CN112961814A

CN112961814A - Construction method of escherichia coli engineering bacteria preferring to efficiently secrete acetic acid and FFA by utilizing xylose

Info

Publication number: CN112961814A
Application number: CN202011442400.XA
Authority: CN
Inventors: 贾晓强; 刘亚茹; 杨松源
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2020-12-08
Filing date: 2020-12-08
Publication date: 2021-06-15

Abstract

The invention relates to a construction method of escherichia coli engineering bacteria which prefers to efficiently secrete acetic acid and FFA by utilizing xylose; coli MG1655 by knocking out ptsG and manZ genes in it to slowly metabolize glucose while preferentially utilizing xylose. Coli MG1655 promotes acetate synthesis by knocking out atpFH, a key gene in the process of oxidizing phosphate. Coli MG1655 knock-out envR gene promotes e.coli to secrete FFA extracellularly. The gene knockout engineering bacteria can synthesize six FFAs (fringe-like enzymes) including n-hexanoic acid, n-heptanoic acid, n-octanoic acid, n-nonanoic acid, n-decanoic acid, n-dodecanoic acid and the like by utilizing xylose, can synthesize 5.5g/L of acetic acid and 0.91g/L of extracellular FFA by taking 20g/L of xylose as a single carbon source in an M9 culture medium, and has obviously improved yield compared with the wild E.coli MG 1655.

Description

Construction method of escherichia coli engineering bacteria preferring to efficiently secrete acetic acid and FFA by utilizing xylose

Technical Field

The invention relates to a construction method of escherichia coli engineering bacteria preferring to efficiently secrete acetic acid and FFA by utilizing xylose, belonging to the field of genetic engineering research.

Background

With the development of society, the problems of energy demand, resource crisis, food shortage and the like are increasingly highlighted, and people are prompted to search for novel energy resources. One third of the land area on earth is covered by plants, forming a large amount of lignocellulosic resources. Lignocellulose mainly comprises cellulose, hemicellulose and lignin, wherein the hemicellulose accounts for about 20-35% of the total amount of the lignocellulose, and the hydrolysate of the lignocellulose is mainly xylose, so that the xylose becomes the second most carbohydrate substance which is only second to glucose in nature. On the basis of the increasingly deep research on the hydrolysis of hemicellulose, xylose is more and more widely applied to agriculture, industry and even scientific research.

Coli (e.coli) is one of the model strains that have been widely studied at present, and its gene background is clear, genetic modification means are abundant, and it should be widely applied to various biological studies. Optimization of recombinant strains for the production of valuable compounds is a major goal of genetic engineering. To this end, various strategies have been devised to construct recombinant strains, including increasing the supply of metabolic precursors, deleting negative regulators, overexpression of positive regulators, and removing precursor consumption and product degradation pathways. Acetic acid is a very promising source of carbon and has been considered as one of the substrates that is expected to reduce the cost of PHA production. Free Fatty Acids (FFA) are free fatty acids that are synthesized by microorganisms under the catalysis of thioesterases (thioesterases) using the fatty acid synthesis pathway intermediary metabolite Acyl-ACP as a substrate. In recent decades, rational metabolic engineering has been widely used to improve the synthesis of FFA and its precursors, but no report has been found on the construction of engineering strains that prefer to produce acetate and FFA with high yield by xylose.

Disclosure of Invention

The invention provides a construction method of escherichia coli engineering bacteria which prefers to efficiently secrete acetic acid and FFA by utilizing xylose.

The invention can slowly metabolize glucose and preferentially utilize xylose by knocking out ptsG and manZ genes in E.coli MG 1655. Coli MG1655 promotes acetate synthesis by knocking out atpFH, a key gene in the process of oxidizing phosphate. The effect of knocking out ptsG and manZ genes on e.coli MG1655 glucose and xylose utilization was verified. Coli MG1655 by knocking out envR gene to promote FFA secretion from escherichia coli to the extracellular. Finally obtaining engineering bacteria EC delta p-m-a-e with four knocked-out genes, and carrying out FFA and acetic acid production tests.

The specific technology of the invention is as follows:

a construction method of escherichia coli engineering bacteria preferring to efficiently secrete acetic acid and FFA by utilizing xylose; coli MG1655 by knocking out ptsG and manZ genes in it to slowly metabolize glucose while preferentially utilizing xylose; promoting acetic acid synthesis by knocking out atpFH (atpFH) key gene in the process of oxidizing phosphoric acid by E.coli MG 1655; promoting escherichia coli to secrete FFA to the outside of cells by knocking out envR gene of E.coli MG 1655; the method comprises the following specific steps:

(1) knocking out ptsG gene from E.coli MG1655 to obtain bacterial strain EC delta p;

(2) knocking out manZ gene from strain EC delta p to obtain strain EC delta p-m;

(3) knocking out atpFH genes from the strain EC delta p-m to obtain a strain EC delta p-m-a;

(4) knocking out envR genes from the strain EC delta p-m-a to obtain a strain EC delta p-m-a-e;

the nucleotide sequence of the ptsG gene in the step (1) is shown as SEQ ID No.1, the nucleotide sequence of the manZ gene in the step (2) is shown as SEQ ID No.2, the nucleotide sequence of the atpFH gene in the step (3) is shown as SEQ ID No.3, and the nucleotide sequence of the envR gene in the step (4) is shown as SEQ ID No. 4.

The gene knockout experiment is a genome traceless operation technology based on lambda-red and I-SceI, and the specific knockout steps are as follows (shown in figure 1):

1) the step (1) of knocking out the ptsG gene from the E.coli MG1655 takes the E.coli MG1655 strain genome as a template, and a ptsG-Af/ptsG-Ar primer and a ptsG-Bf/ptsG-Br primer are utilized to respectively amplify a homologous arm fragment A and a homologous arm fragment B of the upper and lower reaches of the ptsG gene. Using pTKS/CS plasmid as template, primers ptsG-tetf1/ptsG-tetr1 and ptsG-tetf2/ptsG-tetr2 were used to amplify tet1, the upstream fragment and tet2, respectively, of the tetracycline resistance gene. And then using the fragment A, tet1 as a template, using a primer ptsG-Af/ptsG-tetr1 to perform overlap extension PCR to amplify a fragment A-tet1, similarly using the fragments tet2 and B as templates, using a primer ptsG-tetf2/ptsG-Br to perform overlap extension PCR to amplify a fragment tet2-B, finally performing overlap extension PCR again, using A-tet1 and tet2-B as amplification templates, and using ptsG-Af/ptsG-Br as primers to amplify a knock-out fragment A-t-B. The gene knockout principle is shown in fig. 2, and a knockout fragment A-t-B is transferred into E.coli MG1655 by an electric shock transformation method, and a ptsG gene knockout strain E.coli delta p is obtained after two times of screening.

The ptsG gene knockout primer sequence in the step 1) is as follows:

ptsG-Af：TGGCTGTGTTGAAAGGTGTTGCCG

ptsG-Ar：TATCCCTAAGCACAATGCCTCTATTTAGTTAACGTTCTG

ptsG-Bf：AGGGTAATCAGAACGTTAACTAAATAGAGGCATTGTGCTAAAAACGAACAAATGGCAGAG

ptsG-Br：GTCTGCAAGGCGCTCAAGACG

ptsG-tetf1：CAGAACGTTAACTAAATAGAGGCATTGTGCTTAGGGATAACAGGGTAATATTTAC

ptsG-tetr1：GGTAAAGCGATCCCACCACCAGCC

ptsG-tetf2：GCGTGAAGTGGTTCGGTT

ptsG-tetr2：TCGTTTTTAGCACAATGCCTCTATTTAGTTAACGTTCTGATTACCCTGTTATCCCTAC

2) and (2) knocking out the manZ gene from the strain EC delta p, wherein the genome of the E.coli MG1655 strain is used as a template, and primers manZ-Af/manZ-Ar and manZ-Bf/manZ-Br are used for respectively amplifying a manZ gene upstream and downstream homology arm fragment A and a manZ gene downstream and homology arm fragment B. Using pTKS/CS plasmid as template, primers manZ-tetf1/manZ-tetr1 and manZ-tetf2/manZ-tetr2 were used to amplify tet1, which is an upstream fragment of tetracycline resistance gene, and tet2, which is a downstream fragment. And then using the fragment A, tet1 as a template, performing overlap extension PCR by using a primer manZ-Af/manZ-tetr1 to amplify a fragment A-tet1, similarly using the fragments tet2 and B as templates, performing overlap extension PCR by using a primer manZ-tetf2/manZ-Br to amplify a fragment tet2-B, and finally performing overlap extension PCR again, using A-tet1 and tet2-B as amplification templates, and using manZ-Af/manZ-Br as primers to amplify a knock-out fragment A-t-B. The gene knockout principle is shown in figure 2, a knockout fragment A-t-B is transferred into the strain E.coli delta p obtained in the step 1) by an electric shock transformation method, and the manZ gene knockout strain E.coli delta p-m is obtained after two times of screening.

The sequence of the manZ gene knockout primer in the step 2) is as follows:

manZ-Af：TGCAAGCCTTCATCTTCATGG

manZ-Ar：TATCCCTA AGCACAATGCCTCTATTTAGTTAACGTTCTG

manZ-Bf：AGGGTAATCAGAACGTTAACTAAATAGAGGCATTGTGCTAAAAACGAACAAATGGCAGAG

manZ-Br：GTCTGCAAGGCGCTCAAGACG

manZ-tetf1：CAGAACGTTAACTAAATAGAGGCATTGTGCTTAGGGATAACAGGGTAATATTTAC

manZ-tetr1：GGTAAAGCGATCCCACCACCAGCC

manZ-tetf2：GCGTGAAGTGGTTCGGTT

manZ-tetr2：CAACAGTCTTCGCTCACCTGTTAGTCCAGTTCGATTACCCTGTTATCCCTAC

3) and (3) knocking out the atpFH gene from the strain EC delta p-m, wherein the gene group of the E.coli MG1655 strain is used as a template, and primers atpFH-Af/atpFH-Ar and atpFH-Bf/atpFH-Br are used for respectively amplifying an upstream and downstream homologous arm fragment A and a downstream homologous arm fragment B of the atpFH gene. Using pTKS/CS plasmid as a template, the tetracycline resistance gene tet upstream fragment tet1 and downstream fragment tet2 were amplified using primers atpFH-tetf1/atpFH-tetr1 and atpFH-tetf2/atpFH-tetr2, respectively. And then using the fragment A, tet1 as a template, performing overlap extension PCR by using a primer atpFH-Af/atpFH-tetr1 to amplify a fragment A-tet1, similarly using the fragments tet2 and B as templates, using a primer atpFH-tetf2/atpFH-Br to perform overlap extension PCR to amplify a fragment tet2-B, and finally performing overlap extension PCR again, using A-tet1 and tet2-B as amplification templates, and using atpFH-Af/atpFH-Br as primers to amplify a knock-out fragment A-t-B. The gene knockout principle is shown in fig. 2, and the knockout fragment A-t-B is transferred into E.coli delta p-m obtained in the step 8 by an electric shock transformation method, and an atpFH gene knockout strain E.coli delta p-m-a is obtained after two times of screening.

The atpFH gene knockout primer sequence in the step 3) is as follows:

atpFH-Af：TGCAAGCCTTCATCTTCATGG

atpFH-Ar：TATCCCTAAGCACAATGCCTCTATTTAGTTAACGTTCTG

atpFH-Bf：AGGGTAATCAGAACGTTAACTAAATAGAGGCATTGTGCTAAAAACGAACAAATGGCAGAG

atpFH-Br：GTCTGCAAGGCGCTCAAGACG

atpFH-Tetf1：CAGAACGTTAACTAAATAGAGGCATTGTGCTTAGGGATAACAGGGTAATATTAC

atpFH-Tetr1：GGTAAAGCGATCCCACCAGCC

atpFH-Tetf2：GCGTGAAGTGGTTCGGTT

atpFH-Tetr2：TCGTTTTTAGCACAATGCCTCTATTTAGTTAACGTTCTGATTACCCTGTTATCCCTAC

4) and (4) knocking out the envR gene from the strain EC delta p-m-a by taking the genome of the E.coli MG1655 strain as a template, and respectively amplifying an envR gene upstream and downstream homologous arm fragment A and an envR gene downstream and homologous arm fragment B by using primers envR-Af/envR-Ar and envR-Bf/envR-Br. By using pTKS/CS plasmid as a template, primers envR-tetf1/envR-tetr1 and envR-tetf2/envR-tetr2 are used for amplifying a tet1 upstream fragment and a tet2 downstream fragment of the tetracycline resistance gene tet respectively. And then using the fragment A, tet1 as a template, using a primer envR-Af/envR-tetr1 to perform overlap extension PCR to amplify a fragment A-tet1, similarly using the fragments tet2 and B as templates, using a primer envR-tetf2/envR-Br to perform overlap extension PCR to amplify a fragment tet2-B, finally performing overlap extension PCR again, using A-tet1 and tet2-B as amplification templates, using envR-Af/envR-Br as primers, and amplifying a knock-out fragment A-t-B. The gene knockout principle is shown in fig. 2, and a knockout fragment A-t-B is transferred into E.coli delta p-m-a obtained in the step 10 by an electric shock transformation method, and an envR gene knockout strain E.coli delta p-m-a-e is obtained after two times of screening.

The envR gene knockout primer sequence in the step 4) is as follows:

envR-Af：TGCAAGCCTTCATCTTCATGG

envR-Ar：TATCCCTAAGCACAATGCCTCTATTTAGTTAACGTTCTG

envR-Bf：AGGGTAATCAGAACGTTAACTAAATAGAGGCATTGTGCTAAAAACGAACAAATGGCAGAG

envR-Br：GTCTGCAAGGCGCTCAAGACG

envR-tetf1：CAGAACGTTAACTAAATAGAGGCATTGTGCTTAGGGATAACAGGGTAATATTTAC

envR-tetr1：GGTAAAGCGATCCCACCACCAGCC

envR-tetf2：GCGTGAAGTGGTTCGGTT

envR-tetr2：TCGTTTTTAGCACAATGCCTCTATTTAGTTAACGTTCTGATTACCCTGTTATCCCTAC

the nucleotide sequence of the pTKS/CS plasmid in the steps 1) -4) is shown as SEQ ID No.5, the nucleotide sequence of the tetracycline resistance gene tet is shown as SEQ ID No.6, the nucleotide sequence of the upstream fragment tet1 is shown as SEQ ID No.7, and the nucleotide sequence of the downstream fragment tet2 gene is shown as SEQ ID No. 8. The nucleotide sequence of pKTred plasmid is shown as SEQ ID No. 9.

The invention is characterized in that the E.coli MG1655 can preferentially utilize xylose to secrete acetic acid and FFA efficiently under the condition that glucose and xylose exist simultaneously. The sugar utilization capacity of the E.coli MG1655 is regulated by knocking out ptsG and manZ genes of the E.coli MG1655, when the strain EC delta p-m takes glucose as a single carbon source, almost no glucose is consumed after 48h of culture, xylose is preferentially consumed rapidly when a mixed carbon source of glucose and xylose exists, and glucose is slowly metabolized when xylose is consumed until the glucose is consumed, so that knocking out the ptsG and manZ genes of the E.coli MG1655 can enable the glucose to be slowly metabolized by preferentially utilizing xylose when glucose and xylose coexist. The atpFH and envR genes of E.coli MG1655 are knocked out to promote the synthesis capacity of acetic acid and extracellular FFA, the gene knockout engineering bacteria can synthesize six FFAs including n-hexanoic acid, n-heptanoic acid, n-octanoic acid, n-nonanoic acid, n-decanoic acid, n-dodecanoic acid and the like by utilizing xylose, 5.5g/L of acetic acid and 0.91g/L of extracellular FFA can be synthesized by taking 20g/L of xylose as a single carbon source in an M9 culture medium, and the yield is obviously improved compared with that of a wild E.coli MG 1655.

The invention is characterized in that the E.coli MG1655 can improve the acetic acid and FFA secretion capacity while preferring to utilize xylose under the condition of simultaneously existing glucose and xylose.

Drawings

FIG. 1 shows the principle of preparing target gene fragments in gene knock-out;

FIG. 2 shows the principle of the target gene recombination process in gene knock-out;

FIG. 3 shows the sugar utilization of the engineering bacteria EC Δ p-m, Glu is the sugar concentration when glucose is used as a sole carbon source, Xyl is the sugar concentration when xylose is used as a sole carbon source, Mix-Glu is the glucose concentration when a mixed carbon source, and Mix-Xyl is the xylose concentration when a mixed carbon source is used;

FIG. 4 shows the growth of engineering bacteria EC Δ p-m;

FIG. 5 species of engineering bacteria EC Δ p-m-a-e extracellular FFA;

FIG. 6 comparison of the yield of the engineering bacteria EC delta p-m-a-e with the yield of the wild bacteria acetic acid and FFA.

Detailed Description

The original strain Escherichia coli MG1655 is a gift provided by professor of Chentao at Tianjin university, and reagents involved in the embodiments of the present invention are all commercially available products and can be purchased commercially, if no special description is provided. The invention is explained in further detail below with reference to the drawings:

1. culture medium formula and solution formula

LB medium (g/L): 10.0 parts of peptone, 10.0 parts of NaCl, 5.0 parts of yeast extract powder and 7.0-7.2 parts of pH.

M9 mineral salts medium (g/L): na (Na)₂HPO₄·7H₂O 12.8，KH₂PO₄ 3，NH₄Cl 2，NaCl 0.5，MgSO₄0.24, 1mL/L of trace element solution and pH 7.0. The carbon source is added after the conversion of glucose and/or xylose according to the required amount.

Glucose and xylose mother liquor: accurately weighing 100g of glucose and xylose, respectively dissolving in a blue-capped bottle containing 200mL of distilled water (final concentration of sugar is 500g/L), sterilizing with high pressure steam at 115 deg.C for 30min, cooling, and storing at 4 deg.C.

2. Coli electrotransformation competent cells

1) Picking single colony on solid plate culture medium by using a pipettor, inoculating the single colony in a 35mL test tube loaded with 5mL LB culture medium, and culturing at 37 ℃ and 220rpm overnight;

2) 1mL of overnight-cultured bacteria were aspiratedThe solution was inoculated into 250mL Erlenmeyer flask containing 100mL of LB liquid medium and cultured to OD₆₀₀Is between 0.4 and 0.6;

3) placing the shake flask on ice, standing for 10min, pouring the bacterial liquid into a precooled 50mL centrifuge tube, and centrifuging for 5min at 6500rpm and 4 ℃;

4) the supernatant was decanted off and 30mL of precooled ddH was added₂O, blowing and sucking the resuspended thalli;

5) reuse of the precooled ddH, as in step 4 above₂Washing with O and 10% glycerol solution once respectively, and pouring off the supernatant;

6) 2mL of pre-cooled 10% glycerol solution was added to the centrifuge tube, and the resuspended cells were aspirated and dispensed into 100. mu.L/tube and stored at-80 ℃ for later use.

Electrotransformation of E.coli

1) Taking 1 tube of Escherichia coli competent cells, and standing on ice for 10min to melt;

2) adding DNA sample (plasmid or enzyme linked product, DNA amount less than 100ng) into competent cell, and mixing;

3) adding competent cells pre-mixed with DNA into a pre-cooled electric shock cup, and wiping off condensed water on the surface;

4) putting the electric shock cup into an electric rotating instrument, adjusting the PULSE voltage to 1800V, and pressing a PULSE button on the electric rotating instrument;

5) after sounding, taking out the electric shock cup, and adding 1mL of LB culture medium;

6) transferring the bacterial liquid from the electric rotating cup to a 1.5mL centrifuge tube, and culturing for 1h in a shaking table at 37 ℃;

7) centrifuging the bacterial liquid at the room temperature of 4200rpm for 2min, sucking out about 0.9mL of supernatant, resuspending the thalli by using the residual culture solution, and coating the thalli on an LB solid culture medium plate containing corresponding antibiotics;

8) the plate is inverted, and cultured in an incubator at 37 ℃ for 12-16h until colonies grow.

4. Preparation of E.coli recombinant competent cells

1) Introducing the recombinant helper plasmid pTKRed into e.coli according to the method of step 3;

2) colonies into which the recombinant helper plasmid pTKRed had been transferred were picked from the plate using a pipette and transferred into 1 35mL tube containing 5mL of LB medium (spectinomycin resistance). Culturing at 30 deg.C and 220rpm for 14-16 h;

3) 1mL of the suspension was aspirated from the overnight-cultured tube, and inoculated into a 500mL Erlenmeyer flask containing 100mL of LB liquid medium (2mM IPTG, 10g/L glucose, 100. mu.g/mL spectinomycin), and the cells were cultured to OD₆₀₀Is between 0.4 and 0.6;

4) the method of step 3 was used to prepare the electrically transformed cells.

E.coli electrotransformation and screening of positive clones

1) Performing electric transformation by using the electric transformation cells prepared in the step 4 according to the steps 1) to 6) in the step 3;

2) centrifuging at 4200rpm at room temperature for 2min, aspirating about 0.9mL of supernatant, resuspending the cells with the remaining culture medium, plating on LB solid medium plate (2mM IPTG, 20. mu.g/L tetracycline, 100. mu.g/L spectinomycin);

3) carrying out inverted culture on the plate, and culturing at 30 ℃ for 12-16h to grow a bacterial colony;

4) colonies that had successfully integrated were screened by colony PCR from the plates on which the clones grew.

6. Popping tetracycline resistance and deletion pKTRed plasmid

1) Validated positive colonies were picked from the plates and cultured overnight in 5mL tubes of LB liquid medium (2mM IPTG, 0.2% arabinose, 100. mu.g/L spectinomycin);

2) streaking the overnight-cultured bacterial liquid in LB solid medium (2mM IPTG, 0.2% arabinose, 100. mu.g/L spectinomycin);

3) screening colonies which have successfully popped up tetracycline genes from the plate on which the clone grows out by colony PCR;

4) the strain which successfully ejects the tetracycline gene is cultured at 42 ℃, deletion pKTRed plasmid is carried out, and the strain is verified by a medium with spectinomycin resistance.

The present invention will be further described with reference to the following examples.

Example 1 construction of an engineered Escherichia coli that prefers efficient secretion of acetic acid and FFA Using xylose

1. Knock-out of ptsG Gene from E.coli MG1655

The amplification principle of the ptsG gene knock-out fragment is shown in FIG. 1, and the primers used are shown in Table 1. And (3) respectively amplifying a segment A and a segment B of the upstream and downstream homologous arms of the ptsG gene by using a primer ptsG-Af/ptsG-Ar and a primer ptsG-Bf/ptsG-Br by using the E.coli MG1655 strain genome as a template. Using pTKS/CS plasmid as template, primers ptsG-tetf1/ptsG-tetr1 and ptsG-tetf2/ptsG-tetr2 were used to amplify tet1, the upstream fragment and tet2, respectively, of the tetracycline resistance gene. And then using the fragment A, tet1 as a template, using a primer ptsG-Af/ptsG-tetr1 to perform overlap extension PCR to amplify a fragment A-tet1, similarly using the fragments tet2 and B as templates, using a primer ptsG-tetf2/ptsG-Br to perform overlap extension PCR to amplify a fragment tet2-B, finally performing overlap extension PCR again, using A-tet1 and tet2-B as amplification templates, and using ptsG-Af/ptsG-Br as primers to amplify a knock-out fragment A-t-B. The gene knockout principle is shown in fig. 2, and a knockout fragment A-t-B is transferred into E.coli MG1655 by an electric shock transformation method, and a ptsG gene knockout strain E.coli delta p is obtained after two times of screening.

TABLE 1 ptsG Gene knockout primers

2. Knockout of manZ Gene from Strain EC Δ p

The amplification principle of the manZ knock-out fragment is shown in FIG. 1, and the primers used are shown in Table 2. Taking E.coli MG1655 strain genome as template, amplifying manZ-Af/manZ-Ar and manZ-Bf/manZ-Br to obtain manZ gene upstream and downstream homologous arm fragment A and fragment B respectively. Using pTKS/CS plasmid as template, primers manZ-tetf1/manZ-tetr1 and manZ-tetf2/manZ-tetr2 were used to amplify tet1, which is an upstream fragment of tetracycline resistance gene, and tet2, which is a downstream fragment. And then using the fragment A, tet1 as a template, performing overlap extension PCR by using a primer manZ-Af/manZ-tetr1 to amplify a fragment A-tet1, similarly using the fragments tet2 and B as templates, performing overlap extension PCR by using a primer manZ-tetf2/manZ-Br to amplify a fragment tet2-B, and finally performing overlap extension PCR again, using A-tet1 and tet2-B as amplification templates, and using manZ-Af/manZ-Br as primers to amplify a knock-out fragment A-t-B. The gene knockout principle is shown in figure 2, and a knockout fragment A-t-B is transferred into the strain E.coli delta p obtained in the step 7 by an electric shock transformation method, and the manZ gene knockout strain E.coli delta p-m is obtained after two times of screening.

TABLE 2 manZ Gene knockout primers

3. Knockout of atpFH Gene from Strain EC Δ p-m

The amplification principle of the atpFH gene knockout fragment is shown in FIG. 1, and the primers used are shown in Table 3. Coli MG1655 strain genome as template, atpFH-Af/atpFH-Ar and atpFH-Bf/atpFH-Br were used to amplify atpFH gene upstream and downstream homologous arm fragment A and fragment B, respectively. Using pTKS/CS plasmid as a template, the tetracycline resistance gene tet upstream fragment tet1 and downstream fragment tet2 were amplified using primers atpFH-tetf1/atpFH-tetr1 and atpFH-tetf2/atpFH-tetr2, respectively. And then using the fragment A, tet1 as a template, performing overlap extension PCR by using a primer atpFH-Af/atpFH-tetr1 to amplify a fragment A-tet1, similarly using the fragments tet2 and B as templates, using a primer atpFH-tetf2/atpFH-Br to perform overlap extension PCR to amplify a fragment tet2-B, and finally performing overlap extension PCR again, using A-tet1 and tet2-B as amplification templates, and using atpFH-Af/atpFH-Br as primers to amplify a knock-out fragment A-t-B. The gene knockout principle is shown in fig. 2, and the knockout fragment A-t-B is transferred into E.coli delta p-m obtained in the step 8 by an electric shock transformation method, and an atpFH gene knockout strain E.coli delta p-m-a is obtained after two times of screening.

TABLE 3 atpFH Gene knockout primers

4. Knock-out envR gene from strain EC delta p-m-a

The amplification principle of the envR gene knockout fragment is shown in FIG. 1, and the primers used are shown in Table 4. Taking E.coli MG1655 strain genome as template, and amplifying envR-Af/envR-Ar and envR-Bf/envR-Br with primers envR-Af/envR-Br to obtain envR gene upstream and downstream homologous arm fragment A and fragment B respectively. By using pTKS/CS plasmid as a template, primers envR-tetf1/envR-tetr1 and envR-tetf2/envR-tetr2 are used for amplifying a tet1 upstream fragment and a tet2 downstream fragment of the tetracycline resistance gene tet respectively. And then using the fragment A, tet1 as a template, using a primer envR-Af/envR-tetr1 to perform overlap extension PCR to amplify a fragment A-tet1, similarly using the fragments tet2 and B as templates, using a primer envR-tetf2/envR-Br to perform overlap extension PCR to amplify a fragment tet2-B, finally performing overlap extension PCR again, using A-tet1 and tet2-B as amplification templates, using envR-Af/envR-Br as primers, and amplifying a knock-out fragment A-t-B. The gene knockout principle is shown in fig. 2, and a knockout fragment A-t-B is transferred into E.coli delta p-m-a obtained in the step 10 by an electric shock transformation method, and an envR gene knockout strain E.coli delta p-m-a-e is obtained after two times of screening.

TABLE 4 envR Gene knockout primers

Example 2 detection of glucose and xylose utilization by engineering bacteria EC Δ p-m

The engineering strain EC delta p-m obtained in example 1 with ptsG and manZ genes knocked out is cultured, and under the aseptic condition, a single colony of EC delta p-m on a solid culture medium is selected and inoculated in 5mL of LB culture medium, and cultured for 12h at 30 ℃ and 220rpm to obtain a seed culture solution. Inoculating the seed culture solution into a conical flask containing 50mL of M9 culture medium according to 1%, adding a certain amount of glucose or xylose mother liquor into the culture medium according to requirements to prepare a culture medium with glucose as a unique carbon source (20g/L), xylose as a unique carbon source (20g/L) and mixed sugar as carbon sources (10 g/L of glucose and 10g/L of xylose), and culturing at 30 ℃ and 220 rpm. The results are shown in FIGS. 3 and 4. When the strain is cultured by taking glucose as a single carbon source, the concentration of the glucose is slowly reduced in the culture process, a small amount of glucose is consumed after 48 hours, and meanwhile, the biomass is only slightly accumulated, which shows that the glucose metabolism capability of the engineering bacteria is greatly inhibited. When the strain is cultured by taking xylose as a single carbon source, the strain can consume xylose more rapidly, and biomass is accumulated rapidly, which indicates that the strain still has stronger xylose consumption capacity. When the strain was cultured with a mixed carbon source of glucose and xylose, xylose was rapidly consumed during the culture, while glucose was hardly consumed, and only when the xylose concentration was low, it began to be slowly consumed. The above results indicate that simultaneous knock-out of ptsG and manZ genes in e.coli MG1655 can promote its preferential xylose utilization and slow glucose metabolism in the presence of both glucose and xylose.

Example 3 FFA production test of engineering bacteria EC Δ p-m-a-e

The engineering bacteria EC delta p-m-a-e with ptsG, manZ, atpFH and envR genes knocked out and obtained in the final transformation in the embodiment 1 are cultured and tested. Under the aseptic condition, a single colony of EC delta p-m-a-e on a solid culture medium is selected and inoculated in 5mL of LB culture medium, and the culture is carried out for 12h at 30 ℃ and 220rpm to obtain a seed culture solution. Inoculating 1% of the seed culture solution into a conical flask containing 50mL of M9 culture medium, culturing for 60h under aerobic conditions of 30 ℃ and 220rpm, centrifuging the fermentation broth in a centrifuge tube at 12,000rpm for 10min, sucking 2mL of supernatant into the centrifuge tube, adding 200 μ L of acetic acid and 150mg of undecanoic acid as an internal standard, adding 2mL of n-hexane: chloroform (4: 1, v/v) extraction, evaporation of the organic layer to near dryness, dissolution with 1mL chloroform methanol sulfuric acid (10: 8.5: 1.5), sealing, and water bath at 100 deg.C for 1h to methyl-esterify the fatty acid. After methyl esterification, cooling to room temperature, adding 500 μ L of distilled water, shaking, mixing, filtering the organic phase with 0.22 μm filter membrane, and placing the filtrate in a clean centrifuge tube for sample injection. As a result of analyzing the extracellular fatty acid composition using a gas chromatograph, as shown in fig. 5, main components were n-hexanoic acid, n-heptanoic acid, n-octanoic acid, n-nonanoic acid, n-decanoic acid, and n-dodecanoic acid, and these FFAs were all utilized by p.putida to synthesize mcl-PHA. As shown in FIG. 6, compared with wild bacteria, the extracellular FFA yield of the engineering bacteria EC delta p-M-a-e is obviously improved, and when the engineering bacteria EC delta p-M-a-e are cultured for 60 hours in an M9 culture medium under the condition that 20g/L of xylose is a single carbon source, the wild E.coli MG1655 can synthesize 0.62g/L of FFA, and the EC delta p-M-a-e can synthesize 0.91g/L of FFA. It can be seen that the ability of e.coli MG1655 to synthesize acetate and extracellular FFA using xylose was improved by knocking out the envR gene.

EXAMPLE 4 acetic acid production test of engineering bacteria EC Δ p-m-a-e

The engineering bacteria EC delta p-m-a-e with ptsG, manZ, atpFH and envR genes knocked out and obtained in the final transformation in the embodiment 1 are cultured and tested. Under the aseptic condition, a single colony of EC delta p-m-a-e on a solid culture medium is selected and inoculated in 5mL of LB culture medium, and the culture is carried out for 12h at 30 ℃ and 220rpm to obtain a seed culture solution. Inoculating 1% of seed culture solution into a conical flask containing 50mL of M9 culture medium, culturing at 30 ℃ and 220rpm for 60h under the condition that 20g/L of xylose is used as a single carbon source, sucking 2mL of fermentation liquor, placing the fermentation liquor into a centrifuge tube, centrifuging at 12000rpm for 10min, taking supernate, filtering by using a 0.22-micron filter membrane, and measuring the concentration of acetic acid in the fermentation liquor by using a high performance liquid chromatograph, as shown in figure 6, compared with wild bacteria, the extracellular acetic acid yield of the engineering bacteria EC delta p-M-a-e is obviously improved, and in M9 culture medium, under the condition that 20g/L of xylose is used as a single carbon source, culturing for 60h, wild E.coli MG1655 can synthesize 2.4g/L of acetic acid, and EC delta p-M-a-e can synthesize 5.5g/L of FFA. It can be seen that the ability of e.coli MG1655 to synthesize acetate from xylose was improved by knocking out the atpFH gene.

While the methods and techniques of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and/or modifications of the methods and techniques described herein may be made without departing from the spirit and scope of the invention. It is expressly intended that all such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and content of the invention.

Sequence listing

<110> Tianjin university

<120> construction method of escherichia coli engineering bacteria preferring to efficiently secrete acetic acid and FFA by utilizing xylose

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1434

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgtttaaga atgcatttgc taacctgcaa aaggtcggta aatcgctgat gctgccggta 60

tccgtactgc ctatcgcagg tattctgctg ggcgtcggtt ccgcgaattt cagctggctg 120

cccgccgttg tatcgcatgt tatggcagaa gcaggcggtt ccgtctttgc aaacatgcca 180

ctgatttttg cgatcggtgt cgccctcggc tttaccaata acgatggcgt atccgcgctg 240

gccgcagttg ttgcctatgg catcatggtt aaaaccatgg ccgtggttgc gccactggta 300

ctgcatttac ctgctgaaga aatcgcctct aaacacctgg cggatactgg cgtactcgga 360

gggattatct ccggtgcgat cgcagcgtac atgtttaacc gtttctaccg tattaagctg 420

cctgagtatc ttggcttctt tgccggtaaa cgctttgtgc cgatcatttc tggcctggct 480

gccatcttta ctggcgttgt gctgtccttc atttggccgc cgattggttc tgcaatccag 540

accttctctc agtgggctgc ttaccagaac ccggtagttg cgtttggcat ttacggtttc 600

atcgaacgtt gcctggtacc gtttggtctg caccacatct ggaacgtacc tttccagatg 660

cagattggtg aatacaccaa cgcagcaggt caggttttcc acggcgacat tccgcgttat 720

atggcgggtg acccgactgc gggtaaactg tctggtggct tcctgttcaa aatgtacggt 780

ctgccagctg ccgcaattgc tatctggcac tctgctaaac cagaaaaccg cgcgaaagtg 840

ggcggtatta tgatctccgc ggcgctgacc tcgttcctga ccggtatcac cgagccgatc 900

gagttctcct tcatgttcgt tgcgccgatc ctgtacatca tccacgcgat tctggcaggc 960

ctggcattcc caatctgtat tcttctgggg atgcgtgacg gtacgtcgtt ctcgcacggt 1020

ctgatcgact tcatcgttct gtctggtaac agcagcaaac tgtggctgtt cccgatcgtc 1080

ggtatcggtt atgcgattgt ttactacacc atcttccgcg tgctgattaa agcactggat 1140

ctgaaaacgc cgggtcgtga agacgcgact gaagatgcaa aagcgacagg taccagcgaa 1200

atggcaccgg ctctggttgc tgcatttggt ggtaaagaaa acattactaa cctcgacgca 1260

tgtattaccc gtctgcgcgt cagcgttgct gatgtgtcta aagtggatca ggccggcctg 1320

aagaaactgg gcgcagcggg cgtagtggtt gctggttctg gtgttcaggc gattttcggt 1380

actaaatccg ataacctgaa aaccgagatg gatgagtaca tccgtaacca ctaa 1434

<210> 2

<211> 852

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggttgata caactcaaac taccaccgag aaaaaactca ctcaaagtga tattcgtggc 60

gtcttcctgc gttctaacct cttccagggt tcatggaact tcgaacgtat gcaggcactg 120

ggtttctgct tctctatggt accggcaatt cgtcgcctct accctgagaa caacgaagct 180

cgtaaacaag ctattcgccg tcacctggag ttctttaaca cccagccgtt cgtggctgcg 240

ccgattctcg gcgtaaccct ggcgctggaa gaacagcgtg ctaatggcgc agagatcgac 300

gacggtgcta tcaacggtat caaagtcggt ttgatggggc cactggctgg tgtaggcgac 360

ccgatcttct ggggaaccgt acgtccggta tttgcagcac tgggtgccgg tatcgcgatg 420

agcggcagcc tgttaggtcc gctgctgttc ttcatcctgt ttaacctggt gcgtctggca 480

acccgttact acggcgtagc gtatggttac tccaaaggta tcgatatcgt taaagatatg 540

ggtggtggct tcctgcaaaa actgacggaa ggggcgtcta tcctcggcct gtttgtcatg 600

ggggcattgg ttaacaagtg gacacatgtc aacatcccgc tggttgtctc tcgcattact 660

gaccagacgg gcaaagaaca cgttactact gtccagacta ttctggacca gttaatgcca 720

ggcctggtac cactgctgct gacctttgct tgtatgtggc tactgcgcaa aaaagttaac 780

ccgctgtgga tcatcgttgg cttcttcgtc atcggtatcg ctggttacgc ttgcggcctg 840

ctgggactgt aa 852

<210> 3

<211> 602

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ccaatggagc cgccgtttgc ggtcgtacag gacggtcgca agatgtgaaa ccacaagctg 60

ccgtttcagt tttcgacgaa accgcatccc cgctcgatgg cattatttaa gtctgtagtc 120

ggggagggag gaatgtcaag tcgctgttca aataggtgct acagcgacaa tcgtcgaagt 180

aggtgccttg caagctacta gaagagccgc ggtcgttgtc ggtcctatcg ttgaacgaat 240

gcgtcgagaa gtgcccgtgc aaatgcgagc cgaagttaaa ggcggacgcg gacccggtgc 300

taaaatcatg caaggacaag acggagtcga aagcgaagca ggtcttagac gctcgccgca 360

aacaagcgga cgagctacta atggacccga aggcgaaagc gaaaaaagtc gaccagccag 420

cgcgaccgga aacgttccag ttccaggaat acacgagcaa gacgccttcg ttccggcagt 480

cgttaaagaa aaactgcaaa aagctaccga cggtaattac cgccggtatg catgaagtac 540

gtcttgtctt gcttgtcctg tttgcgctac cggaccggct cctaacaacg caattctaag 600

tg 602

<210> 4

<211> 663

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ttacatgaca cttaattcat tcgtttgatg aattaatttc gttatgtttt catctggcat 60

gaacattctt aatacgttat cgaccagagc gggggcttgt ttataaagat cataacccgc 120

catattcatt aaccagtttt gaacaattcc gctgaaggca ccatcaataa taatcatcac 180

aacatctaaa tcgaggttat ttgctacaca accttgttgc tgacacgcct gcaatacttc 240

gcggagagtc tgcggattaa agcccatctt ttcgcgtatc actccctcgg ccagcatctc 300

atcattaaat tcacatttgt gatataagat tttcagcaac gcctgctggc ggggaatttt 360

ggcaatatat tgcaagccga caatcaattt ttcacgcaat tgttgaaacg ggtcatgctc 420

taatccagcc gtcaagtgtt cctggattaa ctcccgcaat gaaggctgtt gcaaccacat 480

ctcattaaac agttgagtct tgttttcgaa gtgccagtag atagcgccac gcgtaacgtt 540

agcggcgtcg gcaatgtcgt tgagcgtcgt cttgcttacg ccatgctgcg caaactgggc 600

gatggcagtt tcaatcagtt cttgccgggt cttcagagct tcggctttgg ttctttttgc 660

cat 663

<210> 5

<211> 3251

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tacggcccca aggtccaaac ggtgataggg ataacagggt aatatttacg ttgacaccac 60

ctttcgcgta tggcatgata gcgcccggaa gagagtcaat tcagggtggt gaatatgaat 120

agttcgacaa agatcgcatt ggtaattacg ttactcgatg ccatggggat tggccttatc 180

atgccagtct tgccaacgtt attacgtgaa tttattgctt cggaagatat cgctaaccac 240

tttggcgtat tgcttgcact ttatgcgtta atgcaggtta tctttgctcc ttggcttgga 300

aaaatgtctg accgatttgg tcggcgccca gtgctgttgt tgtcattaat aggcgcatcg 360

ctggattact tattgctggc tttttcaagt gcgctttgga tgctgtattt aggccgtttg 420

ctttcaggga tcacaggagc tactggggct gtcgcggcat cggtcattgc cgataccacc 480

tcagcttctc aacgcgtgaa gtggttcggt tggttagggg caagttttgg gcttggttta 540

atagcggggc ctattattgg tggttttgca ggagagattt caccgcatag tccctttttt 600

atcgctgcgt tgctaaatat tgtcactttc cttgtggtta tgttttggtt ccgtgaaacc 660

aaaaatacac gtgataatac agataccgaa gtaggggttg agacgcaatc aaattcggtg 720

tacatcactt tatttaaaac gatgcccatt ttgttgatta tttatttttc agcgcaattg 780

ataggccaaa ttcccgcaac ggtgtgggtg ctatttaccg aaaatcgttt tggatggaat 840

agcatgatgg ttggcttttc attagcgggt cttggtcttt tacactcagt attccaagcc 900

tttgtggcag gaagaatagc cactaaatgg ggcgaaaaaa cggcagtact gctcgaattt 960

attgcagata gtagtgcatt tgccttttta gcgtttatat ctgaaggttg gttagatttc 1020

cctgttttaa ttttattggc tggtggtggg atcgctttac ctgcattaca gggagtgatg 1080

tctatccaaa caaagagtca tgagcaaggt gctttacagg gattattggt gagccttacc 1140

aatgcaaccg gtgttattgg cccattactg tttactgtta tttataatca ttcactacca 1200

atttgggatg gctggatttg gattattggt ttagcgtttt actgtattat tatcctgcta 1260

tcaatgacct tcatgttgac ccctcaagct caggggagta aacaggagac aagtgcttag 1320

tagggataac agggtaatga tggcgcctca tccctgaagc caaggcatca aataaaacga 1380

aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt gaacgctctc 1440

ctgagtagga caaatccgcc gccctagacc taggggatat attccgcttc ctcgctcact 1500

gactcgctac gctcggtcgt tcgactgcgg cgagcggaaa tggcttacga acggggcgga 1560

gatttcctgg aagatgccag gaagatactt aacagggaag tgagagggcc gcggcaaagc 1620

cgtttttcca taggctccgc ccccctgaca agcatcacga aatctgacgc tcaaatcagt 1680

ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctggc ggctccctcg 1740

tgcgctctcc tgttcctgcc tttcggttta ccggtgtcat tccgctgtta tggccgcgtt 1800

tgtctcattc cacgcctgac actcagttcc gggtaggcag ttcgctccaa gctggactgt 1860

atgcacgaac cccccgttca gtccgaccgc tgcgccttat ccggtaacta tcgtcttgag 1920

tccaacccgg aaagacatgc aaaagcacca ctggcagcag ccactggtaa ttgatttaga 1980

ggagttagtc ttgaagtcat gcgccggtta aggctaaact gaaaggacaa gttttggtga 2040

ctgcgctcct ccaagccagt tacctcggtt caaagagttg gtagctcaga gaaccttcga 2100

aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa gagattacgc gcagaccaaa 2160

acgatctcaa gaagatcatc ttattaatca gataaaatat ttctagattt cagtgcaatt 2220

tatctcttca aatgtagcac ctgaagtcag ccccatacga tataagttgt tactagtgct 2280

tggattctca ccaataaaaa acgcccggcg gcaaccgagc gttctgaaca aatccagatg 2340

gagttctgag gtcattactg gatctatcaa caggagtcca agcgagctcg atatcaaatt 2400

acgccccgcc ctgccactca tcgcagtact gttgtaattc attaagcatt ctgccgacat 2460

ggaagccatc acagacggca tgatgaacct gaatcgccag cggcatcagc accttgtcgc 2520

cttgcgtata atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc atattggcca 2580

cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa aacatattct 2640

caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca tcttgcgaat 2700

atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat gaaaacgttt 2760

cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc accagctcac 2820

cgtctttcat tgccatacgg aattccggat gagcattcat caggcgggca agaatgtgaa 2880

taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag gccgtaatat 2940

ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc tcaaaatgtt 3000

ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt ttctccattt 3060

tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacgcccggt agtgatctta 3120

tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat tttcgccaga 3180

tatcgacgtc taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac 3240

gaggcccttt c 3251

<210> 6

<211> 1209

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgaatagtt cgacaaagat cgcattggta attacgttac tcgatgccat ggggattggc 60

cttatcatgc cagtcttgcc aacgttatta cgtgaattta ttgcttcgga agatatcgct 120

aaccactttg gcgtattgct tgcactttat gcgttaatgc aggttatctt tgctccttgg 180

cttggaaaaa tgtctgaccg atttggtcgg cgcccagtgc tgttgttgtc attaataggc 240

gcatcgctgg attacttatt gctggctttt tcaagtgcgc tttggatgct gtatttaggc 300

cgtttgcttt cagggatcac aggagctact ggggctgtcg cggcatcggt cattgccgat 360

accacctcag cttctcaacg cgtgaagtgg ttcggttggt taggggcaag ttttgggctt 420

ggtttaatag cggggcctat tattggtggt tttgcaggag agatttcacc gcatagtccc 480

ttttttatcg ctgcgttgct aaatattgtc actttccttg tggttatgtt ttggttccgt 540

gaaaccaaaa atacacgtga taatacagat accgaagtag gggttgagac gcaatcaaat 600

tcggtgtaca tcactttatt taaaacgatg cccattttgt tgattattta tttttcagcg 660

caattgatag gccaaattcc cgcaacggtg tgggtgctat ttaccgaaaa tcgttttgga 720

tggaatagca tgatggttgg cttttcatta gcgggtcttg gtcttttaca ctcagtattc 780

caagcctttg tggcaggaag aatagccact aaatggggcg aaaaaacggc agtactgctc 840

gaatttattg cagatagtag tgcatttgcc tttttagcgt ttatatctga aggttggtta 900

gatttccctg ttttaatttt attggctggt ggtgggatcg ctttacctgc attacaggga 960

gtgatgtcta tccaaacaaa gagtcatgag caaggtgctt tacagggatt attggtgagc 1020

cttaccaatg caaccggtgt tattggccca ttactgttta ctgttattta taatcattca 1080

ctaccaattt gggatggctg gatttggatt attggtttag cgttttactg tattattatc 1140

ctgctatcaa tgaccttcat gttgacccct caagctcagg ggagtaaaca ggagacaagt 1200

gcttagtag 1209

<210> 7

<211> 1036

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

tagggataac agggtaatat ttacgttgac accacctttc gcgtatggca tgatagcgcc 60

cggaagagag tcaattcagg gtggtgaata tgaatagttc gacaaagatc gcattggtaa 120

ttacgttact cgatgccatg gggattggcc ttatcatgcc agtcttgcca acgttattac 180

gtgaatttat tgcttcggaa gatatcgcta accactttgg cgtattgctt gcactttatg 240

cgttaatgca ggttatcttt gctccttggc ttggaaaaat gtctgaccga tttggtcggc 300

gcccagtgct gttgttgtca ttaataggcg catcgctgga ttacttattg ctggcttttt 360

caagtgcgct ttggatgctg tatttaggcc gtttgctttc agggatcaca ggagctactg 420

gggctgtcgc ggcatcggtc attgccgata ccacctcagc ttctcaacgc gtgaagtggt 480

tcggttggtt aggggcaagt tttgggcttg gtttaatagc ggggcctatt attggtggtt 540

ttgcaggaga gatttcaccg catagtccct tttttatcgc tgcgttgcta aatattgtca 600

ctttccttgt ggttatgttt tggttccgtg aaaccaaaaa tacacgtgat aatacagata 660

ccgaagtagg ggttgagacg caatcaaatt cggtgtacat cactttattt aaaacgatgc 720

ccattttgtt gattatttat ttttcagcgc aattgatagg ccaaattccc gcaacggtgt 780

gggtgctatt taccgaaaat cgttttggat ggaatagcat gatggttggc ttttcattag 840

cgggtcttgg tcttttacac tcagtattcc aagcctttgt ggcaggaaga atagccacta 900

aatggggcga aaaaacggca gtactgctcg aatttattgc agatagtagt gcatttgcct 960

ttttagcgtt tatatctgaa ggttggttag atttccctgt tttaatttta ttggctggtg 1020

gtgggatcgc tttacc 1036

<210> 8

<211> 845

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gcgtgaagtg gttcggttgg ttaggggcaa gttttgggct tggtttaata gcggggccta 60

ttattggtgg ttttgcagga gagatttcac cgcatagtcc cttttttatc gctgcgttgc 120

taaatattgt cactttcctt gtggttatgt tttggttccg tgaaaccaaa aatacacgtg 180

ataatacaga taccgaagta ggggttgaga cgcaatcaaa ttcggtgtac atcactttat 240

ttaaaacgat gcccattttg ttgattattt atttttcagc gcaattgata ggccaaattc 300

ccgcaacggt gtgggtgcta tttaccgaaa atcgttttgg atggaatagc atgatggttg 360

gcttttcatt agcgggtctt ggtcttttac actcagtatt ccaagccttt gtggcaggaa 420

gaatagccac taaatggggc gaaaaaacgg cagtactgct cgaatttatt gcagatagta 480

gtgcatttgc ctttttagcg tttatatctg aaggttggtt agatttccct gttttaattt 540

tattggctgg tggtgggatc gctttacctg cattacaggg agtgatgtct atccaaacaa 600

agagtcatga gcaaggtgct ttacagggat tattggtgag ccttaccaat gcaaccggtg 660

ttattggccc attactgttt actgttattt ataatcattc actaccaatt tgggatggct 720

ggatttggat tattggttta gcgttttact gtattattat cctgctatca atgaccttca 780

tgttgacccc tcaagctcag gggagtaaac aggagacaag tgcttagtag ggataacagg 840

gtaat 845

<210> 9

<211> 10726

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ctgctcgcgc aggctgggtg ccaagctctc gggtaacatc aaggcccgat ccttggagcc 60

cttgccctcc cgcacgatga tcgtgccgtg atcgaaatcc agatccttga cccgcagttg 120

caaaccctca ctgatccgca tgcttatgac aacttgacgg ctacatcatt cactttttct 180

tcacaaccgg cacggaactc gctcgggctg gccccggtgc attttttaaa tacccgcgag 240

aaatagagtt gatcgtcaaa accaacattg cgaccgacgg tggcgatagg catccgggtg 300

gtgctcaaaa gcagcttcgc ctggctgata cgttggtcct cgcgccagct taagacgcta 360

atccctaact gctggcggaa aagatgtgac agacgcgacg gcgacaagca aacatgctgt 420

gcgacgctgg cgatatcaaa attgctgtct gccaggtgat cgctgatgta ctgacaagcc 480

tcgcgtaccc gattatccat cggtggatgg agcgactcgt taatcgcttc catgcgccgc 540

agtaacaatt gctcaagcag atttatcgcc agcagctccg aatagcgccc ttccccttgc 600

ccggcgttaa tgatttgccc aaacaggtcg ctgaaatgcg gctggtgcgc ttcatccggg 660

cgaaagaacc ccgtattggc aaatattgac ggccagttaa gccattcatg ccagtaggcg 720

cgcggacgaa agtaaaccca ctggtgatac cattcgcgag cctccggatg acgaccgtag 780

tgatgaatct ctcctggcgg gaacagcaaa atatcacccg gtcggcaaac aaattctcgt 840

ccctgatttt tcaccacccc ctgaccgcga atggtgagat tgagaatata acctttcatt 900

cccagcggtc ggtcgataaa aaaatcgaga taaccgttgg cctcaatcgg cgttaaaccc 960

gccaccagat gggcattaaa cgagtatccc ggcagcaggg gatcattttg cgcttcagcc 1020

atacttttca tactcccgcc attcagagaa gaaaccaatt gtccatattg catcagacat 1080

tgccgtcact gcgtctttta ctggctcttc tcgctaacca aaccggtaac cccgcttatt 1140

aaaagcattc tgtaacaaag cgggaccaaa gccatgacaa aaacgcgtaa caaaagtgtc 1200

tataatcacg gcagaaaagt ccacattgat tatttgcacg gcgtcacact ttgctatgcc 1260

atagcatttt tatccataag attagcggat cctacctgac gctttttatc gcaactctct 1320

actgtttctc catacccgtt tttttgggaa ttcgagctct aaggaggtta taaaaaatgc 1380

atcaaaaaaa ccaggtaatg aacctgggtc cgaactctaa actgctgaaa gaatacaaat 1440

cccagctgat cgaactgaac atcgaacagt tcgaagcagg tatcggtctg atcctgggtg 1500

atgcttacat ccgttctcgt gatgaaggta aaacctactg tatgcagttc gagtggaaaa 1560

acaaagcata catggaccac gtatgtctgc tgtacgatca gtgggtactg tccccgccgc 1620

acaaaaaaca acgtgttaac cacctgggta acctggtaat cacctggggc gcccagactt 1680

tcaaacacca agctttcaac aaactggcta acctgttcat cgttaacaac aaaaaaacca 1740

tcccgaacaa cctggttgaa aactacctga ccccgatgtc tctggcatac tggttcatgg 1800

atgatggtgg taaatgggat tacaacaaaa actctaccaa caaatcgatc gtactgaaca 1860

cccagtcttt cactttcgaa gaagtagaat acctggttaa gggtctgcgt aacaaattcc 1920

aactgaactg ttacgtaaaa atcaacaaaa acaaaccgat catctacatc gattctatgt 1980

cttacctgat cttctacaac ctgatcaaac cgtacctgat cccgcagatg atgtacaaac 2040

tgccgaacac tatctcctcc gaaactttcc tgaaataagc atgcccgttc catacagaag 2100

ctgggcgaac aaacgatgct cgccttccag aaaaccgagg atgcgaacca cttcatccgg 2160

ggtcagcacc accggcaagc gccgcgacgg ccgaggtctt ccgatctcct gaagccaggg 2220

cagatccgtg cacagcacct tgccgtagaa gaacagcaag gccgccaatg cctgacgatg 2280

cgtggagacc gaaaccttgc gctcgttcgc cagccaggac agaaatgcct cgacttcgct 2340

gctgcccaag gttgccgggt gacgcacacc gtggaaacgg atgaaggcac gaacccagtg 2400

gacataagcc tgttcggttc gtaagctgta atgcaagtag cgtatgcgct cacgcaactg 2460

gtccagaacc ttgaccgaac gcagcggtgg taacggcgca gtggcggttt tcatggcttg 2520

ttatgactgt ttttttgggg tacagtctat gcctcgggca tccaagcagc aagcgcgtta 2580

cgccgtgggt cgatgtttga tgttatggag cagcaacgat gttacgcagc agggcagtcg 2640

ccctaaaaca aagttaaaca tcatgaggga agcggtgatc gccgaagtat cgactcaact 2700

atcagaggta gttggcgtca tcgagcgcca tctcgaaccg acgttgctgg ccgtacattt 2760

gtacggctcc gcagtggatg gcggcctgaa gccacacagt gatattgatt tgctggttac 2820

ggtgaccgta aggcttgatg aaacaacgcg gcgagctttg atcaacgacc ttttggaaac 2880

ttcggcttcc cctggagaga gcgagattct ccgcgctgta gaagtcacca ttgttgtgca 2940

cgacgacatc attccgtggc gttatccagc taagcgcgaa ctgcaatttg gagaatggca 3000

gcgcaatgac attcttgcag gtatcttcga gccagccacg atcgacattg atctggctat 3060

cttgctgaca aaagcaagag aacatagcgt tgccttggta ggtccagcgg cggaggaact 3120

ctttgatccg gttcctgaac aggatctatt tgaggcgcta aatgaaacct taacgctatg 3180

gaactcgccg cccgactggg ctggcgatga gcgaaatgta gtgcttacgt tgtcccgcat 3240

ttggtacagc gcagtaaccg gcaaaatcgc gccgaaggat gtcgctgccg actgggcaat 3300

ggagcgcctg ccggcccagt atcagcccgt catacttgaa gctagacagg cttatcttgg 3360

acaagaagaa gatcgcttgg cctcgcgcgc agatcagttg gaagaatttg tccactacgt 3420

gaaaggcgag atcaccaagg tagtcggcaa ataatgtcta acaattcgtt caagccgacg 3480

ccgcttcgcg gcgcggctta actcaagcgt tagatgcact aagcacataa ttgctcacag 3540

ccaaactatc aggtcaagtc tgcttttatt atttttaagc gtgcataata agccctacac 3600

aaattgggag atatatcatg aaaggctggc tttttcttgt tatcgcaata gttggcgaag 3660

taatcgcaac atccgcatta aaatctagcg agggctttac taagctcctg cagagatctg 3720

aattccctag agagacgaaa gtgattgcgc ctacccggat attatcgtga ggatgcgtca 3780

tcgccattgc tccccaaata caaaaccaat ttcagccagt gcctcgtcca ttttttcgat 3840

gaactccggc acgatctcgt caaaactcgc catgtacttt tcatcccgct caatcacgac 3900

ataatgcagg ccttcacgct tcatacgcgg gtcatagttg gcaaagtacc aggcattttt 3960

tcgcgtcacc cacatgctgt actgcacctg ggccatgtaa gctgacttta tggcctcgaa 4020

accaccgagc cggaacttca tgaaatcccg ggaggtaaac gggcatttca gttcaaggcc 4080

gttgccgtca ctgcataaac catcgggaga gcaggcggta cgcatacttt cgtcgcgata 4140

gatgatcggg gattcagtaa cattcacgcc ggaagtgaat tcaaacaggg ttctggcgtc 4200

gttctcgtac tgttttcccc aggccagtgc tttagcgtta acttccggag ccacaccggt 4260

gcaaacctca gcaagcaggg tgtggaagta ggacattttc atgtcaggcc acttctttcc 4320

ggagcggggt tttgctatca cgttgtgaac ttctgaagcg gtgatgacgc cgagccgtaa 4380

tttgtgccac gcatcatccc cctgttcgac agctctcaca tcgatcccgg tacgctgcag 4440

gataatgtcc ggtgtcatgc tgccaccttc tgctctgcgg ctttctgttt caggaatcca 4500

agagctttta ctgcttcggc ctgtgtcagt tctgacgatg cacgaatgtc gcggcgaaat 4560

atctgggaac agagcggcaa taagtcgtca tcccatgttt tatccagggc gatcagcaga 4620

gtgttaatct cctgcatggt ttcatcgtta accggagtga tgtcgcgttc cggctgacgt 4680

tctgcagtgt atgcagtatt ttcgacaatg cgctcggctt catccttgtc atagatacca 4740

gcaaatccga aggccagacg ggcacactga atcatggctt tatgacgtaa catccgtttg 4800

ggatgcgact gccacggccc cgtgatttct ctgccttcgc gagttttgaa tggttcgcgg 4860

cggcattcat ccatccattc ggtaacgcag atcggatgat tacggtcctt gcggtaaatc 4920

cggcatgtac aggattcatt gtcctgctca aagtccatgc catcaaactg ctggttttca 4980

ttgatgatgc gggaccagcc atcaacgccc accaccggaa cgatgccatt ctgcttatca 5040

ggaaaggcgt aaatttcttt cgtccacgga ttaaggccgt actggttggc aacgatcagt 5100

aatgcgatga actgcgcatc gctggcatca cctttaaatg ccgtctggcg aagagtggtg 5160

atcagttcct gtgggtcgac agaatccatg ccgacacgtt cagccagctt cccagccagc 5220

gttgcgagtg cagtactcat tcgttttata cctctgaatc aatatcaacc tggtggtgag 5280

caatggtttc aaccatgtac cggatgtgtt ctgccatgcg ctcctgaaac tcaacatcgt 5340

catcaaacgc acgggtaatg gattttttgc tggccccgtg gcgttgcaaa tgatcgatgc 5400

atagcgattc aaacaggtgc tggggcaggc ctttttccat gtcgtctgcc agttctgcct 5460

ctttctcttc acgggcgagc tgctggtagt gacgcgccca gctctgagcc tcaagacgat 5520

cctgaatgta ataagcgttc atggctgaac tcctgaaata gctgtgaaaa tatcgcccgc 5580

gaaatgccgg gctgattagg aaaacaggaa agggggttag tgaatgcttt tgcttgatct 5640

cagtttcagt attaatatcc attttttata agcgtcgact gtttcctgtg tgaaattgtt 5700

atccgctcac aattccacac attatacgag ccggaagcat aaagtgtaaa gcctggggtg 5760

cctaatgagt gagaattcgg atctcgacgg gatgttgatt ctgtcatggc atatccttac 5820

aacttaaaaa agcaaaaggg ccgcagatgc gacccttgtg tatcaaacaa gacgattaaa 5880

aatcttcgtt agtttctgct acgccttcgc tatcatctac agagaaatcc ggcgttgagt 5940

tcgggttgct cagcagcaac tcacgtactt tcttctcgat ctctttcgcg gtttccgggt 6000

tatctttcag ccaggcagtc gcattcgctt taccctgacc gatcttctca cctttgtagc 6060

tgtaccacgc gcctgctttc tcgatcagct tctcttttac gcccaggtca accagttcgc 6120

cgtagaagtt gataccttcg ccgtagagga tctggaattc agcctgttta aacggcgcag 6180

cgattttgtt cttcaccact ttcacgcggg tttcgctacc caccacgttt tcgccctctt 6240

tcaccgcgcc gatacgacgg atgtcgagac gaacagaggc gtagaatttc agcgcgttac 6300

caccggtagt ggtttccggg ttaccgaaca tcacaccaat tttcatacgg atctggttga 6360

tgaagatcag cagcgtgttg gactgcttca ggttacccgc cagcttacgc atcgcctggc 6420

tcatcatacg tgccgcaagg cccatgtgag agtcgccgat ttcgccttcg atttccgctt 6480

tcggcgtcag tgccgccacg gagtcaacga cgataacgtc tactgcgcca gaacgcgcca 6540

gggcgtcaca gatttccagt gcctgctcgc cggtgtccgg ctgggagcac agcaggttgt 6600

cgatatcgac gcccagttta cgtgcgtaga ttgggtccag cgcgtgttca gcatcgataa 6660

acgcacaggt tttaccttca cgctgcgctg cggcgatcac ctgcagcgtc agcgtggttt 6720

taccggaaga ttccggtccg tagatttcga cgatacggcc catcggcaga ccacctgccc 6780

caagcgcgat atccagtgaa agcgaaccgg tagagatggt ttccacatcc atggaacggt 6840

cttcacccag gcgcatgatg gagcctttac caaattgttt ctcaatctgg cccagtgctg 6900

ccgccaacgc tttctgtttg ttttcgtcga tagccatttt tactcctgtc atgccgggta 6960

ataccggata gtcaatatgt tctgttgaag caattatact gtatgctcat acagtatcaa 7020

gtgttttgta gaaattgttg ccacaaggtc tgcaatgcat acgcagtagc ctgacgacgc 7080

accgcatcac ggtcgccgct gaagcatccc gccgggtaat gccttcaccg cgggcagtgg 7140

caaaagcaaa ccagacggtg ccgacaggct tctctagatc cgcctacctt tcacgagttg 7200

cgcagtttgt ctgcaagact ctatgagaag cagataagcg ataagtttgc tcaacatctt 7260

ctcgggcata agtcggacac catggcatca cagtatcgtg atgacagagg cagggagtgg 7320

gacaaaattg aaatcaaata atgattttat tttgactgat agtgacctgt tcgttgcaac 7380

aaattgataa gcaatgccaa gcttggcact ggctgatcag ctagctcact gcccgctttc 7440

cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc 7500

ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg 7560

attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc 7620

cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc 7680

ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat 7740

ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat 7800

gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc 7860

ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg 7920

cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa 7980

tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt 8040

gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc 8100

cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg 8160

cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga 8220

caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga 8280

cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc 8340

cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt 8400

ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata 8460

agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct 8520

gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc accattcgat 8580

gctagcccat gggtatggac agttttccct ttgatatgta acggtgaaca gttgttctac 8640

ttttgtttgt tagtcttgat gcttcactga tagatacaag agccataaga acctcagatc 8700

cttccgtatt tagccagtat gttctctagt gtggttcgtt gtttttgcgt gagccatgag 8760

aacgaaccat tgagatcata cttactttgc atgtcactca aaaattttgc ctcaaaactg 8820

gtgagctgaa tttttgcagt taaagcatcg tgtagtgttt ttcttagtcc gttacgtagg 8880

taggaatctg atgtaatggt tgttggtatt ttgtcaccat tcatttttat ctggttgttc 8940

tcaagttcgg ttacgagatc catttgtcta tctagttcaa cttggaaaat caacgtatca 9000

gtcgggcggc ctcgcttatc aaccaccaat ttcatattgc tgtaagtgtt taaatcttta 9060

cttattggtt tcaaaaccca ttggttaagc cttttaaact catggtagtt attttcaagc 9120

attaacatga acttaaattc atcaaggcta atctctatat ttgccttgtg agttttcttt 9180

tgtgttagtt cttttaataa ccactcataa atcctcatag agtatttgtt ttcaaaagac 9240

ttaacatgtt ccagattata ttttatgaat ttttttaact ggaaaagata aggcaatatc 9300

tcttcactaa aaactaattc taatttttcg cttgagaact tggcatagtt tgtccactgg 9360

aaaatctcaa agcctttaac caaaggattc ctgatttcca cagttctcgt catcagctct 9420

ctggttgctt tagctaatac accataagca ttttccctac tgatgttcat catctgagcg 9480

tattggttat aagtgaacga taccgtccgt tctttccttg tagggttttc aatcgtgggg 9540

ttgagtagtg ccacacagca taaaattagc ttggtttcat gctccgttaa gtcatagcga 9600

ctaatcgcta gttcatttgc tttgaaaaca actaattcag acatacatct caattggtct 9660

aggtgatttt aatcactata ccaattgaga tgggctagtc aatgataatt actagtcctt 9720

ttcctttgag ttgtgggtat ctgtaaattc tgctagacct ttgctggaaa acttgtaaat 9780

tctgctagac cctctgtaaa ttccgctaga cctttgtgtg ttttttttgt ttatattcaa 9840

gtggttataa tttatagaat aaagaaagaa taaaaaaaga taaaaagaat agatcccagc 9900

cctgtgtata actcactact ttagtcagtt ccgcagtatt acaaaaggat gtcgcaaacg 9960

ctgtttgctc ctctacaaaa cagaccttaa aaccctaaag gcttaagtag caccctcgca 10020

agctcggttg cggccgcaat cgggcaaatc gctgaatatt ccttttgtct ccgaccatca 10080

ggcacctgag tcgctgtctt tttcgtgaca ttcagttcgc tgcgctcacg gctctggcag 10140

tgaatggggg taaatggcac tacaggcgcc ttttatggat tcatgcaagg aaactaccca 10200

taatacaaga aaagcccgtc acgggcttct cagggcgttt tatggcgggt ctgctatgtg 10260

gtgctatctg actttttgct gttcagcagt tcctgccctc tgattttcca gtctgaccac 10320

ttcggattat cccgtgacag gtcattcaga ctggctaatg cacccagtaa ggcagcggta 10380

tcatcaacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 10440

agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 10500

atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca 10560

cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag 10620

ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac 10680

ccacgctcac cggctccaga tttatcagca ataaaccagc cagccg 10726

Claims

1. the construction method of the Escherichia coli engineering bacteria that prefers to utilize xylose to efficiently secrete acetic acid and FFA; it is characterized in that, comprises the steps:

(1) Knock out the ptsG gene from E. coli MG1655 to obtain strain ECΔp;

(2) Knock out the manZ gene from strain ECΔp to obtain strain ECΔp-m;

(3) Knock out the atpFH gene from strain ECΔp-m to obtain strain ECΔp-m-a;

(4) Knock out the envR gene from strain ECΔp-m-a to obtain strain ECΔp-m-a-e.

2. The method of claim 1, wherein the nucleotide sequence of the ptsG gene is SEQ ID No. 1; the nucleotide sequence of the manZ gene is SEQ ID No. 2; the nucleotide sequence of the atpFH gene is SEQ ID No. 2 ID No.3; the nucleotide sequence of the envR gene is SEQ ID No.4.

3. method as claimed in claim 1, is characterized in that, gene knockout is based on λ-red and I-SceI genome traceless operation technology; Concrete knockout step is as follows:

(1) Using the genome of E.coli MG1655 strain as a template, the upstream and downstream homology arm fragment A and fragment B of the target gene were amplified respectively;

(2) Using the pTKS/CS plasmid as a template, amplify the upstream fragment tet1 and the downstream fragment tet2 of the tetracycline resistance gene tet, respectively;

(3) Take fragment A and tet1 as templates again, carry out overlapping extension PCR to amplify fragment A-tet1, and also use fragment tet2 and B as templates, carry out overlapping extension PCR to amplify fragment tet2-B;

(4) Carry out overlap extension PCR again, and use A-tet1 and tet2-B as amplification templates to amplify the knockout fragment A-t-B;

(5) The knockout fragment A-t-B was transformed into Escherichia coli by electroporation method;

(6) The tetracycline resistance and deletion pKTRed plasmid was popped up, and the target gene knockout strain was obtained after two screenings.

4. The method of claim 3, wherein the pTKS/CS plasmid is the nucleotide sequence shown in SEQ ID No.5; the tetracycline resistance gene tet is the nucleotide sequence shown in SEQ ID No.6; The nucleotide sequence of the upstream fragment tet1 is shown in SEQ ID No. 7; the nucleotide sequence of the downstream fragment tet2 gene is shown in SEQ ID No. 8; the nucleotide sequence of the pKTRed plasmid is shown in SEQ ID No. 9.

5. method as claimed in claim 3 is characterized in that, in described step 1), ptsG gene knockout primer sequence is as follows:

ptsG-Af: TGGCTGTGTTGAAAGGTGTTGCCG

ptsG-Ar: TATCCCTAAGCACAATGCCTCTATTTAGTTAACGTTCTG

ptsG-Bf: AGGGTAATCAGAACGTTAACTAAATAGAGGCATTGTGCTAAAAACGAACAAATGGCAGAG

ptsG-Br: GTCTGCAAGGCGCTCAAGACG

ptsG-tetf1: CAGAACGTTAACTAAATAGAGGCATTGTGCTTAGGGATAACAGGGTAATATTTAC

ptsG-tetr1: GGTAAAGCGATCCCACCACCAGCC

ptsG-tetf2: GCGTGAAGTGGTTCGGTT

ptsG-tetr2: TCGTTTTTAGCACAATGCCTCTATTTAGTTAACGTTCTGATTACCCTGTTATCCCCTAC.

6. method as claimed in claim 3, is characterized in that, in described step 2), manZ gene knockout primer sequence is as follows:

manZ-Af:TGCAAGCCTTCATCTTCATGG

manZ-Ar: TATCCCTAAGCACAATGCCTCTATTTAGTTAACGTTCTG

manZ-Bf: AGGGTAATCAGAACGTTAACTAAATAGAGGCATTGTGCTAAAAACGAACAAATGGCAGAG

manZ-Br: GTCTGCAAGGCGCTCAAGACG

manZ-tetf1: CAGAACGTTAACTAAATAGAGGCATTGTGCTTAGGGATAACAGGGTAATATTTAC

manZ-tetr1: GGTAAAGCGATCCCACCACCAGCC

manZ-tetf2: GCGTGAAGTGGTTCGGTT

manZ-tetr2: CAACAGTCTTCGCTCACCTGTTAGTCCAGTTCGATTACCCTGTTATCCCCTAC.

7. method as claimed in claim 3 is characterized in that, in described step 3), atpFH gene knockout primer sequence is as follows:

atpFH-Af:TGCAAGCCTTCATCTTCATGG

atpFH-Ar: TATCCCTAAGCACAATGCCTCTATTTAGTTAACGTTCTG

atpFH-Bf: AGGGTAATCAGAACGTTAACTAAATAGAGGCATTGTGCTAAAAACGAACAAATGGCAGAG

atpFH-Br: GTCTGCAAGGCGCTCAAGACG

atpFH-Tetf1: CAGAACGTTAACTAAATAGAGGCATTGTGCTTAGGGATAACAGGGTAATATTAC

atpFH-Tetr1: GGTAAAGCGATCCCACCAGCC

atpFH-Tetf2: GCGTGAAGTGGTTCGGTT

atpFH-Tetr2:TCGTTTTTAGCACAATGCCTCTATTTAGTTAACGTTCTGATTACCCTGTTATCCCCTAC.

8. The method of claim 3, wherein the envR gene knockout primer sequence in the step 4) is as follows:

envR-Af:TGCAAGCCTTCATCTTCATGG

envR-Ar: TATCCCTAAGCACAATGCCTCTATTTAGTTAACGTTCTG

envR-Bf: AGGGTAATCAGAACGTTAACTAAATAGAGGCATTGTGCTAAAAACGAACAAATGGCAGAG

envR-Br: GTCTGCAAGGCGCTCAAGACG

envR-tetf1: CAGAACGTTAACTAAATAGAGGCATTGTGCTTAGGGATAACAGGGTAATATTTAC

envR-tetr1: GGTAAAGCGATCCCACCACCAGCC

envR-tetf2:GCGTGAAGTGGTTCGGTT

envR-tetr2: TCGTTTTTAGCACAATGCCTCTATTTAGTTAACGTTCTGATTACCCTGTTATCCCCTAC.