WO2013086907A1

WO2013086907A1 - Genetic engineering strain for producing succinic acid by using glucose and method for producing acid by fermenting the strain

Info

Publication number: WO2013086907A1
Application number: PCT/CN2012/083891
Authority: WO
Inventors: 姜岷; 梁丽亚; 刘嵘明; 苟冬梅; 张常青; 马江锋; 陈可泉; 韦萍; 欧阳平凯
Original assignee: 南京工业大学
Priority date: 2011-12-13
Filing date: 2012-10-31
Publication date: 2013-06-20
Also published as: CN102533626A

Abstract

A genetic engineering strain for producing succinic acid by using glucose and a method for producing acid by fermenting the strain are provided. The genetic engineering strain for producing succinic acid is E.coli strain BA205, deposited as CCTCC NO: M2011447. The construction process comprises: using the E.Coli strain lacking activities of lactate dehydrogenase (LDH) gene and pyruvate formate lyase (PFL) gene as the original strain, knockouting phosphoenolpyruvate carboxylase (PPC) gene by using homologous recombination technology, and over co-expressing phosphoenolpyruvate carboxylated kinase gene and nicotinic acid phosphoribosyl transferase. The method for producing succinic acid using the genetic engineering strain employs two-stage fermentation. With the biomass being increased in aerobic stage, and with acid being produced by fermentation in anaerobic stage.

Description

Genetically engineered strain producing succinic acid by using glucose and fermenting acid producing method thereof

Technical field

The invention belongs to the field of bioengineering technology, and relates to a genetic engineering strain for producing succinic acid by using glucose and a method for fermenting acid production thereof, in particular to a recombinant strain of succinic acid which is highly efficient in utilizing glucose growth and fermented by the strain. The method of acid.

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 Anaewbiospirillum succiniciproducens, Actinobacillus succinogenes, Mannheimia succiniciproducens recombinant Corynebacterium glutamicum and recombinant Escherichia coli. 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.coU 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.

The existing construction of recombinant succinic acid recombinant Escherichia coli mainly includes key enzymes in the production pathway of inactivated byproducts (such as pyruvate formate lyase and lactate dehydrogenase), and enhances the enzyme in the succinic acid synthesis pathway (such as phosphoenol). The activity of pyruvate carboxylase) and exogenous introduction can lead to the synthesis of succinic acid enzymes (such as pyruvate carboxylase) to increase its glucose utilization rate and production rate. Among them, E. _CO / NZNl ll was simultaneously inactivated by pyruvate formate lyase and lactate dehydrogenase, NADH could not be regenerated into NAD ^{+ in time} , and the intracellular coenzyme NAD(H) imbalance was observed (NADH/NAD+ ratio More than 2), eventually the strain can not use glucose under anaerobic conditions. Its spontaneous mutant E. AFPll mutates the ptsG gene in the glucose-specific transport system, reduces the rate of NADH production in the EMP pathway, restores the NAD(H) balance, and enables the strain to utilize glucose under anaerobic conditions. And the product is mainly succinic acid. During the aerobic anaerobic two-stage fermentation of AFP111, the mass yield of succinic acid reaches 96%, and the production intensity is 1.21 gL^h^. Therefore, in the high-yield E. coli strain During the construction process, ensuring the balance of intracellular coenzyme NAD(H) is one of the key factors for the high yield of succinic acid in recombinant E. coli.

The biosynthesis and decomposition pathway of NAD(H) in Escherichia coli is shown in Figure 1. There are three main genes involved in its synthesis. (pncB, nadD, nadE), there are two major genes involved in catabolism (yjaD, yrfE), and NAD+ and NADH have more than 300 transformation reactions with each other. Related studies have shown that the use of DNA recombination technology to transform the NAD(H) biosynthetic pathway is an effective means to increase the total amount of NAD(H). San et al. (Metab Eng, 2002, 4: 238-247; Metab Eng, 2002, 4: 182-192) by overexpressing niacin to phosphoribosyl ribose during the study of the effects of cofactor regulation on E. coli metabolic flux distribution The total amount of NAD(H) in the kinase (NAPRTaseM吏 increased by 41.7%; Heuser et al. (Eng Life Sci, 2007, 7: 343-353) by overexpressing niacin-transphosphoryl ribokinase and NAD synthase, or simultaneously These two enzymes increased the total amount of intracellular NAD(H) by more than 2 times and applied it to the process of enzymatic conversion of (R)-methyl-3-hydroxybutylamine, making NAD(H) The amount is no longer a limiting factor, thus improving the efficiency of enzyme conversion. Numerous scientific practices have also proved that the use of fermentation regulation can effectively regulate the ratio of NAD(H) to NADH/NAD+, thereby effectively improving substrate utilization and product production. Level. Fermentation using Sciccha yces cerevisiae TMB3001 (Biotechnol Bioeng, 2002, 78: 172-178) and Fusarium oxysporum (J Biosci Bioeng, 2004, 97: 299-304. Enzyme Micro Technol, 2005, 36: 100-106) Adding acetoin as an exogenous electron in the process of producing ethanol from xylose Body, effectively increasing the intracellular content of NAD ^+, to improve the ethanol yield; San et al. (; Metab Eng, 2002, 4 : 182-192) during 1,2-propanediol production in E. coli using, found In the 0.1 h- ¹ dilution anaerobic culture system, the intracellular NADH/NAD+ ratio increased from 0.51 (gluconic acid) to 0.75 (glucose) and 0.94 (sorbitol) as the carbon source decreased. And the ratio of the central metabolic stream product ethanol (consumption of 2 mol NADH) to acetic acid (non-consumption of NADH) is 0.29, 1 and 3.62, respectively. Niacin phosphoribosyltransferase is the rate-limiting step enzyme in the synthesis of NAD(H) and ATP participation is required (see Figure 1).

In Escherichia coli, phosphoenolpyruvate produces oxaloacetate by phosphoenolpyruvate carboxylase, and no ATP is formed in this process, but in Bfld//^ subtiUs, phosphoenolpyruvate is The formation of oxaloacetate by phosphoenolpyruvate carboxylation kinase, during which ATP is produced, and Millard et al. overexpress E. coli ppc and pck in E. coli, and found that overexpression of ppc can make succinic acid As the main product of mixed acid fermentation, and the yield is 3.5 times higher than that of the original strain, the overexpression has no effect on the fermentation result, but in the / ψί·deficient strain, the overexpression can increase the yield of succinic acid. The prior patent application of the inventor team of the present application relates to a genetically engineered strain capable of producing succinic acid by using xylose fermentation, and carrying out the patent deposit of the strain, patent application number 201110380396.3, application date November 25, 2011, preservation of biological materials No. CCTCC NO : M2011207. This strain can efficiently utilize xylose to produce succinic acid, but cannot produce succinic acid by glucose fermentation.

In the absence of lactate dehydrogenase gene, pyruvate formate lyase gene and phosphoenolpyruvate carboxylase gene activity, and overexpressing phosphoenolpyruvate carboxylase kinase strain E. coli BA204 as the starting strain, After overexpressing niacin phosphoribosyltransferase, a genetically engineered strain capable of efficiently utilizing glucose growth and producing succinic acid is obtained. Summary of the invention

The object of the present invention is to provide a genetically engineered strain capable of efficiently utilizing glucose growth and producing succinic acid, and a method for constructing the same, and using the anaerobic fermentation of the strain to produce succinic acid, and the method for constructing the strain is simple and convenient, and the construction method is obtained. The fermentation method of the strain is simple and feasible, easy to industrialize, and has the purpose of strong acid production, thereby greatly reducing production cost and improving economic efficiency.

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

1. The present invention provides a strain of a genetically engineered succinic acid producing strain, which is classified as Escherichia coli BA205 (with the accession number CCTCC NO: M 2011447).

2. The method for constructing Escherichia coli BA205 according to the present invention, characterized in that a strain lacking lactate dehydrogenase (LDH) gene and pyruvate formate lyase (PFL) gene activity is used as a starting strain, The homologous recombination technique knocks out the phosphoenolpyruvate carboxylase (PPC) gene and overexpresses the phosphoenolpyruvate carboxylase and nicotinic acid phosphoribosyltransferase to obtain efficient glucose utilization. Escherichia coli BA205.

Further, the specific construction steps are as follows:

(1) E.raW NZNl ll strain lacking lactate dehydrogenase gene (WM) and pyruvate formate lyase gene pflB activity was used as the starting strain, and the phosphoenolpyruvate carboxylase (PPC) gene was knocked out. Obtaining competent strains lacking both WM, pflB and ppc;

(2) synthesizing a pair of primers with a cleavage site at the 5' end, and using the Bfld//^^brifc genomic DNA as a template to purify the amplified gene, and expressing the plasmid pTr _C 99a with the enzyme designed by the primer The restriction enzyme was digested with restriction enzymes and ligated to obtain the recombinant plasmid pTrc99a-pd;

(3) A pair of primers carrying the same restriction site at the 5' end were synthesized, and the recombinant plasmid pT _rC 99a-pd was constructed by using the E. coli K12 genomic DNA as a template to purify the amplified / cfi gene. Primer-designed cleavage site-consistent enzyme digestion, ligation to obtain recombinant plasmid

(4) introducing the recombinant plasmid pT _rC 99 _a -p -/ _Cj B into the competent strain obtained in the step (1) to obtain a positive transformant;

(5) Using the positive transformants of step (4) to co-express phosphoenolpyruvate carboxylase and nicotinic acid phosphoribosyltransferase to restore their ability to metabolize glucose under anaerobic conditions, and obtain efficient utilization of glucose metabolism. Succinic acid genetically engineered bacteria Escherichia coli BA205.

3. A method for producing succinic acid by fermentation of Escherichia coli BA205 according to the present invention, characterized in that a two-stage fermentation method is employed, the biomass is increased in the aerobic phase, and the acid is produced in the anaerobic phase.

Further, the specific steps are as follows.

Escherichia coli BA205 was inoculated into the aerobic fermentation medium in aerobic culture at a 1% (v/v) inoculum. When the oxygen cultured cells OD _{6 () Q} to 0.4 to 0.6 were induced with 0.3 mM IPTG to OD _{6 (K)} = 3, 10% of the inoculum was transferred to the anaerobic fermentation medium in the anaerobic fermentation medium.

The aerobic stage fermentation medium is a conventional medium for aerobic culture of E. coli produced in the prior art; the anaerobic stage fermentation medium is a succinic acid large intestine producing glucose as a carbon source. Fermentation medium for bacilli.

The beneficial effects of the present invention are as follows: a strain lacking lactate dehydrogenase gene, pyruvate formate lyase gene and phosphoenolpyruvate carboxylase gene activity, and overexpressing phosphoenolpyruvate carboxylase kinase BA204 is the starting strain, and after overexpressing nicotinic acid phosphoribosyltransferase, the genetically engineered bacteria capable of efficiently utilizing glucose growth and producing succinic acid can be obtained, which overcomes the defect that the original BA204 strain cannot utilize glucose, and enhances the adaptation range of the strain.

DRAWINGS

Figure 1 Biosynthesis and decomposition pathway of NAD(H) in Escherichia coli.

Figure 2 Construction map of the recombinant plasmid pTrc99a-p.

Figure 3. Construction map of recombinant plasmid pT c99a-pck-pncB.

Figure 4 Agarose gel electrophoresis identification of the PCR product pck.

Figure 5 Agarose gel electrophoresis identification of PCR product /rncfi.

Figure 6. Double restriction enzyme digestion map of recombinant plasmid pTrc99a-p.

Figure 7. Double restriction enzyme digestion map of recombinant plasmid pTrc99a-pck-pncB.

The microorganism of the present invention is classified as Escherichia coli BA205 (Escherichia coli BA205), and the preservation date is December 7, 2011. The depository unit is called the China Center for Type Culture Collection, referred to as CCTCC, and the address of the depository: China. Wuhan University Wuhan; Deposit No.: CCTCC NO: M 2011447.

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, purchased 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 ATCC 23857.

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

The originating strain E.coW NZNl l l ( CGSC#: 7726 ) of the present invention has two sources:

(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. The biological material is obtained free of charge; and the applicant 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 Chinese Patent Application No. 96198547.X, Application Date 1996.10.31, Authorization Date January 1, 2003, Authorization Publication No. CN1097632C.

Example 1

This example illustrates the knockout of the phosphoenolpyruvate carboxylase ppc gene in the starting strain NZN111 by homologous recombination technique to obtain a process for eliminating apramycin resistant strains.

1. Using LB medium, Escherichia coli NZN111 was incubated at 37 ° C under aerobic conditions to OD _6QQ = 0.4 to 0.6 to prepare an electroporation competent state.

2. Transfer plasmid pKD46 to 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.

3. 10 mM L-arabinose was added to the LB medium, and the plasmid pKD46 was induced to express the lambda recombinase at 30 ° C to prepare an electroporation competent state.

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 sequences were as follows:

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

5, -ATGAACGAACAATAT

GGGATCCGTCGACC-3 '.

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

5, -AGCACGAGGGTTTGC

GCTGGAGCTGCTTC-3 '.

Reaction system: Upstream and downstream primers with homology arms (100 pmol L) 0.5 μM each, template DNA (100 ng/μΐ^ 0.5 μ^, lOxbuffer 5 μ ^, dNTPs (10 mM) each 1 L; DMSO (100 %) 2.5 μΙ·, Pyrobest DNA polymerase (2.5 U^L) 1 μΙ·, ddH ₂ 0 36/35.5 μΙ·, 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 which has been induced to express λ recombinase, and coated on LB plate with apramycin to screen positive recombinants, and PCR identification, electropherogram As shown in Figure 3.

6. The positive recombinant is made into a competent state, and then pCP20, which can induce the expression of FLP recombinase, can be induced to amplify the apramycin resistance after heat-expressing FLP recombinase at 42 °C. Using a pair of plates, parallel-spotting, strains capable of growing on non-resistant plates, but not growing on resistant plates, were extremely resistant to knockout. Example 2

This example illustrates the construction of an expression plasmid for over-expression of phosphoenolpyruvate carboxylase and nicotinic acid phosphoribosyltransferase to restore the ability of the recombinant strain to metabolize glucose under anaerobic conditions, and to obtain a strain.

1. Construction of the pTrc99a-p plasmid, the process comprising:

(1) synthesizing primers with restriction sites for ^cl and Xb d,

Upstream primer: 5'- CGAGCTCATGAACTCAGTTGATTTGACCG -3Ό

Downstream primer: 5'- GCTCTAGAGCATTCCGTCAATTAAAACAAG -3Ό

(2) PCR amplification of the target gene fragment using Bfld/Z ^ fc genomic DNA as template. The reaction conditions were: 94 ° C, 5 min; (94 ° C 45 s, 53 ° C 45 s, 72 ° C 100 s , 35 cycles); 72 °C, 10 min. After purifying the amplified gene, the expression plasmid was digested with pTrc99a and ligated with ^c MTbfll, respectively, to obtain the recombinant plasmid pTrc99a-p.

2. Excessive expression of an expression plasmid for phosphoenolpyruvate carboxylase and niacin phosphoribosyltransferase, the process comprising:

(1) Synthesizing primers with Hind III restriction sites in both upstream and downstream primers.

Upstream primer: 5'- CCCAAGCTTATGACACAATTCGCTTCTCCTG-3

Downstream bow I: 5 ' -CCCAAGCTTCACTTGTCCACCCGTAAATGG-3 '.

(2) The target gene fragment was amplified by PCR using E. coli K12 series as template. The reaction conditions were: 94 ° C, 5 min; (94 °C 45 s, 55 °C 45 s, 72 °C 1 min, 35 Cycle); 72 ° C, 10 min. After purifying the amplified / cfi gene, the plasmid ^rc99a-pck Hind III was digested and ligated to obtain the recombinant plasmid pTrc99a-pd -/S.

3,

-/^ί·β was introduced into Example 1 while lacking the competent strains of WM, ρ/Ζβ and /, and the obtained positive transformant was the newly constructed strain coli BA205 of the present invention.

Example 3 This example illustrates the overexpression of a newly constructed recombinant colonic strain coli BA205 and Example 1 obtained. The comparison of the total amount of NAD(H) and the ratio of NADH/NAD+ in the apramycin-resistant strains, and the comparison of the sugar-consuming and acid-producing abilities of the two fermentation processes.

When the plasmid pTn^-pck-pncB was introduced, the over-expression of phosphoenolpyruvate carboxylase and nicotinic acid phosphoribosyltransferase in the apramycin-resistant strain restored the redox balance of the recombinant bacteria under anaerobic conditions. The total amount of NAD(H) is significantly improved, and the ability to metabolize glucose under anaerobic conditions is also restored, while the main product is succinic acid, which is free of accumulation of formic acid and lactic acid.

Escherichia coli BA205 was inoculated into the aerobic fermentation medium in aerobic culture at a 1% (v/v) inoculum. When the aerobic cultured cells were OD _{6 (K)} to 0.4 to 0.6 with 0.3 mM IPTG. When induced to OD _{6 (K)} = 3, 10% of the inoculum was transferred to the anaerobic fermentation medium in the anaerobic fermentation medium.

The aerobic stage fermentation medium described above is a conventional medium for aerobic culture of Escherichia succinate in the prior art; the medium in this embodiment is: LB medium.

The anaerobic stage fermentation medium is a fermentation medium for producing succinic acid Escherichia coli which uses glucose as a carbon source. The medium in this example is as follows.

The anaerobic serum bottle fermentation medium was: LB + glucose (20 g / L) + basic magnesium carbonate 0.48 g + + Amp (ampicillin 5 (^ g / mL) + 0.3 mM IPTG + 0.5 mM NA (nicotinic acid).

The results of various parameters after anaerobic serum bottle culture are shown in Table 1.

Table 1 Results of various parameters after anaerobic serum bottle culture

Time DCW NAD+ NADH sugar-consuming succinic acid strain

(h) (g/L) ( g/L )

(g/L) (mmol/g) (mmol/g)

The apramycin resistant strain of Example 1 48 0. 444 3.004 1.924 0 0

E.coli BA205 48 3.214 9.517 2.8 12 15 8

Claims

Rights request

1. A strain of succinic acid genetically engineered bacteria, classified as Escherichia coli BA205 (Escherichia coli BA205), with a registration number of CCTCCM 2011447.

The method for constructing Escherichia coli BA205 according to claim 1, characterized in that the strain lacking the lactate dehydrogenase gene and the pyruvate formate lyase gene activity is used as a starting strain, and the homologous strain is used. Recombinant technology knocks out the phosphoenolpyruvate carboxylase gene and overexpresses phosphoenolpyruvate carboxylase and niacin phosphoribosyltransferase to obtain efficient use of glucose to produce succinic acid Heisella BA205.

3. The method of constructing Escherichia coli BA205 according to claim 1, wherein the specific construction steps are as follows:

(1) The E NZNlll strain lacking the lactate dehydrogenase gene and the pyruvate formate lyase gene activity was used as the starting strain, and the phosphoenolpyruvate carboxylase gene was knocked out to obtain the simultaneous lack of ΜΜ, /^β and PPC. Competent strain;

(2) A pair of primers with a cleavage site at the 5' end were synthesized, and the amplified gene was purified by using Bad/Z ^ brifc genomic DNA as a template. The expression plasmid pTr _C 99a was digested with the primer. Site-consistent enzyme digestion, ligation to obtain recombinant plasmid pTrc99a-pd;

(3) A pair of primers with the same restriction site at the 5' end were synthesized, and the recombinant plasmid pTrc99a-p was constructed with primers after purification of the amplified pncB gene using E. coli K12 genomic DNA as a template. The enzyme cleavage site is consistent with the enzyme digestion and ligation to obtain the recombinant plasmid pTrc99a-p-/S;

(5) Using the positive transformants of step (4) to co-express phosphoenolpyruvate carboxylase and niacin phosphoribosyltransferase to restore their ability to metabolize glucose under anaerobic conditions, and obtain efficient utilization of glucose metabolism. Succinic acid genetically engineered bacteria Escherichia coli BA205.

A method for producing succinic acid by fermentation of Escherichia coli BA205 according to claim 1, characterized in that a two-stage fermentation method is employed, the biomass is increased in the aerobic phase, and the acid is produced in the anaerobic phase.

5. The method according to claim 4, characterized in that Escherichia coli BA205 is inoculated into the aerobic fermentation medium in aerobic culture at a 1% (v/v) inoculum, when the aerobic culture cells are cultured. When OD _6QQ to 0.4 to _{0.6 was} induced with 0.3 mM IPTG to OD _{6 (K)} = 3, 10% of the inoculum was transferred to the anaerobic fermentation medium in the anaerobic fermentation medium.

6. The method according to claim 4, wherein the anaerobic stage fermentation medium is a fermentation medium for producing Succinic acid succinate using glucose as a carbon source.