tgaccgtaca taacctgctg 540

aaaattgaag tgcaaacgct ggacggcgag gcactgacgc ttggatcgga cgcgctggat 600

tcacctggtt ttgacctgct ggcgctgttc accggatcgg aaggtatgct cggcgtgacc 660

accgaagtga cggtaaaact gctgccgaag ccgcccgtgg cgcgggttct gttagccagc 720

tttgactcgg tagaaaaagc cggacttgcg gttggtgaca tcatcgccaa tggcattatc 780

cccggcgggc tggagatgat ggataacctg tcgatccgcg cggcggaaga ttttattcat 840

gccggttatc ccgtcgacgc cgaagcgatt ttgttatgcg agctggacgg cgtggagtct 900

gacgtacagg aagactgcga gcgggttaac gacatcttgt tgaaagcggg cgcgactgac 960

gtccgtctgg cacaggacga agcagagcgc gtacgtttct gggccggtcg caaaaatgcg 1020

ttcccggcgg taggacgtat ctccccggat tactactgca tggatggcac catcccgcgt 1080

cgcgccctgc ctggcgtact ggaaggcatt gcccgtttat cgcagcaata tgatttacgt 1140

gttgccaacg tctttcatgc cggagatggc aacatgcacc cgttaatcct tttcgatgcc 1200

aacgaacccg gtgaatttgc ccgcgcggaa gagctgggcg ggaagatcct cgaactctgc 1260

gttgaagttg gcggcagcat cagtggcgaa catggcatcg ggcgagaaaa aatcaatcaa 1320

atgtgcgccc agttcaacag cgatgaaatc acgaccttcc atgcggtcaa ggcggcgttt 1380

gaccccgatg gtttgctgaa ccctgggaaa aacattccca cgctacaccg ctgtgctgaa 1440

tttggtgcca tgcatgtgca tcacggtcat ttacctttcc ctgaactgga gcgtttc 1497

<210> 10

<211> 4792

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

aatgtgcctg tcaaatggac gaagcaggga ttctgcaaac cctatgctac tccgtcaagc 60

cgtcaattgt ctgattcgtt accaattatg acaacttgac ggctacatca ttcacttttt 120

cttcacaacc ggcacggaac tcgctcgggc tggccccggt gcatttttta aatacccgcg 180

agaaatagag ttgatcgtca aaaccaacat tgcgaccgac ggtggcgata ggcatccggg 240

tggtgctcaa aagcagcttc gcctggctga tacgttggtc ctcgcgccag cttaagacgc 300

taatccctaa ctgctggcgg aaaagatgtg acagacgcga cggcgacaag caaacatgct 360

gtgcgacgct ggcgatatca aaattgctgt ctgccaggtg atcgctgatg tactgacaag 420

cctcgcgtac ccgattatcc atcggtggat ggagcgactc gttaatcgct tccatgcgcc 480

gcagtaacaa ttgctcaagc agatttatcg ccagcagctc cgaatagcgc ccttcccctt 540

gcccggcgtt aatgatttgc ccaaacaggt cgctgaaatg cggctggtgc gcttcatccg 600

ggcgaaagaa ccccgtattg gcaaatattg acggccagtt aagccattca tgccagtagg 660

cgcgcggacg aaagtaaacc cactggtgat accattcgcg agcctccgga tgacgaccgt 720

agtgatgaat ctctcctggc gggaacagca aaatatcacc cggtcggcaa acaaattctc 780

gtccctgatt tttcaccacc ccctgaccgc gaatggtgag attgagaata taacctttca 840

ttcccagcgg tcggtcgata aaaaaatcga gataaccgtt ggcctcaatc ggcgttaaac 900

ccgccaccag atgggcatta aacgagtatc ccggcagcag gggatcattt tgcgcttcag 960

ccatactttt catactcccg ccattcagag aagaaaccaa ttgtccatat tgcatcagac 1020

attgccgtca ctgcgtcttt tactggctct tctcgctaac caaaccggta accccgctta 1080

ttaaaagcat tctgtaacaa agcgggacca aagccatgac aaaaacgcgt aacaaaagtg 1140

tctataatca cggcagaaaa gtccacattg attatttgca cggcgtcaca ctttgctatg 1200

ccatagcatt tttatccata agattagcgg atcctacctg acgcttttta tcgcaactct 1260

ctactgtttc tccatacccg ttttttgggc taacaggagg aattaaccat gggtacctct 1320

catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagcctcga gggtagatct 1380

ggtactagtg gtgaattcgg tgagctcggt ctgcagctgg tgccgcgcgg cagccaccac 1440

caccaccacc actaatacag attaaatcag aacgcagaag cggtctgata aaacagaatt 1500

tgcctggcgg cagtagcgcg gtggtcccac ctgaccccat gccgaactca gaagtgaaac 1560

gccgtagcgc cgatggtagt gtggggtctc cccatgcgag agtagggaac tgccaggcat 1620

caaataaaac gaaaggctca gtcgaaagac tgggcctttc gtcgaccaga cccgccataa 1680

aacgccctga gaagcccgtg acgggctttt cttgtattat gggtagtttc cttgcatgaa 1740

tccataaaag gcgcctgtag tgccatttac ccccattcac tgccagagcc gtgagcgcag 1800

cgaactgaat gtcacgaaaa agacagcgac tcaggtgcct gatggtcgga gacaaaagga 1860

atattcagcg atttgcccga gcttgcgagg gtgctactta agcctttagg gttttaaggt 1920

ctgttttgta gaggagcaaa cagcgtttgc gacatccttt tgtaatactg cggaactgac 1980

taaagtagtg agttatacac agggctggga tctattcttt ttatcttttt ttattctttc 2040

tttattctat aaattataac cacttgaata taaacaaaaa aaacacacaa aggtctagcg 2100

gaatttacag agggtctagc agaatttaca agttttccag caaaggtcta gcagaattta 2160

cagataccca caactcaaag gaaaaggtct agtaattatc attgactagc ccatctcaat 2220

tggtatagtg attaaaatca cctagaccaa ttgagatgta tgtctgaatt agttgttttc 2280

aaagcaaatg aactagcgat tagtcgctat gacttaacgg agcatgaaac caagctaatt 2340

ttatgctgtg tggcactact caaccccacg attgaaaacc ctacaaggaa agaacggacg 2400

gtatcgttca cttataacca atacgctcag atgatgaaca tcagtaggga aaatgcttat 2460

ggtgtattag ctaaagcaac cagagagctg atgacgagaa ctgtggaaat caggaatcct 2520

ttggttaaag gctttgagat tttccagtgg acaaactatg ccaagttctc aagcgaaaaa 2580

ttagaattag tttttagtga agagatattg ccttatcttt tccagttaaa aaaattcata 2640

aaatataatc tggaacatgt taagtctttt gaaaacaaat actctatgag gatttatgag 2700

tggttattaa aagaactaac acaaaagaaa actcacaagg caaatataga gattagcctt 2760

gatgaattta agttcatgtt aatgcttgaa aataactacc atgagtttaa aaggcttaac 2820

caatgggttt tgaaaccaat aagtaaagat ttaaacactt acagcaatat gaaattggtg 2880

gttgataagc gaggccgccc gactgatacg ttgattttcc aagttgaact agatagacaa 2940

atggatctcg taaccgaact tgagaacaac cagataaaaa tgaatggtga caaaatacca 3000

acaaccatta catcagattc ctacctacgt aacggactaa gaaaaacact acacgatgct 3060

ttaactgcaa aaattcagct caccagtttt gaggcaaaat ttttgagtga catgcaaagt 3120

aagcatgatc tcaatggttc gttctcatgg ctcacgcaaa aacaacgaac cacactagag 3180

aacatactgg ctaaatacgg aaggatctga ggttcttatg gctcttgtat ctatcagtga 3240

agcatcaaga ctaacaaaca aaagtagaac aactgttcac cgttagatat caaagggaaa 3300

actgtcgata tgcacagatg aaaacggtgt aaaaaagata gatacatcag agcttttacg 3360

agtttttggt gcatttaaag ctgttcacca tgaacagatc gacaatgtaa cagatgaaca 3420

gcatgtaaca cctaatagaa caggtgaaac cagtaaaaca aagcaactag aacatgaaat 3480

tgaacacctg agacaacttg ttacagctca acagtcacac atagacagcc tgaaacaggc 3540

gatgctgctt atcgaatcaa agctgccgac aacacgggag ccagtgacgc ctcccgtggg 3600

gaaaaaatca tggcaattct ggaagaaata gcgctttcag ccggcaaacc tgaagccgga 3660

tctgcgattc tgataacaaa ctagcaacac cagaacagcc cgtttgcggg cagcaaaacc 3720

cgcggccgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc 3780

atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt 3840

caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct 3900

cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt 3960

tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt 4020

tttccaatga tgagcacttt taaagttctg ctatgtgata cactattatc ccgtattgac 4080

gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac 4140

tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct 4200

gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg 4260

aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg 4320

gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca 4380

atgccaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa 4440

caattaatag actgaatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt 4500

ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc 4560

attgcagcac tggggccaga tggtaagcgc tcccgtatcg tagttatcta caccacgggg 4620

agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt 4680

aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt 4740

catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatcgcat gc 4792

Claims

1. Recombinant escherichia coli, characterized in that: compared with a receptor Escherichia coli, the recombinant Escherichia coli has the advantages that the expression quantity of the gene of the protein A in the recombinant Escherichia coli is increased, and/or the content of the protein A is increased, and/or the activity of the protein A is increased, and/or the expression quantity of the gene of the protein B in the recombinant Escherichia coli is reduced, and/or the content of the protein B is reduced, and/or the activity of the protein B is reduced;

the protein A is selected from at least one of the following:

A1) malic acid thiokinase;

A2) malyl-coa lyase;

A3) glyoxylate reductase;

A4) isocitrate dehydrogenase kinase/phosphatase;

A5) isocitrate lyase;

the protein B is selected from at least one of the following:

B1) NAD-dependent-malic enzyme;

B2) NADP-dependent-malic enzyme;

B3) a malate synthase A;

B4) glyoxylate pathway transcription repressing factor;

B5) glycolate oxidase.

2. The construction method of the recombinant escherichia coli is characterized by comprising the following steps:

the construction method comprises the following steps of increasing the expression quantity of a protein A gene and/or the content of the protein A and/or the activity of the protein A in receptor Escherichia coli, and/or reducing the expression quantity of a protein B gene and/or the content of the protein B and/or the activity of the protein B in the receptor Escherichia coli:

the protein A is selected from at least one of the following:

A1) malic acid thiokinase;

A2) malyl-coa lyase;

A3) glyoxylate reductase;

A4) isocitrate dehydrogenase kinase/phosphatase;

A5) isocitrate lyase;

the protein B is selected from at least one of the following:

B1) NAD-dependent-malic enzyme;

B2) NADP-dependent-malic enzyme;

B3) a malate synthase A;

B4) glyoxylate pathway transcription repressing factor;

B5) glycolate oxidase.

3. The construction method according to claim 2, wherein: the construction method is realized by introducing the gene of the protein A into recipient Escherichia coli; the acceptor escherichia coli is an escherichia coli mutant or wild type escherichia coli;

specifically, the wild type Escherichia coli is Escherichia coli K12.

4. The method for constructing Escherichia coli according to claim 3, wherein the Escherichia coli mutant is obtained by modifying the genome of wild type Escherichia coli with the following modifications of all, any four, any three, any two or any one of m1) -m 5):

m1) knocking out genes of NAD dependent-malic enzyme;

m2) knocking out the gene of NADP dependent-malic enzyme;

m3) knocking out the gene of malate synthase A;

m4) knocking out the gene of the transcription repressing factor of the glyoxylate pathway;

m5) knocking out the gene of glycolate oxidase.

5. The recombinant Escherichia coli according to claim 1, or the construction method according to any one of claims 2 to 4, wherein:

the gene of the malic acid thiokinase is from methylococcus capsulatus, and the gene of the malyl-CoA lyase is from rhodobacter sphaeroides.

6. The recombinant Escherichia coli according to claim 1, or the construction method according to any one of claims 2 to 4, wherein:

the glyoxylate reductase is C1) or C2):

C1) protein coded by DNA molecule shown in SEQ ID No. 2;

the isocitrate lyase is represented by C3) or C4):

the isocitrate dehydrogenase kinase/phosphatase is represented by C5) or C6):

the malate thiokinase is C7) or C8):

the malyl-coenzyme A lyase is represented by C9) or C10):

the NAD-dependent malic enzyme is C11) or C12):

C11) protein coded by DNA molecule shown in SEQ ID No. 5;

C12) a protein having 90% or more identity and function identity to the protein represented by C11) obtained by substitution and/or deletion and/or addition of one or more amino acid residues of the protein represented by C11); the NADP dependent-malic enzyme is C13) or C14):

C13) protein coded by DNA molecule shown in SEQ ID No. 6;

the malate synthase A is C15) or C16):

C15) protein coded by DNA molecule shown in SEQ ID No. 7;

the glyoxylate pathway transcription inhibitor is C17) or C18):

C17) protein coded by DNA molecule shown in SEQ ID No. 8;

the glycolate oxidase is represented by C19) or C20):

C19) protein coded by DNA molecule shown in SEQ ID No. 9;

7. The recombinant Escherichia coli according to claim 1, or the construction method according to any one of claims 2 to 4, wherein:

the gene of the glyoxylate reductase is shown as D1) or D2):

D1) the coding sequence is DNA molecule shown in SEQ ID No. 2;

the isocitrate lyase gene is shown in D3) or D4) as follows:

the isocitrate dehydrogenase kinase/phosphatase gene is shown in D5) or D6) as follows:

the gene of the malate thiokinase is shown as D7) or D8):

the gene of the malyl-CoA lyase is shown as D9) or D10):

the gene of the NAD dependent-malic enzyme is shown as D11) or D12):

D11) the coding sequence is DNA molecule shown in SEQ ID No. 5;

D12) DNA molecule which is obtained by substituting and/or deleting and/or adding one or more nucleotides in SEQ ID No.5 and has the same function with the 1 st to 1695 th positions of SEQ ID No. 5;

the gene of the NADP dependent-malic enzyme is shown as D13) or D14):

D13) the coding sequence is DNA molecule shown in SEQ ID No. 6;

the gene of the malate synthase A is shown as D15) or D16):

D15) the coding sequence is DNA molecule shown in SEQ ID No. 7;

the genes of the glyoxylate pathway transcription inhibitor are shown as D17) or D18):

D17) the coding sequence is DNA molecule shown in SEQ ID No. 8;

the gene of the glycolate oxidase is shown in the following D19) or D20):

D19) the coding sequence is DNA molecule shown in SEQ ID No. 9;

8. A recombinant Escherichia coli constructed by the method according to any one of claims 2 to 7.

9. Use of the recombinant E.coli of any one of claims 1 or 5 to 8 for the preparation of glycolic acid.

10. A process for the preparation of glycolic acid characterized by: the method comprises using the recombinant Escherichia coli of any one of claims 1 or 5-8 to catalyze a glucose reaction to obtain glycolic acid.