CN104046577A

CN104046577A - Gene engineering bacterium for producing malic acid and construction and application thereof

Info

Publication number: CN104046577A
Application number: CN201410129479.9A
Authority: CN
Inventors: 姜岷; 任心怡; 马江锋; 张敏; 朱婧; 李凤
Original assignee: Nanjing Tech University
Current assignee: Nanjing Tech University
Priority date: 2014-04-01
Filing date: 2014-04-01
Publication date: 2014-09-17

Abstract

The invention provides a malic acid producing genetic engineering bacterium Escherichia coli BA043, which is classified and named as Escherichia coli BA043 (Escherichia coli BA 043), and the preservation registration number is CCTCCNO: m2014034. The invention also provides a construction method of the strain and a method for producing malic acid by fermentation, which leads recombinant escherichia coli to grow by utilizing glucose metabolism and reduce the generation of byproduct pyruvic acid by knocking out or inactivating fumarase and fumaric reductase and co-expressing exogenous pyruvate carboxylase and nicotinic acid phosphoribosyltransferase in excess so as to greatly improve the yield and production intensity of malic acid.

Description

Malic acid gene engineering and construction and application thereof are produced in one strain

Technical field

The invention belongs to technical field of bioengineering, relate to a kind of produce oxysuccinic acid Recombinant organism strain and structure and application, be specifically related to a kind of structure of glucose production malic acid gene engineering Escherichia coli BA043 and method of producing oxysuccinic acid thereof efficiently utilized.

Background technology

Oxysuccinic acid, is again 2-hydroxy-butanedioic acid, is one of tricarboxylic acid cycle member in organism, and molecular formula is C ₄h ₆0 ₅, in molecule, have a chiral carbon atom, thereby have 3 kinds of existence forms, be i.e. D-malic acid, L MALIC ACID and DL-oxysuccinic acid.L-type oxysuccinic acid is extensively present in occurring in nature, and the oxysuccinic acid of chemosynthesis is the mixture of L-type and D-type.Because L MALIC ACID can directly be absorbed by organism, and D-malic acid is non-physiologically active, need to, through the oxydo-reductase effect of the racemase of liver and kidney, change into L MALIC ACID and just can be utilized, become a kind of demand therefore produce the oxysuccinic acid of single opticity.L MALIC ACID is colourless crystallization or powder, frank tart flavour slightly with irritating, and sour intensity is equivalent to 1.2 times of left and right of citric acid, relative density 1.595,100 DEG C of fusing points, moisture-sensitive, soluble in water, be slightly soluble in ethanol and ether, be heated to 180 DEG C and can lose water and become fumaric acid or toxilic acid.L MALIC ACID is because its special physicochemical property are widely used in food, medicine, chemical industry and other industry, and has irreplaceability.

Because L MALIC ACID is extensively present in fruits and vegetables, the oxysuccinic acid obtaining in early days extracts from fruit, but this method output is limited, is not suitable for industrial production.The production method of oxysuccinic acid has chemical synthesis, direct fermentation (claiming again one-step fermentation), two-step fermentation, enzyme transforming process and direct extraction method, or adopts coproduction L MALIC ACID and lactic acid and immobilized cell coproduction L MALIC ACID and aspartic acid production technique.Chemical synthesis technology maturation, cost is lower, but cannot obtain the L MALIC ACID of single opticity, has limited its application at food and medicine trade, and meanwhile, this method is higher to equipment requirements, consumes fossil energy, to environment.What research was more at present is to utilize enzyme transforming process and microbe fermentation method.Enzyme transforming process is to utilize FURAMIC ACID that fumaric acid is converted into oxysuccinic acid, but this method yields poorly, cost is high.By contrast, fermentation rule has more advantage.If be used for the microbial host mould that fermentation prepares oxysuccinic acid, the wherein flavus Aspergillus flavus research object of attaching most importance to.According to report, Takao etc. first use rhizopus arrhizus (Rhizopus ahirrizus) or zhizopchin (Rhizopu chinensis) that saccharine material is changed into fumaric acid, and then with one of Pichia membranaefaciens (Pichamembrane aefaciens faciens), proteus vulgaris (Proteus vulgaris) and paecilomyces varioti (Paeciomyces varioti), fumaric acid is changed into L MALIC ACID.Domesticly also prepare aspect L MALIC ACID and study for a long period of time at fermentation method, as people's (Food Additives Used in China such as Shandong food friend's ferment engineering experiment chamber Liu Jianjuns, 2003, 3:52～56) be devoted to for many years direct fermentation and produce the research of L MALIC ACID, and separation screening from soil, a mutagenic obtained strain directly utilizes saccharine material to produce the superior strain Aspergillus flavus HA5800 of L MALIC ACID, through condition optimizing, 7000 liters of fermentor tanks produce acid and reach 8.68%, glucose acid invert ratio reaches 83.5%, fermentation period 112 hours, for L MALIC ACID industrialization is laid a good foundation.

Utilize fermentation method to produce oxysuccinic acid and can utilize renewable resources to generate oxysuccinic acid, not only break away from the dependence to petrochemical industry resource, cost is low, pollute little, environmental friendliness, and can absorb during the fermentation a large amount of CO ₂, opened up the new way that greenhouse gases carbonic acid gas utilizes.Because intestinal bacteria are because of plurality of advantages such as genetic background is clear, growth quick, culture condition is simple, security good, easy transformations easy to operate, successfully produce as the product such as lactic acid, succsinic acid, therefore there is the realistic feasibility of realizing oxysuccinic acid good quality and high output suitability for industrialized production through transformation.

Improving colibacillary fermentation and acid ability has multiple approach, comprises the several different methods such as bacterial strain screening, physics and chemistry mutagenesis, genetic engineering means, fermentation control.The people such as Soon Ho Hong have reported the method for producing succsinic acid genetic engineering bacterium that builds, principle is pyruvate formate-lyase gene (pflB) and the lactate dehydrogenase gene (ldhA) knocking out in wild intestinal bacteria, reduce and even do not produce secondary thing formic acid, lactic acid, acetic acid and ethanol etc., make more metabolism stream flow to succsinic acid (Biotechnol Bioeng, 2001,74:89～95).On this basis, if the FUM in downstream leg and FRD are knocked out, oxysuccinic acid capable of blocking flows to fumaric acid and then generates the reaction of succinic acid, the form accumulation that product can oxysuccinic acid.

The granted patent ZL200810023410.2(denomination of invention previous according to this team is " the new high yield oxysuccinic acid engineering bacteria building and the method for producing oxysuccinic acid thereof "), obtain a strain and knock out three of pflB, ldhA, fumB simultaneously and strike bacterial strain Escherichia coli JM127.Under anaerobic, this bacterial strain reverts to the approach of fumaric acid and is controlled from oxysuccinic acid, but after genetic modification, the required coenzyme NAD of this bacterial strain metabolism ⁺regeneration be subject to impact, consumption sugar ability and the key enzyme work of several places of bacterial strain are decreased, limited to a certain extent the acid producing ability of bacterial strain.

The biosynthesizing of NAD in intestinal bacteria (H) and decomposition approach as shown in Figure 2, relate to its synthetic gene and mainly contain three (pncB, nadD, nadE), and relate to catabolic gene and mainly contain two (yjaD, yrfE), and NAD ⁺reach more than 300 with NADH conversion reaction each other.Correlative study shows, utilizes DNA recombinant technology to transform the effective means that NAD (H) biosynthetic pathway is raising NAD (H) total amount.The people such as San (Metab Eng, 2002,4:238-247; Metab Eng, 2002,4:182-192) in research cofactor regulates and controls the influence process of Metabolism of E. coli flow point cloth, make NAD in born of the same parents (H) total amount improve 41.7% by overexpression nicotinic acid phosphoribosyl transferase; The people such as Heuser (Eng Life Sci, 2007,7:343-353) by overexpression nicotinic acid phosphoribosyl transferase and NAD synthase, or express this two enzymes simultaneously, NAD (H) total amount in bacterial strain born of the same parents has been improved more than 2 times, and apply it in synthetic (R)-methyl-3-hydroxyl butylamine process of enzymatic conversion, make the amount of NAD (H) no longer become limiting factor, thereby improved the efficiency of enzymatic conversion.E.coli JM127 due to while inactivation pyruvate formate-lyase, serum lactic dehydrogenase and FURAMIC ACID, NADH can not be regenerated as NAD in time ⁺, cause the imbalance (NADH/NAD of coenzyme NAD (H) in born of the same parents ⁺ratio exceedes 2), finally cause bacterial strain under anaerobic condition can not utilize glucose.Therefore,, in high yield oxysuccinic acid coli strain building process, guarantee that the balance of coenzyme NAD (H) in born of the same parents is one of key factor of the high succinic acid-producing of recombination bacillus coli.

In addition, owing to having lacked pyruvate formate-lyase gene pflB and lactate dehydrogenase gene ldhA, cause a large amount of accumulation of pyruvic acid, the transhipment of feedback inhibition PTS sugar transport system to glucose, make thalline under anaerobic can not utilize glucose, and growth is suppressed.The people such as Vemuri introduce the pyc gene in R.etli in NZN111, and result pyruvic acid has reduced 3 times, and succinic acid output has increased by 52%.The people such as Yue Fang side are the pyc gene in overexpression subtilis in JM1307, and the output of result succinic acid has increased by 1.9 times, if the amount of pyruvic acid also has and can overcome deficiency from molecular level simultaneously, the growth that recovers bacterial strain consumes sugared ability.Introduce the pyruvate carboxylase gene in B.subtilis by external source, made the pyruvic acid of accumulation flow to oxaloacetic acid, removed the feedback inhibition phenomenon of pyruvic acid, and then accumulation oxysuccinic acid.Pyruvate carboxylase PYC can catalysis pyruvic acid fixation of C O ₂generate oxaloacetic acid, this enzyme of overexpression can increase TCA reduction arm carbon circulation, thereby is expected to improve the output of oxysuccinic acid.

Summary of the invention

The object of the invention is to the deficiency for existing bacterial strain, engineering strain and construction process thereof that a kind of energy high yield oxysuccinic acid is provided, can further reduce the generation of downstream product, recovers the utilization to glucose, reduce the accumulation of pyruvic acid, and utilize this strain fermentation to produce oxysuccinic acid.

In order to realize the object of the invention, the present invention by the following technical solutions:

One, the invention provides a strain and produce malic acid gene engineering bacterial strain, its Classification And Nomenclature is colon bacillus BA043(Escherichia coli BA043), its preservation registration number is CCTCC NO:M2014034.

Two, the present invention provides the construction process of above-mentioned colon bacillus BA043 simultaneously, the bacterial strain NZN111 that it is characterized in that lacking lactate dehydrogenase gene (ldhA), pyruvate formate-lyase gene (pflB) is starting strain, utilize homologous recombination technique further to knock out fumarase gene (fumB) and fumaric reductase gene (frdABCD) activity, and coexpression external source pyruvate carboxylase gene (pyc) and nicotinic acid phosphoribosyltransferase gene (pncB), obtain producing the colon bacillus BA043 of oxysuccinic acid.

Further, described concrete construction step is as follows:

(1) taking the bacterial strain E.coli NZN111 that lacks lactate dehydrogenase gene (ldhA), pyruvate formate-lyase gene (pflB) as starting strain, knock out fumarase gene (fumB) and fumaric reductase gene (frdABCD) wherein, lacked the competence bacterial strain of ldhA, pflB, fumB and frdABCD simultaneously;

(2) purifying amplifies pyruvate carboxylase gene (pyc), builds the expression plasmid that obtains expressing pyruvate carboxylase;

(3) purifying amplifies nicotinic acid phosphoribosyltransferase gene (pncB), is connected on the described recombinant plasmid of step (2), builds the expression plasmid that obtains excessive coexpression pyruvate carboxylase and nicotinic acid phosphoribosyltransferase;

(4) plasmid obtaining in step (3) is imported to the competence bacterial strain obtaining in step (1), obtain positive transformant;

(5) the excessive coexpression pyruvate carboxylase of positive transformant and the nicotinic acid phosphoribosyltransferase that utilize step (4) to obtain, recover its under anaerobic ability of metabolizable glucose, simultaneously, reduce the accumulation of pyruvic acid, obtain utilizing glucose metabolism to produce malic acid gene engineering colon bacillus BA043.

Three, utilize the method for colon bacillus BA043 fermentative production oxysuccinic acid of the present invention, it is characterized in that adopting two stage fermentation modes, the aerobic stage is improved biomass, anaerobic stages fermentation and acid, and further, concrete steps are as follows:

Colon bacillus BA043 is accessed triangular flask from cryopreservation tube by 1% (v/v) inoculum size, when aerobic is cultivated thalline OD ₆₀₀to 3～4 left and right, concentrate 3 times to OD ₆₀₀=9 left/right rotations are connected in the serum bottle of 30mL LB, add the IPTG of 0.3mM as inductor simultaneously, and 0.5mM nicotinic acid is as NAD (H) substrate, and 30 DEG C, 170rpm, fermentation 24h.

Fermentation results shows that the recombination bacillus coli Escherichia coli BA043 building can accumulate oxysuccinic acid in a large number, fumaric acid, succinic acid accumulate less, compare starting strain, the accumulation of Escherichia coli BA043 oxysuccinic acid increases, and by product output of pyruvic acid reduces.

Meanwhile, above-mentioned aerobic stage fermentation substratum is that in prior art, aerobic is cultivated the colibacillary conventional medium of succinic acid-producing; Anaerobic stages fermention medium is the succinic acid-producing intestinal bacteria fermention medium taking glucose as carbon source.

Beneficial effect of the present invention is to utilize genetic engineering means, intestinal bacteria is carried out to the strengthening of target product approach, and making metabolism stream is how to flow to oxysuccinic acid.The strain fermentation method simple possible that structure obtains, while utilizing this bacterial strain to carry out two stage fermentation experiments, has good effect to accumulation oxysuccinic acid.

Brief description of the drawings

In Fig. 1 intestinal bacteria, contain the anaerobism mixed acid fermentation approach of PYC approach.

The biosynthesizing of NAD in Fig. 2 intestinal bacteria (H) and decomposition approach.

The electrophoresis qualification figure of Fig. 3 linear DNA fragment.

The electrophoresis qualification figure (knocking out FUM) of Fig. 4 homologous recombination positive recombinant.

The electrophoresis qualification figure of Fig. 5 linear DNA fragment.

The electrophoresis qualification figure (knocking out FRD) of Fig. 6 homologous recombination positive recombinant.

The agarose gel electrophoresis figure of Fig. 7 PCR product pyc.

The structure collection of illustrative plates of Fig. 8 recombinant plasmid pTrc99a-pyc.

The single endonuclease digestion qualification figure of Fig. 9 recombinant plasmid pTrc99a-pyc.

The agarose gel electrophoresis figure of Figure 10 PCR product pncB.

The structure collection of illustrative plates of Figure 11 recombinant plasmid pTrc99a-pyc-pncB.

The double digestion qualification figure of Figure 12 recombinant plasmid pTrc99a-pyc-pncB.

The Classification And Nomenclature of microorganism of the present invention is colon bacillus BA043(Escherichia coli BA043), preservation date is on January 19th, 2014, depositary institution's full name is Chinese Typical Representative culture collection center, referred to as CCTCC, address is: China. Wuhan. and Wuhan University, deposit number: CCTCC NO:M2014034.

Embodiment

Further illustrating the present invention below in conjunction with drawings and Examples, is only the explanation as typical case, is not limitation of the invention.

The explanation in the source of biomaterial of the present invention:

1, plasmid source:

(1) pIJ773: obtain from the azure professor of Shao of Nanjing Normal University place;

(2) pKD46, pCP20: purchased from Introvegen company;

(3) pTrc99a: purchased from Introvegen company;

2, genomic templates source: GENE BANK.

3, starting strain: there are two places in the source of the competence bacterial strain of E.coli NZN111:

（1）Biotechnol?Bioeng,2001,74:89～95。Applicant is first by finding the above-mentioned document source of this biomaterial, and to have contacted utterer be the David P.Clark professor of Univ Chicago USA, and its this biomaterial of gifting of mail requests, and freely obtained this biomaterial; And applicant ensured in 20 years, to provide this biomaterial to the public from the application's day;

(2) this biomaterial also discloses and obtains the authorization in the patent documentation of Chinese patent (application number 96198547.X, applying date 1996.10.31 authorize day on January 1st, 2003, Granted publication CN1097632C).

4, the design of primer and synthetic: designed, designed outer Si Rui covered with gold leaf biotech company are synthetic.

Embodiment 1

The present embodiment explanation utilizes homologous recombination technique to knock out fumarase gene fumB in parent intestinal bacteria NZN111, the process of the apramycin resistant strain that is eliminated.

Utilize homologous recombination technique to knock out FURAMIC ACID (FUM) gene:

1, utilize LB substratum, under 37 DEG C, aerobic conditions, cultivate intestinal bacteria NZN111 to OD ₆₀₀=0.4～0.6, be prepared into electricity and turn competence.

2, plasmid pKD46 electricity is proceeded to competent intestinal bacteria NZN111, electric shock condition is: 200 Ω, 25 μ F, electric shock voltage 2.3kV, electric shock time 4～5ms.After electric shock, rapidly thalline is added to the SOC substratum of precooling 1mL, the LB culture medium flat plate of coating band penbritin (Amp) after 150r/min, 30 DEG C of cultivation 1h filters out positive transformant NZN111 (pKD46).

3, in LB substratum, add the L-arabinose of 10mM, at 30 DEG C, induce plasmid pKD46 to give expression to λ recombinase, make electricity and turn competence.

4, taking both sides, the apramycin resistance gene with FRT site is template, utilizes High fidelity PCR amplification system, taking plasmid pIJ773 as template, and designs the amplimer of two ends with FUM homologous fragment, amplifies linear DNA homologous fragment, and primer sequence is as follows:

Upstream band homology arm primer H1-P1. underscore is homologous fragment:

5'- CGGCACGCCATTTTCGAATAACAAATACAGAGTTACAGGCTGGAAG CTATTCCGGGGATCCGTCGACC-3’

Downstream band homology arm primer H2-P2, underscore is homologous fragment:

5'- TTACTTAGTGCAGTTCGCGCACTGTTTGTTGACGATTTGCTGGAAGA ATGTAGGCTGGAGCTGCTTC-3’

Reaction system: the each 0.5 μ L of upstream and downstream primer (100pmol/ μ L) with homology arm; Template DNA (100ng/ μ L) 0.5 μ L; 10 × buffer5 μ L; The each 1 μ L of dNTPs (10mM); DMSO (100%) 2.5 μ L; Pyrobest archaeal dna polymerase (2.5U/ μ L) 1 μ L; ddH ₂o36 μ L; Cumulative volume 50 μ L.

Reaction conditions: 94 DEG C, 2min; (94 DEG C, 45sec; 50 DEG C, 45sec; 72 DEG C, 90sec; 10 circulations); (94 DEG C, 45sec; 55 DEG C, 45sec; 72 DEG C, 90sec; 15 circulations); 72 DEG C, 5min.

The qualification of linear DNA fragment is as Fig. 3.

5, electricity turns the linear DNA fragment NZN111 of abduction delivering λ recombinase (PKD46) competence extremely, and coats with the LB flat screen of apramycin and select positive recombinant, and has carried out PCR qualification, and electrophorogram as shown in Figure 4.

6, positive recombinant be prepared into pour into after competence can abduction delivering FLP recombinase plasmid pCP20, after FLP recombinase is expressed in 42 DEG C of heat shocks, can eliminate apramycin resistance.Utilize pair of plates, carry out parallel point sample, can on non-resistant flat board, grow, but the bacterium that can not grow in resistant panel is the bacterial strain that knocks out resistance, is lacked ldhA simultaneously, the competence bacterial strain of pflB and fumB.

Embodiment 2

The present embodiment explanation parent intestinal bacteria NZN111 is knocked on the basis of fumarase gene fumB, utilizes homologous recombination technique further to knock out fumaric reductase gene frdABCD, the process of the apramycin resistant strain that is eliminated.

Utilize homologous recombination technique to knock out fumaric reductase (FRD) gene:

1, utilize LB substratum, under 37 DEG C, aerobic conditions, cultivate and lack ldhA, the intestinal bacteria of pflB and fumB are to OD ₆₀₀=0.4～0.6, be prepared into electricity and turn competence.

2, plasmid pKD46 electricity is proceeded in the competence bacterial strain of above-mentioned steps 1, electric shock condition is: 200 Ω, 25 μ F, electric shock voltage 2.3kV, electric shock time 4～5ms.After electric shock, rapidly thalline is added to the SOC substratum of precooling 1mL, the LB culture medium flat plate of coating band penbritin (Amp) after 150r/min, 30 DEG C of cultivation 1h filters out positive transformant.

4, taking both sides, the apramycin resistance gene with FRT site is template, utilizes High fidelity PCR amplification system, taking plasmid pIJ773 as template, and designs the amplimer of two ends with FRD homologous fragment, amplifies linear DNA homologous fragment, and primer sequence is as follows:

Upstream band homology arm primer H1 '-P1 '. underscore is homologous fragment:

5'- GTGCAAACCTTTCAAGCCGATCTTGCCATTGTAGGCGCCGGTGGCG CGATTCCGGGGATCCGTCGACC-3’

Downstream band homology arm primer H2 '-P2 ', underscore is homologous fragment:

5'- TTACTTAGTGCAGTTCGCGCACTGTTTGTTGACGATTTGCTGGAAGA ATGTAGGCTGGAGCTGCTTC-3’

The qualification of linear DNA fragment is as Fig. 5.

5, when electricity turns linear DNA fragment to own abduction delivering λ recombinase, lack ldhA, the competence of pflB and fumB, and coat with the LB flat screen of apramycin and select positive recombinant, and having carried out PCR qualification, electrophorogram is as shown in Figure 6.

6, positive recombinant be prepared into pour into after competence can abduction delivering FLP recombinase plasmid pCP20, after FLP recombinase is expressed in 42 DEG C of heat shocks, can eliminate apramycin resistance.Utilize pair of plates, carry out parallel point sample, can on non-resistant flat board, grow, but the bacterium that can not grow in resistant panel is the bacterial strain that knocks out resistance, thereby lacked ldhA simultaneously, pflB, the bacterial strain of fumB and frdABCD.Embodiment 3

The building process of the expression plasmid pTrc99a-pyc of the present embodiment explanation external source pyruvate carboxylase.

1, the synthetic primer with NcoI and PstI restriction enzyme site:

Upstream primer: 5 '-CATGCCATGGTCAGCTGATGAGAAACGTCGAGAAG-3 '

Downstream primer: 5 '-AAAACTGCAGGGTCATCTCTTCAAAGCCAAAACGA-3 '

2, taking Lactococcus lactis cremoris NZ9000 genomic dna as template, pcr amplification goal gene fragment, reaction conditions is: 94 DEG C, 5min; (94 DEG C of 45s, 53 DEG C of 45s, 72 DEG C of 300s, 35 circulations); 72 DEG C, 10min, the agarose gel electrophoresis figure of PCR product pyc is as shown in Figure 7.After the pyc gene that purifying amplifies, use NcoI, PstI double digestion with expression plasmid pTrc99a simultaneously, then connect and obtain recombinant plasmid pTrc99a-pyc, construction of recombinant plasmid collection of illustrative plates as shown in Figure 8.Fig. 9 has shown the single endonuclease digestion qualification result of recombinant plasmid pTrc99a-pyc.

Embodiment 4

The present embodiment explanation builds the expression plasmid pTrc99a-pyc-pncB of coexpression pyruvate carboxylase and nicotinic acid phosphoribosyltransferase, in reducing pyruvic acid accumulation, recover the under anaerobic ability of metabolizable glucose of recombinant bacterial strain, obtain the process of bacterial strain Escherichia coli BA043.

1, the expression plasmid that builds excessive coexpression pyruvate carboxylase and nicotinic acid phosphoribosyltransferase, its process comprises:

(1) synthetic upstream and downstream primer is all with the primer of NcoI restriction enzyme site,

Upstream primer: 5 '-CATGCCATGGGAAAGGTGGCATATGGTGTGATCGG-3 '

Downstream primer: 5 '-CATGCCATGGCGGCTACAGGCACAACGCTCATAAT-3

(2) taking e. coli k12 series as template, pcr amplification goal gene fragment, reaction conditions is: 94 DEG C, 5min; (94 DEG C of 45s, 63 DEG C of 45s, 72 DEG C of 96s, 35 circulations); 72 DEG C, 10min, the agarose gel electrophoresis figure of PCR product pncB is as shown in figure 10.After the pncB gene that purifying amplifies, with plasmid pTrc99a-pyc simultaneously with NcoI single endonuclease digestion, be connected and obtain recombinant plasmid pTrc99a-pyc-pncB, construction of recombinant plasmid collection of illustrative plates is as shown in figure 11.Figure 12 has shown the double digestion qualification result of recombinant plasmid pTrc99a-pyc-pncB.

2, plasmid pTrc99a-pyc-pncB is imported to the competent cell that simultaneously lacks lactate dehydrogenase gene (ldhA), pyruvate formate-lyase gene (pflB), FURAMIC ACID gene (fumB), fumaric reductase gene (frdABCD), the positive transformant of acquisition is new structure bacterial strain Escherichia coli BA043 of the present invention.

Embodiment 5

The new recombination bacillus coli BA043 building of the present embodiment explanation and the contrast of starting strain NZN111 fermentation and acid ability.

Intestinal bacteria Escherichia coli BA043 can oxysuccinic acid the generation of blocking-up downstream product, simultaneously, due to excessive coexpression pyruvate carboxylase and nicotinic acid phosphoribosyltransferase, recover the redox equilibrium of recombinant bacterium under anaerobic condition, the total amount of NAD (H) is significantly improved, also recovered the ability of metabolizable glucose under anaerobic condition, can accumulate in a large number oxysuccinic acid simultaneously.Adopt two stage fermentation modes, it is characterized in that by 1% (v/v) inoculum size from cryopreservation tube access triangular flask, when aerobic is cultivated thalline OD ₆₀₀to 3～4 left and right, concentrate 3 times to OD ₆₀₀=9 left/right rotations are connected in the serum bottle of 30mL LB, add the IPTG of 0.3mM as inductor simultaneously, and 0.5mM nicotinic acid is as NAD (H) substrate, and 30 DEG C, 170rpm, fermentation 24h.

Aerobic stage substratum is: LB+Amp (penbritin 100 μ g/mL)

Anaerobic stages substratum is: LB+ glucose (20g/L)+magnesium basic carbonate (15g/L)+Amp (100 μ g/mL)+0.3mM IPTG+0.5mM NA(nicotinic acid)

Fermentation results is in table 1.

The result comparison of table 1Escherichia coli BA043 and starting strain fermentation and acid

Note: ND represents not detect.

Claims

1. A strain of malic acid-producing genetic engineering bacteria, which is classified as Escherichia coli BA043 ( Escherichia coli BA043), and its deposit registration number is CCTCC NO: M 2014034.

2. The construction method of Escherichia coli BA043 according to claim 1, characterized in that the strain NZN111, which lacks the lactate dehydrogenase gene ( ldhA ) and the pyruvate formate lyase gene ( pflB ), is used as the starting strain, and the Homologous recombination technology further knocked out fumarase gene ( fumB ) and fumarate reductase gene ( frdABCD ) activities, and co-expressed exogenous pyruvate carboxylase gene ( pyc ) and nicotinic acid phosphoribosyltransferase gene ( pncB ) to obtain malate-producing Escherichia coli BA043.

3. construction method as claimed in claim 2, its concrete construction steps are as follows:

Step 1: Knock out the fumarase gene ( fumB ) and fumarate reduction in Escherichia coli strain NZN111 lacking the activity of lactate dehydrogenase gene ( ldhA ) and pyruvate formate lyase gene ( pflB ) as the starting strain Enzyme gene ( frdABCD ) to obtain a competent strain lacking ldhA , pflB , fumB and frdABCD activities at the same time;

Step 2, purifying and amplifying the pyruvate carboxylase gene ( pyc ), and constructing an expression plasmid expressing pyruvate carboxylase;

Step 3, purifying and amplifying the nicotinic acid phosphoribosyltransferase gene ( pncB ), connecting it to the recombinant plasmid described in step 2, and constructing an expression plasmid co-expressing pyruvate carboxylase and nicotinic acid phosphoribosyltransferase;

Step 4, introducing the plasmid obtained in step 3 into the competent cells obtained in step 1 to obtain positive transformants;

Step five, using the positive transformants of step four to co-express pyruvate carboxylase and nicotinic acid phosphoribosyltransferase to restore their ability to metabolize glucose under anaerobic conditions, and at the same time, reduce the accumulation of pyruvate to obtain malic acid-producing Genetically engineered bacteria Escherichia coli BA043.

4. Utilize the method for producing malic acid by Escherichia coli BA043 fermentation described in claim 1, it is characterized in that adopting two-stage fermentation mode, the aerobic stage increases biomass, and anaerobic stage fermentation produces acid.

5. the method for producing malic acid by Escherichia coli BA043 fermentation according to claim 4, is characterized in that: the inoculum size of 1% by volume is inserted in the Erlenmeyer flask from cryopreservation tube for aerobic cultivation, when aerobic Cultivate the bacteria with OD ₆₀₀ to about 3~4, concentrate 3 times to about OD ₆₀₀ =9, transfer to a 30mL LB serum bottle, add 0.3 mM IPTG as inducer, 0.5mM niacin as NAD(H) base anaerobic fermentation.