WO2013010483A1 - Construction method of escherichia coli genetically engineered bacteria producing succinic acid by xylose metabolism - Google Patents

Abstract

Provided is a construction method of an escherichia coli genetically engineered bacteria producing succinic acid by xylose metabolism and the method for producing succinic acid by fermentation using the bacteria. The ATP biosynthesis pathway of escherichia coli is modified by means of molecular biology and the enzyme activity related to the pathway is over-expressed; thus the total ATP amount within escherichia coli cells is effectively increased such that the recombinant escherichia coli is able to grow using xylose metabolism, and the succinic acid synthesis efficiency is thereby significantly improved.

Description

 Method for constructing genetically engineered bacteria producing succinic acid by using xylose metabolism

Technical field

 The invention belongs to the field of bioengineering technology, and relates to a method for constructing a genetically engineered bacterium of succinic acid using xylose metabolism.

Background technique

 Succinic acid (Succinic acid), also known as succinic acid, is widely used in the pharmaceutical, pesticide, dye, fragrance, paint, food and plastic industries. As a C4 platform compound, it can be used to synthesize 1,4-butanediol, tetrahydrofuran, Organic chemicals such as γ-butyrolactone and polybutylene succinate (PBS) biodegradable materials are considered by the US Department of Energy to be one of the 12 most valuable biorefining products in the future.

The production method of succinic acid mainly includes chemical synthesis method and microbial fermentation method, and uses microbial fermentation method to convert renewable resources (glucose, xylose, etc.), due to wide source and low price of raw materials, low pollution, environmental friendliness, and in the fermentation process. It can absorb fixed C0 ₂ , which can effectively alleviate the greenhouse effect and open up a new way of utilizing greenhouse gas carbon dioxide. This year has become a research hotspot. The production strain of succinic acid is mainly concentrated in Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes ^ Mannheimia succiniciproducens ^ recombinant Corynebacterium glutamicum and recombinant E.coIi. Although the production of succinic acid by wild strains has obtained a higher product concentration, the culture medium cost is higher, and by-products such as formic acid and acetic acid accumulate more, hindering the industrialization process. E.coli has been widely used in recent years to obtain excellent strains of succinic acid production due to its clear genetic background, easy operation, easy regulation, simple medium requirements and rapid growth.

Although the _CO /wild strain produces succinic acid, although higher product concentration is obtained, the culture medium cost is higher, and by-products such as formic acid and acetic acid accumulate more, so the lactate can be knocked out or inactivated. Hydrogenase (LDH) gene and pyruvate formate lyase (PFL) gene activity to reduce by-product formation. However, the knockdown of the PFL gene results in the inability of the strain to produce acetic acid, thereby reducing the ATP accompanying the production of acetic acid, eventually leading to the inability of recombinant E. coli to utilize xylose metabolic growth and to produce succinic acid.

Corn cob is a common waste in agricultural production. Because its composition contains a lot of cellulose, its hydrolyzate is a good sustainable green carbon source for microbial fermentation, but its hydrolyzate contains high The concentration of xylose, therefore E. coli NZN111 can not be fermented using corn cob hydrolysate to produce succinic acid. Jiang Yan et al. prepared corn cob liquid with a ratio of 1:5 (mass to volume ratio) of corn cob, material size 250~380μηι, H ₂ S0 ₄ dosage 3% (volume fraction), hydrolysis temperature 126 °C, The reaction time was 215 h, and the corncob multi-component sugar solution was detoxified and desalted by activated carbon adsorption and Ca(OH) ₂ neutralization. The total sugar concentration was 50 g/L, and xylose accounted for more than 80%. .

 Straw straw is an important class of renewable biomass resources. At present, most of them are discarded except for the use in the paper industry, which wastes resources and pollutes the environment. Its main components are cellulose, hemicellulose and lignin, so its hydrolyzate is a good sustainable green carbon source for microbial fermentation, but its hydrolyzate contains high concentration of xylose, so it can not E. coli strains using xylose could not be fermented by straw straw hydrolysate to produce succinic acid. Tao Wenqi et al. treated straw straw with dilute sulfuric acid at 121 °C for 1 h, and treated 20 g/L NaOH at 121 °C for 1 h. The total mass concentration of both glucose and xylose is about 50 g/L.

 Bagasse is the main component left after sugar cane sugar production. Therefore, its hydrolyzate is a good sustainable green carbon source for microbial fermentation, but its hydrolyzate contains high concentration of xylose. Therefore, the E. coli strain which cannot utilize xylose can not be fermented to produce succinic acid by using straw straw hydrolysate, and about 50% of cellulose can be obtained by pulverization and alkali/oxidation pretreatment to obtain a total sugar mass of 50 g/L, wherein wood Sugar accounts for more than 80%.

rato fc中， 磷酸烯醇式丙酮酸是通过磷酸烯醇式丙酮酸羧 化激酶生成草酰乙酸的， 在此过程中有 ATP的生成， 并且 Millard等在大肠杆菌中过量表达 E. coli ppc和 pck, 研究发现过量表达/ 可以使琥珀酸作为混合酸发酵的主要产物， 且产量 较出发菌株提高 3.5倍， 而过量表达 对发酵结果没有影响， 但在^ C缺陷菌株中， 的 过量表达能够提高琥珀酸的产量。 In Escherichia coli phosphoenolpyruvate, oxaloacetate is produced by phosphoenolpyruvate carboxylase, in the process without ATP formation, but in

In rato fc, phosphoenolpyruvate produces oxaloacetate by phosphoenolpyruvate carboxylation kinase, in which ATP is produced, and Millard et al. overexpress E. coli ppc and E. coli. Pck, the study found that overexpression / can make succinic acid as the main product of mixed acid fermentation, and yield The expression was 3.5 times higher than that of the original strain, and the overexpression had no effect on the fermentation result, but in the C-deficient strain, the overexpression could increase the yield of succinic acid.

Summary of the invention

 The technical aim of the present invention is to provide a method for constructing an Escherichia coli strain based on ATP biosynthesis system, and the method for constructing the strain is simple and convenient, and the fermentation method of the constructed strain is simple and feasible, easy to industrialize, and capable of producing acid. The purpose is to greatly reduce production costs and improve economic efficiency.

 In order to achieve the object of the present invention, the present invention adopts the following technical solutions.

 A method for constructing a succinic acid Escherichia coli genetically engineered bacterium by utilizing xylose metabolism, comprising the steps of:

(1) The .cc^'NZNlll strain lacking the lactate dehydrogenase gene pyruvate formate lyase gene (Ζβ) activity was used as the starting strain, and the phosphoenolpyruvate carboxylase (PPC) gene was knocked out. Competent strains lacking WM, pflB and PPC;

 (2) Purification of the phosphoenolpyruvate carboxylase pck gene, or purification and amplification of the phosphoenolpyruvate carboxylase pck~) gene, and selection of the malic enzyme G/) gene, One of three genes, the malate dehydrogenase mdh gene or the pyruvate carboxylase (pyc) gene, is constructed to overexpress phosphoenolpyruvate carboxylase alone or to overexpress phosphoenolpyruvate. a carboxylated kinase, and an expression plasmid selected from one of three enzymes, malic enzyme, malate dehydrogenase or pyruvate carboxylase;

 (3) introducing the plasmid described in the step (2) into the competent strain obtained in the step (1) to obtain a positive transformant;

(4) Excessive overexpression of phosphoenolpyruvate carboxylase by positive transformants of step (3), or overexpression of phosphoenolpyruvate carboxylase, and dehydrogenation from malic enzyme and malate One of the three enzymes, enzyme or pyruvate carboxylase, restores its ability to metabolize xylose under anaerobic conditions, and obtains genetically engineered bacteria producing succinic acid by utilizing xylose metabolism.

 Specifically, the above method described in the present invention can be subdivided into the following specific methods.

 A. Excessive expression of phosphoenolpyruvate carboxylase to obtain Escherichia coli BA204 capable of efficiently utilizing xylose growth and producing succinic acid:

 The E.coli NZN111 strain lacking lactate dehydrogenase gene ldhA and pyruvate formate lyase gene pflB activity was used as the starting strain, and the phosphoenolpyruvate carboxylase (PPC) gene was knocked out, which resulted in the lack of WM. Competent strains of pflB and PPC;

 A pair of primers with a restriction enzyme site at the 5' end were synthesized, and the amplified gene was purified by using BadU s^rifc genomic DNA as a template. The expression plasmid pTrc99a was doubled with the enzyme cleavage site designed by the primer. Digestion, ligation to obtain recombinant plasmid pTrc99a- ct;

 The plasmid ρΤκ99ίΐ-;7^ was introduced to eliminate the apramycin resistance, and the competent state of the NZN111 strain of the phosphoenolpyruvate carboxylase (PPC) gene was knocked out. The positive transformant obtained was Escherichia coli BA204. ;

 Escherichia coli BA204 was used to overexpress phosphoenolpyruvate carboxylase to restore its ability to metabolize xylose under anaerobic conditions. The constructed map of the recombinant plasmid ρΤκ^^-ρ is shown in Fig. 3.

 B. Excessive co-expression of phosphoenolpyruvate carboxylase and malic enzyme to obtain Escherichia coli BA205 which can efficiently utilize xylose to grow and produce succinic acid:

 The E. coli NZN111 strain lacking the lactate dehydrogenase gene (intestine') and the pyruvate formate lyase gene (pflB) activity was used as the starting strain, and the phosphoenolpyruvate carboxylase (PPC) gene was knocked out. Simultaneous lack of competent strains of WM, pflB and PPC;

A pair of primers carrying the same restriction site at the 5' end were synthesized, and the amplified ppk gene was purified by using ^^7/^^^' genomic DNA as a template, and the recombinant plasmid pTr _C 99a- o was constructed. The enzyme is digested with the enzyme cleavage site designed by the primer, and the recombinant plasmid pTrc99a-Ji h -; jd:; The competent transformant obtained by introducing the plasmid pT _rC 99a-^ l-pd to eliminate apramycin resistance, knocked out the NZN1 11 strain of the decanoic pyruvate carboxylase (PPC) gene, and obtained the positive transformant That is Escherichia coli BA205;

 Escherichia coli BA205 was used to co-express phosphoenolpyruvate carboxylase and malic enzyme to restore its ability to metabolize xylose under anaerobic conditions. The construction map of the recombinant plasmid ρΤ3⁄49 - -/ Λ is shown in Fig. 4.

 C. Excessive co-expression of phosphoenolpyruvate carboxylase and malate dehydrogenase enables efficient use of xylose to produce succinic acid to obtain Escherichia coli BA206:

 The E.coli NZN111 strain lacking lactate dehydrogenase gene (WM) and pyruvate formate lyase gene (ρ/?) was used as the starting strain, and the phosphoenolpyruvate carboxylase (PPC) gene was knocked out. Obtaining competent strains lacking both WM, pflB and PPC;

 A pair of primers carrying the same restriction site at the 5' end were synthesized, and the amplified ppk gene was purified by using ^^'3⁄4« ^^7^ genomic DNA as a template, and the constructed recombinant plasmid pTrc99a-TMft was used. The enzyme was digested with the enzyme cleavage site designed by the primer, and the recombinant plasmid pTrc99a-wtt i-;?d was obtained;

The plasmid pT _rc 99 _a -TM3⁄4-;7 _C t was introduced before the apramycin resistance was eliminated, and the competent state of the NZN1 11 strain of the phosphoenolpyruvate carboxylase (PPC) gene was knocked out. The positive transformant is Escherichia coli BA206;

 Escherichia coli BA206 was used to co-express phosphoenolpyruvate carboxylase and malate dehydrogenase to restore its ability to metabolize xylose under anaerobic conditions. The construction map of the recombinant plasmid ρΎκ99 dh-pck is shown in Fig. 5.

 D. Excessive co-expression of the acid-enolpyruvate carboxylase and pyruvate carboxylase enables the efficient use of xylose to produce succinic acid to obtain Escherichia coli BA207:

 The E.coli NZN111 strain lacking lactate dehydrogenase gene IdhA and pyruvate formate lyase gene ipflB activity was used as the starting strain, and the phosphoenolpyruvate carboxylase (PPC) gene was knocked out, which resulted in the lack of WM and pflB. Competent strains of PPC;

A pair of primers carrying the same restriction site at the 5' end were synthesized and purified by using ad3⁄4«TMM genomic DNA as a template; after the * gene, the constructed recombinant plasmid pT _rc 99 _a -;? _yc The recombinant plasmid pTrc99a-/^c-pcJt was obtained by single enzyme digestion and ligation with the enzyme cleavage site designed by the primer;

 The plasmid ρΤ ^^-ρ^-ρ was introduced before the introduction of the NZN1 11 strain which amplifies the resistance to apramycin, and the phosphoenolpyruvate carboxylase PPC gene was knocked out. The positive transformant obtained was Escherichia coli BA207;

Escherichia coli BA207 was used to co-express phosphoenolpyruvate carboxylase and pyruvate carboxylase to restore its ability to metabolize xylose under anaerobic conditions. The constructed map of the recombinant plasmid ρΤκ;99 - ^- _Ρ is shown in Fig. 6.

 The beneficial effects of the invention are:

First, in the anaerobic fermentation process, a large amount of by-products such as acetic acid which are toxic to the strain are produced, so that the two-stage fermentation method is considered, the aerobic phase increases the biomass, and the anaerobic phase performs the acidogenic fermentation. Membrane separation technology can also be selectively employed to achieve the purpose of isolating the cells, and then used for anaerobic fermentation. The specific steps are as follows: using a two-stage fermentation mode, the bacteria liquid stored in the cryotube-80°C cryotube is applied to the plate containing ampicillin, and the single colony grown on the plate is picked into the test tube of 5 ml LB medium, 1%. (v/v) Inoculum size was added to the flask, when aerobic cultured cells were OD ₆ . . When induced to 0.3 mM IPTG to about OD _6M = 3 at about 0.8-1.0, the anaerobic fermentation was carried out in a serum bottle at 10% of the inoculum. The results of the fermentation indicated that the newly constructed genetically engineered bacteria associated with the E. coli ATP biosynthesis pathway restored anaerobic conditions by £^π·ί3⁄4·α coli BA 204, Escherichia coli BA 205, Escherichia coli BA 206 and Escherichia coli BA 207. The ability to metabolize xylose, and the efficient use of xylose to produce succinic acid.

 Secondly, the present invention modifies the ATP biosynthesis pathway of Escherichia coli by molecular biological means, increases the supply of ATP, supplements the energy supply in the xylose transport and metabolism process, enables the recombinant Escherichia coli to continue to utilize xylose growth and efficiently synthesize it. The method of diacid has not been disclosed, and this application will greatly advance the progress and development of the succinic acid industry.

DRAWINGS Figure 1. Electrophoretic identification of a linear DNA fragment.

 Figure 2. Electrophoretic identification of homologous recombinant positive recombinants.

 Figure 3. Construction map of the recombinant plasmid pTrc99a-p.

 Figure 4. Construction map of recombinant plasmid pTrc99a--/^.

 Figure 5. Construction map of the recombinant plasmid pTrc99a-m^-prtt.

 Figure 6. Construction of recombinant plasmid ρΤκ;99 - }^-ρ^.

 Figure 7 Agarose gel electrophoresis identification of the PCR product pck.

 Figure 8. Identification of the recombinant plasmid ρΤΐΐ99&-ρ by single and double digestion.

 Figure 9. Single-double digestion map of recombinant plasmid pTrc99a-^ 4-p.

Figure 10 is a single double-digestion identification map of the recombinant plasmid pTrc99a-mi- _P (^.

 Figure 11 is a single-double digestion map of the recombinant plasmid pTrc99a-;^c-pd.

detailed description

 The following examples are illustrative of the invention but are not intended to limit the invention.

 The source of the apramycin resistance gene of the present invention is: pIJ773, obtained from Professor Shao Weilan of Nanjing Normal University. The source of the plasmid capable of inducing expression of the lambda recombinase of the present invention is: pKD46, available from Introvegen. The source of the plasmid capable of inducing the production of FLP recombinase according to the present invention is: pCP20, purchased from Introvegen. The source of the Bacillus subtilis genome of the present invention is: purchased from the China Center for Type Culture Collection.

 The source of pTrc99a for the expression plasmid of the present invention is: purchased from Introvegen.

 The starting strain of the present invention: Ε. The source of the competent strain of ΝΖΝ111 has two places:

 (1) Biotechnol Bioeng, 2001, 74: 89-95. The applicant first consulted the source of the above-mentioned literature on the biological material, and contacted the publisher, Professor David P. Clark of the University of Chicago, and requested the gift of the biological material, and obtained the biological material free of charge; The person guarantees that the biological material will be distributed to the public within 20 years from the date of this application;

 (2) The biological material is also disclosed and authorized in the patent documents of the Chinese patent (Application No. 96198547.X, Application Date 1996.10.31, Authorization Date January 1, 2003, Authorization Bulletin No. CN1097632C).

 The design and synthesis of the primers of the present invention: self-designed and outsourced synthesis by Kingsray Biotech.

 Example 1

 This example illustrates the construction of an expression plasmid overexpressing phosphoenolpyruvate carboxylase to restore the ability of the recombinant strain to metabolize xylose under anaerobic conditions to obtain the strain Escherichia coli BA204.

 1. The .co/ NZNl ll strain lacking lactate dehydrogenase gene (WM) and pyruvate formate lyase gene pflB~) was used as the starting strain, and the phosphoenolpyruvate carboxylase (PPC) gene was knocked out. A competent strain lacking both WM, pflB and PPC was obtained.

 Knockout of phosphoenolpyruvate carboxylase (PPC) gene by homologous recombination technique: using a high-fidelity PCR amplification system with an apramycin resistance gene flanked by FRT sites as a template Amplification primers with PPC homologous fragments at both ends, and linear DNA homologous fragments were successfully amplified;

 A plasmid capable of inducing expression of λ recombinase is introduced into the starting strain NZN111, so that after electroporation into a linear DNA fragment, the exonuclease inside the cell can be inhibited, the decomposition of the linear fragment can be prevented, and homologous recombination can be performed at the same time. Positive screening for positive recombinants; introduction of a plasmid capable of inducing the production of FLP recombinase, after induction, using a pair of plates, parallel spotting, capable of growing on non-resistant plates, but not growing on resistant plates The resistant NZN111 strain has been knocked out extremely.

 Specific method:

(1) Escherichia coli NZN111 was cultured under aerobic conditions at 37 ° C under an aerobic condition to an OD ₆ QQ = 0.4 to 0.6 to prepare an electrotransformed competent state. (2) The plasmid pKD46 was electrotransferred into competent E. coli NZN111. The electric shock conditions are: 200 Ω, 25 μΡ, electric shock voltage 2.3 kV, and electric shock time 4 to 5 ms. After electroshock, the cells were quickly added to pre-cooled 1 mL SOC medium, cultured at 150 r/min and 30 °C for 1 h, and then plated on LB medium plates with ampicillin (amp) to screen positive transformants E. coli NZN111. (pKD46).

 (3) 10 mM L-arabinose was added to LB medium, and plasmid pKD46 was induced to express λ recombinase at 30 ° C to prepare electroporation.

 (4) Using the apramycin resistance gene with FRT locus on both sides as a template, using high-fidelity PCR amplification system, using plasmid PIJ773 as a template, and designing amplification primers with PPC homologous fragments at both ends , a linear DNA homologous fragment was amplified, and the primer sequence was as follows:

 The homologous arm primer H1-P1 is upstream and the underline is a homologous fragment:

GGGATCCGTCGACC-3 '.

 Downstream with the homology arm primer Η2-Ρ2, underlined as a homologous fragment:

GCTGGAGCTGCTTC-3 '.

Reaction system: Upstream and downstream primers with homology arms (100 ριηοΐ/μί) 0.5 μ^ each, template DNA (100 ng/μί) 0.5 μί; lO bufFer 5 μί; dNTPs (10 mM) each 1 L; DMSO (100 %) 2.5 μ^, Pyrobest DNA polymerase (2.5 U/L) l L; ddH ₂ 0 36/35.5 μί; total volume 50 L.

Reaction conditions: 94 ° C, 2 min _; (94 ° C 45 sec; 50 ° C 45 sec; 72 ° C 90 sec; 10 cycles); (94 ° C 45 sec; 50 ° C 45 sec; 72 ° C 90 sec; 15 cycles); 72 °C, 5 min.

 The identification of linear DNA fragments is shown in Figure 2.

 (5) electrotransformed linear DNA fragment to E. coli NZN1 ll (pKD46) competent form that has been induced to express λ recombinase, and coated on LB plate with apramycin to screen positive recombinants, and PCR identification, electrophoresis The figure is shown in Figure 3.

 (6) After the positive recombinant was made into a competent state, pCP20, which can induce the expression of FLP recombinase, was inverted, and the resistance to apramycin was eliminated after heat-excited FLP recombinase at 42 °C. Using a pair of plates, parallel-like, can grow on non-resistant plates, but can not grow on resistant plates, which are extremely resistant to knockout.

 2. Constructing an expression plasmid overexpressing phosphoenolpyruvate carboxylase, the process comprising:

 (1) synthesizing primers with ^JCI and Xbal restriction sites,

 Upstream primer: 5,- CGAGCTCATGAACTCAGTTGATTTGACCG -3';

 Downstream primer: 5'- GCTCTAGAGCATTCCGTCAATTAAAACAAG -3Ό

 (2) PCR amplification of the target gene fragment using the «7/^ «/^7 genomic DNA as a template, the reaction conditions are: 94 ° C, 5 min; (94 ° C 45 s, 55 ° C 45 s, 72 ° C 100 s, 35 cycles); 72 °C, 10 min. The purified d gene and the expression plasmid pTrc99a were digested with ^icl and βΙ, respectively, and ligated to obtain the recombinant plasmid pTrc99a-/^Jt. The agarose gel electrophoresis identification of the PCR product is shown in Figure 7; the double-digestion electrophoresis identification of the plasmid pTrc99a-pdt is shown in Figure 8.

3. The importation of the plasmid pT _rC 99 _a -p _C fe to eliminate the apramycin resistance, the competent state of the NZN111 strain which has been knocked out by the phosphoenolpyruvate carboxylase (PPC) gene, and the positive transformation obtained The child is Escherichia coli BA204.

 Example 2

 This example illustrates the construction of an expression plasmid co-expressing phosphoenolpyruvate carboxylase and malic enzyme to restore the ability of the recombinant strain to metabolize xylose under anaerobic conditions, and the strain Escherichia coli BA205 was obtained.

1. In the absence of lactate dehydrogenase gene UdhA, pyruvate formate lyase gene pjm activity. / ΝΖΝ111 The strain was a starting strain, and the phosphoenolpyruvate carboxylase (PPC) gene was knocked out to obtain a competent strain lacking WM, pflB and PPC (the same as in Example 1).

 2. Constructing an expression plasmid co-expressing phosphoenolpyruvate carboxylase and malic enzyme, the process comprising:

(1) synthesizing primers with HindUl cleavage sites upstream and downstream,

 Upstream primer: 5'- CCCAAGCTTATGAACTCAGTTGATTTGACCG -3 ';

 Downstream primer: 5'-CCCAAGCTTGCATTCCGTCAATTAAAACAAG-3'.

(2) The target gene fragment was amplified by PCR using Ba ZZM MMfo genomic DNA as template. The reaction conditions were: 94 ° C, 5 min _; (94 ° C 45 s, 55 ° C 45 s, 72 ° C 100 s, 35 Cycles); 72 °C, 10 min. The purified amplified gene and the expression plasmid pTrc99a- were digested with H dlll and ligated, respectively, to obtain a recombinant plasmid pTrc99a-^cA-pd. A single-double digestion map of the recombinant plasmid pTrc99a- / -p is shown in Figure 9.

3. The plasmid pTr _C 99a- /b -p _C t was introduced before the apramycin resistance was eliminated, and the competent state of the NZN 111 strain of the phosphoenolpyruvate carboxylase (PPC) gene was knocked out. The positive transformant is Escherichia coll BA205.

 Example 3

 This example illustrates the construction of an expression plasmid co-expressing phosphoenolpyruvate carboxylase and malate dehydrogenase to restore the ability of the recombinant strain to metabolize xylose under anaerobic conditions, and the strain Escherichia coli BA206 was obtained.

 1. The .co/ NZNl ll strain lacking lactate dehydrogenase gene UdhA and pyruvate formate lyase gene pflB~) was used as the starting strain, and the phosphoenolpyruvate carboxylase (PPC) gene was knocked out. At the same time, the competent strains of MM, pflB and PPC were lacking (same as in Example 1).

 2. Constructing an expression plasmid co-expressing phosphoenolpyruvate carboxylase and malate dehydrogenase, the process comprising:

(1) synthesizing primers with H U1 cleavage sites upstream and downstream,

 Upstream primer: 5'- CCCAAGCTTATGAACTCAGTTGATTTGACCG -3 ';

 Downstream primer: 5'-CCCAAGCTTGCATTCCGTCAATTAAAACAAG-3'.

(2) Using Bod/to raMfc genomic DNA as a template, PCR amplification of the target gene fragment was carried out at 94 ° C for 5 min _; (94 ° C for 45 s, 55 ° C for 45 s, 72 ° C for 100 s, 35 cycles); 72 °C, 10 min. The purified and amplified plasmid pTrc99a-TMft was digested with H'«i/III and ligated, respectively, to obtain the recombinant plasmid pTrc99a-w3⁄4^-pd. A single double digestion map of the recombinant plasmid pTrc99a-mi*-pd is shown in FIG.

3. The competent strain of NZN 111 strain which has been knocked out of the phosphoenolpyruvate carboxylase (PPC) gene before the plasmid pT _rC 99 _a - ift-p was introduced, and the positive transformation was obtained. The child is Escherichia coli BA206.

 Example 4

 This example illustrates the construction of an expression plasmid co-expressing phosphoenolpyruvate carboxylase and pyruvate carboxylase to restore the ability of the recombinant strain to metabolize xylose under anaerobic conditions, and the strain Escherichia coli BA207 was obtained.

 1. The strain lacking lactate dehydrogenase gene (WM) and pyruvate formate lyase gene pflB was used as the starting strain, and the phosphoenolpyruvate carboxylase (PPC) gene was knocked out. At the same time, the competent strains of WM, pflB and PPC were lacking (same as in Example 1).

 2. Constructing an expression plasmid co-expressing phosphoenolpyruvate carboxylase and pyruvate carboxylase, the process comprising:

(1) synthesizing primers with H m cleavage sites upstream and downstream,

 Upstream primer: 5,- CCCAAGCTTATGAACTCAGTTGATTTGACCG -3 ';

 Downstream primer: 5'-CCCAAGCTTGCATTCCGTCAATTAAAACAAG-3'.

(2) PCR amplification of the target gene fragment using BadZto ^ Mfo genomic DNA as a template, the reaction conditions are: 94 ° C, 5 min _; (94 °C 45 s, 55 °C 45 s, 72 °C 100 s, 35 cycles); 72 °C, 10 min. The purified pd gene and the expression plasmid pTrc99a-/?yc were digested with H^ffll and ligated, respectively, to obtain the recombinant plasmid pTrc99a-ro (^d. Recombinant plasmid pTrc99a-/^c-/7 The identification map is shown in Figure 11.

 3. Except for the apramycin resistance before introduction of the plasmid pTrc ^-ro -prtt, the competent state of the NZN 111 strain having the phosphoenolpyruvate carboxylase (PPC) gene knocked out, and the obtained positive transformant is For Escherichia coll BA207.

 Example 5

 This example illustrates the comparison of the acidogenic ability of the newly constructed recombinant Escherichia coli BA204 of Example 1 with the starting strain Escherichia coli NZNl l1.

E. coli _e n' _C /u' _fl BA204 can efficiently utilize xylose fermentation and accumulate a large amount of succinic acid. It adopts a two-stage fermentation method, which is characterized by 1% (v/v) inoculum and access from the cryopreservation tube. In the flask, when the aerobic cultured cells OD _{6 (K)} to 0.4 to 0.6 were induced with 0.3 mM IPTG to OD _6Q . When it is around 3, it is transferred to the serum bottle for anaerobic fermentation according to the inoculation amount of 10%, and fermentation is carried out for 48 hours.

 The aerobic phase medium is: LB + Amp (ampicillin 50 g / mL).

 The anaerobic phase medium was: LB + xylose (20 g/L) + basic magnesium carbonate 0.48 g + Amp (ampicillin 50 g / mL) ten 0.3 mM IPTG.

 The fermentation results are shown in Table 1.

 Table 1 Comparison of the results of fermentation of acid produced by Escherichia coli BA204 and starting strains

 Note: ND means not detected.

 Example 6

 This example illustrates the comparison of the newly constructed recombinant Escherichia coli BA204 with the fermentation ability of the starting strain Escherichia coli NZNl l1.

Escherichia coli BA204 can efficiently utilize corncob hydrolysate fermentation and accumulate a large amount of succinic acid. It adopts a two-stage fermentation method, which is characterized by 1% (vΛ inoculum from the cryotube to the flask, when aerobic The cultured OD _{6 was} induced to 0.3 mM IPTG to about OD _{6 (K)} = 3 to about 0.4 to 0.6, and then transferred to a serum bottle for anaerobic fermentation at a dose of 10%, and fermented for 48 hours.

 The aerobic phase medium is: LB + Amp (ampicillin 50 g / mL).

 The anaerobic phase medium was: LB+ corncob hydrolysate (total sugar 20 g/L) + basic magnesium carbonate 0.48 g+Amp (ampicillin 50 g/mL) + 0.3 mM IPTG.

 The fermentation results are shown in Table 2.

 Table 2 Comparison of the results of fermentation of Escherichia coli BA204 with starting strains

0 7.02 16 ND ND ND ND ND ND

0 7.02 16 ND ND ND ND ND ND

BA204

 48 6.55 0 ND 14.4 0.5 ND 0.5 ND Note: ND means not detected.

 Example 7

 This example illustrates the comparison of the newly constructed recombinant Escherichia coli BA204 with the fermentation ability of the starting strain Escherichia coli NZN1 11.

Escherichia coli BA204 can efficiently ferment the straw straw hydrolysate and accumulate a large amount of succinic acid. The two-stage fermentation method is characterized by 1% (v/v) inoculum from the cryotube into the flask. When aerobic cultured cells OD _6Q . It was induced to OD _6Q with 0.3 mM IPTG to about 0.4 to _0.6 . When it is around 3, it is transferred to the serum bottle for anaerobic fermentation according to the inoculation amount of 10%, and fermentation is carried out for 48 hours.

 The aerobic phase medium is: LB + Amp (ampicillin 50 g / mL).

 The anaerobic phase medium was: LB + straw straw hydrolysate (total sugar 20 g / L) + basic magnesium carbonate 0.48 g + Amp (ampicillin 50 g / mL) + 0.3 mM IPTG.

 The fermentation results are shown in Table 3.

 Table 3 Comparison of the results of fermentation of Escherichia coli BA204 with starting strains

 Note: ND means not detected.

 Example 8

 This example illustrates the comparison of the newly constructed recombinant Escherichia coli BA204 with the fermentation ability of the starting strain Escherichia coli NZN1 11.

Escherichia coli BA204 can efficiently utilize the sugarcane residue hydrolyzate fermentation and accumulate a large amount of succinic acid. It adopts a two-stage fermentation method, which is characterized by 1% (vA inoculum is introduced into the flask from the cryotube, when aerobic The cultured OD _{6 was} induced to 0.3 mM to 0.3 mM IPTG to OD ₆ . When the concentration was about 3, 10% of the inoculum was transferred to the serum bottle for anaerobic fermentation, and fermentation was carried out for 48 h.

 The aerobic phase medium is: LB + Amp (ampicillin 50 g / mL).

 The anaerobic stage medium was: LB + bagasse hydrolysate (total sugar 20 g/L basic magnesium carbonate 0.48 g + Amp (ampicillin 50 g/mL) + 0.3 mM IPTG.

 The fermentation results are shown in Table 4.

 Table 4 Comparison of the results of fermentation of Escherichia coli BA204 with starting strains

注: ND表示未检测到,

Note: ND means not detected,

 Example 9

 This example illustrates the comparison of the newly constructed recombinant Escherichia coli BA205 with the fermentation ability of the original strain Escherichia coli NZN1 11.

E. coli cA _e n' _C /ui _CO « BA205 is able to efficiently utilize xylose fermentation and accumulate a large amount of succinic acid. It is a two-stage fermentation method characterized by a 1% (v/v) inoculum from the frozen storage tube. In a triangular flask, when the aerobic cultured OD _{6 (K) is} about 0.4 to _{0.6 and} induced with 0.3 mM IPTG to OD _6QQ = 3, the anaerobic fermentation is transferred to the serum bottle by 10% of the inoculum. Fermentation for 48 h.

 The aerobic phase medium is: LB + Amp (ampicillin 50 g / mL).

 The anaerobic phase medium was: LB + xylose (20 g/L) + basic magnesium carbonate 0.48 g + Amp (ampicillin 50 g / mL) ten 0.3 mM IPTG.

 The fermentation results are shown in Table 5.

 Note: ND means not detected.

 Example 10

 This example illustrates the comparison of the newly constructed recombinant Escherichia coli BA205 with the fermentation ability of the original strain Escherichia coli NZN1 11.

Escherichia coli BA205 can efficiently utilize corncob hydrolysate fermentation and accumulate a large amount of succinic acid. It adopts a two-stage fermentation method, which is characterized by 1% (v/v) inoculum and is inserted into the flask from the cryotube. When aerobic cultured cells OD ₆ . . When induced by 0.3 mM IPTG to about OD _{6 (K)} = 3 to about 0.4 to 0.6, 10% of the inoculum was transferred to a serum bottle for anaerobic fermentation, and fermentation was carried out for 48 hours.

 The aerobic phase medium is: LB + Amp (ampicillin 50 g / mL).

 The anaerobic phase medium was: LB+ corncob hydrolysate (total sugar 20 g/L basic magnesium carbonate 0.48 g+Amp (ampicillin 50 g/mL) + 0.3 mM IPTG.

 The fermentation results are shown in Table 6.

 Table 6 Comparison of the results of fermentation of Escherichia coli BA205 with starting strains

Note: ND means no _E is detected.

Example 11 This example illustrates the comparison of the newly constructed recombinant Escherichia coli BA205 with the fermentation ability of the original strain Escherichia coli NZN1 11.

Escherichia coli BA205 can efficiently ferment the straw straw hydrolysate and accumulate a large amount of succinic acid. The two-stage fermentation method is characterized by 1% (v/v) inoculum from the frozen tube into the flask. When aerobic cultured cells OD ₆ . _Q to 0.4 to 0.6 was induced to OD ₆ with 0.3 mM IPTG. _{When Q} = 3 or so, transfer to the serum bottle for anaerobic fermentation according to the inoculation amount of 10%, and ferment for 48 hours.

 The aerobic phase medium is: LB + Amp (ampicillin 50 g / mL).

 The anaerobic phase medium was: LB + straw straw hydrolysate (total sugar 20 g / L) + basic magnesium carbonate 0.48 g + Amp (ampicillin 50 g / mL) + 0.3 mM IPTG.

 The fermentation results are shown in Table 7.

 Table 7 Comparison of the results of fermentation of Escherichia coli BA205 with starting strains

 Note: ND means not detected.

 Example 12

 This example illustrates the comparison of the newly constructed recombinant Escherichia coli BA205 with the fermentation ability of the original strain Escherichia coli NZN1 11.

Escherichia coli BA205 can efficiently utilize the sugarcane residue hydrolyzate fermentation and accumulate a large amount of succinic acid. It adopts a two-stage fermentation method, which is characterized by 1% (vA inoculum from the cryotube into the flask, when aerobic The cultured OD _{6 was} induced to 0.3 mM to 0.3 mM IPTG to OD ₆ . When the concentration was about 3, 10% of the inoculum was transferred to the serum bottle for anaerobic fermentation, and fermentation was carried out for 48 h.

 The aerobic phase medium is: LB + Amp (ampicillin 50 g / mL).

 The anaerobic stage medium was: LB + bagasse hydrolysate (total sugar 20 g/L basic magnesium carbonate 0.48 g + Amp (ampicillin 50 g/mL) + 0.3 mM IPTG.

 The fermentation results are shown in Table 8.

 Table 8 Comparison of the results of fermentation of Escherichia coli BA205 with starting strains

 Note: ND means not detected.

 Example 13

This example illustrates the comparison of the newly constructed recombinant Escherichia coli BA206 with the fermentation ability of the original strain Escherichia coli NZN1 11. Escherichia coli c/i _e n'c/ 'ii _CO / BA206 can efficiently utilize xylose fermentation and accumulate a large amount of succinic acid, using a two-stage fermentation method, which is characterized by a 1% (v/v) inoculum from the frozen The storage tube is connected to the triangular flask. When the aerobic cultured bacterium OD _{6 (K) is} about 0.4~0.6 and induced with 0.3 mM IPTG to OD _6M = 3, the inoculum is 10% transferred to the serum bottle. Oxygen fermentation, fermentation for 48 h.

 The aerobic phase medium is: LB + Amp (ampicillin 50 g / mL).

 The anaerobic phase medium was: LB + xylose (20 g/L) + basic magnesium carbonate 0.48 g + Amp (ampicillin 50 g/mL) + 0.3 mM IPTG.

 The fermentation results are shown in Table 9.

 Table 9 Comparison of the results of fermentation of Escherichia coli BA206 with starting strains

 Note: ND means not detected.

 Example 14

 This example illustrates the comparison of the newly constructed recombinant Escherichia coli BA206 with the fermentation ability of the starting strain Escherichia coli NZN1 11.

Escherichia coli BA206 can efficiently utilize corncob hydrolysate fermentation and accumulate a large amount of succinic acid. It adopts a two-stage fermentation method, which is characterized by 1% (v/v) inoculum and is inserted into the flask from the cryotube. When aerobic cultured cells OD ₆ . . It was induced to OD ₆ with 0.3 mM IPTG to about 0.4 to 0.6. . When it is around 3, it is transferred to the serum bottle for anaerobic fermentation according to the inoculation amount of 10%, and fermentation is carried out for 48 hours.

 The aerobic phase medium is: LB + Amp (ampicillin 50 g / mL).

 The anaerobic phase medium was: LB + corn cob hydrolysate (total sugar 20 g/L) + basic magnesium carbonate 0.48 g + Amp (ampicillin 50 g/mL) ten 0.3 mM IPTG.

 The fermentation results are shown in Table 10.

 Note: ND means not detected.

 Example 15

 This example illustrates the comparison of the newly constructed recombinant Escherichia coli BA206 with the fermentation ability of the starting strain Escherichia coli NZN1 11.

Escherichia coli BA206 can efficiently ferment the straw straw hydrolysate and accumulate a large amount of succinic acid. It adopts a two-stage fermentation method, which is characterized by 1% (v/v) inoculum from the cryotube into the flask. Aerobic culture Body OD ₆ . _Q to 0.4 to 0.6 was induced to OD ₆ with 0.3 mM IPTG. . When it is around 3, it is transferred to the serum bottle for anaerobic fermentation according to the inoculation amount of 10%, and fermentation is carried out for 48 hours.

 The aerobic phase medium is: LB + Amp (ampicillin 50 g / mL).

 The anaerobic phase medium was: LB + straw straw hydrolysate (total sugar 20 g / L) + basic magnesium carbonate 0.48 g + Amp (ampicillin 50 g / mL) ten 0.3 mM IPTG.

 The fermentation results are shown in Table 11.

 Note: ND means not detected.

 Example 16

 This example illustrates the comparison of the newly constructed recombinant Escherichia coli BA206 with the fermentation ability of the starting strain Escherichia coli NZN1 11.

Escherichia coli BA206 can efficiently utilize the sugarcane residue hydrolyzate fermentation and accumulate a large amount of succinic acid. It adopts a two-stage fermentation method, which is characterized by 1% (vA inoculum is introduced into the flask from the cryotube, when aerobic The cultured OD _{6 was} induced to 0.3 mM to 0.3 mM IPTG to OD ₆ . When the concentration was about 3, 10% of the inoculum was transferred to the serum bottle for anaerobic fermentation, and fermentation was carried out for 48 h.

 The aerobic phase medium is: LB + Amp (ampicillin 50 g / mL).

 The anaerobic stage medium was: LB + bagasse hydrolysate (total sugar 20 g/L basic magnesium carbonate 0.48 g + Amp (ampicillin 50 g/mL) + 0.3 mM IPTG.

 The fermentation results are shown in Table 12.

 Note: ND means not detected.

 Example 17

 This example illustrates the comparison of the newly constructed recombinant Escherichia coli BA207 with the fermentation ability of the original strain Escherichia coli NZN1 11.

E. coli _e n' _C /u' _fl BA207 can efficiently utilize xylose fermentation and accumulate a large amount of succinic acid. It adopts a two-stage fermentation method, which is characterized by 1% (v/v) inoculum and access from the cryotube. In the flask, when the aerobic cultured cells OD _{6Q ()} to 0.4 to 0.6 were induced with 0.3 mM IPTG to OD _6Q . When it is around 3, it is transferred to the serum bottle for anaerobic fermentation according to the inoculation amount of 10%, and fermentation is carried out for 48 hours. The aerobic phase medium was: LB + Amp (ampicillin 50 g/mL).

 The anaerobic phase medium was: LB + xylose (20 g / L) + basic magnesium carbonate 0.48 g + Amp (ampicillin: 50 g / mL) + 0.3 mM IPTG.

 The fermentation results are shown in Table 13.

 Table 13 Comparison of the results of fermentation of Escherichia coli BA207 with starting strains

 Note: ND means not detected.

 Example 18

 This example illustrates the comparison of the newly constructed recombinant Escherichia coli BA207 with the fermentation ability of the original strain Escherichia coli NZN1 11.

Escherichia coli BA207 can efficiently utilize corncob hydrolysate fermentation and accumulate a large amount of succinic acid. It adopts a two-stage fermentation method, which is characterized by 1% (v/v) inoculum and is inserted into the flask from the cryotube. When aerobic cultured cells OD ₆ . _Q to 0.4 to 0.6 was induced to OD ₆ with 0.3 mM IPTG. . When it is around 3, it is transferred to the serum bottle for anaerobic fermentation according to the inoculation amount of 10%, and fermentation is carried out for 48 hours.

 The aerobic phase medium is: LB + Amp (ampicillin 50 g / mL).

 The anaerobic phase medium was: LB+ corncob hydrolysate (total sugar 20 g/L) decahydrate magnesium carbonate 0.48 g+Amp (ampicillin 50 g/mL) + 0.3 mM IPTG.

 The fermentation results are shown in Table 14.

 Note: ND means not detected.

 Example 19

 This example illustrates the comparison of the newly constructed recombinant Escherichia coli BA207 with the fermentation ability of the starting strain Escherichia coli NZNl l l.

Escherichia coli BA207 can efficiently ferment the straw straw hydrolysate and accumulate a large amount of succinic acid. It adopts a two-stage fermentation method, which is characterized by 1% (vA inoculum from the cryotube to the flask, when aerobic The cultured OD _{6 was} induced to 0.3% with IPTG of 0.3 mM to about OD ₆ . When _Q = 3, 10% of the inoculum was transferred to a serum bottle for anaerobic fermentation, and fermentation was carried out for 48 h.

 The aerobic phase medium is: LB + Amp (ampicillin 50 g / mL).

The anaerobic stage medium is: LB+ straw straw hydrolysate (total sugar 20 g/L) + basic magnesium carbonate 0.48 g+Amp (ammonium chloride) Mycin 50 g/mL) +0.3 mM IPTGc

 The fermentation results are shown in Table 15.

 Note: ND means not detected.

 Example 20

 This example illustrates the comparison of the newly constructed recombinant Escherichia coli BA207 with the fermentation ability of the starting strain Escherichia coli NZNl l l.

Escherichia coli BA207 can efficiently utilize the sugarcane residue hydrolyzate fermentation and accumulate a large amount of succinic acid. It adopts a two-stage fermentation method, which is characterized by 1% (vA inoculum is introduced into the flask from the cryotube, when aerobic The cultured OD _{6 was} induced to 0.3 mM to 0.3 mM IPTG to OD ₆ . When the concentration was about 3, 10% of the inoculum was transferred to the serum bottle for anaerobic fermentation, and fermentation was carried out for 48 h.

 The aerobic phase medium is: LB + Amp (ampicillin 50 g / mL).

 The anaerobic stage medium was: LB + bagasse hydrolysate (total sugar 20 g/L basic magnesium carbonate 0.48 g + Amp (ampicillin 50 g/mL) + 0.3 mM IPTG.

 The fermentation results are shown in Table 16.

Note: ND means no _ε detected

Claims

Rights request 1. A method for constructing a genetically engineered Escherichia coli bacteria that utilizes xylose metabolism to produce succinic acid, which is characterized by comprising the following steps: (1) Taking an Escherichia coli strain lacking lactate dehydrogenase gene and pyruvate formate lyase gene activity as the starting strain, knocking out the phosphoenolpyruvate carboxylase gene, and obtaining the feeling of lacking ldhA, pflB and PPC at the same time State strains; (2) Separately purify and amplify the phosphoenolpyruvate carboxykinase gene, or purify and amplify the phosphoenolpyruvate carboxykinase gene, and select from the malic enzyme gene, malate dehydrogenase gene or One of the three genes, pyruvate carboxylase gene, was constructed to overexpress phosphoenolpyruvate carboxykinase alone, or to overexpress phosphoenolpyruvate carboxykinase, and to select from malic enzyme, An expression plasmid for one of the three enzymes, malate dehydrogenase or pyruvate carboxylase; (3) Introduce the plasmid described in step (2) into the competent strain obtained in step (1) to obtain positive transformants; (4) Utilize the positive transformant of step (3) to overexpress phosphoenolpyruvate carboxykinase alone, or to overexpress phosphoenolpyruvate carboxykinase, as well as malic enzyme and malate dehydrogenase. Or one of the three enzymes, pyruvate carboxylase, to restore its ability to metabolize xylose under anaerobic conditions, and obtain genetically engineered bacteria that can utilize xylose to metabolize succinic acid.