CN112359005A

CN112359005A - Escherichia coli engineering bacterium with improved acid stress capability and application thereof

Info

Publication number: CN112359005A
Application number: CN202011258998.7A
Authority: CN
Inventors: 张娟; 杨谨华; 堵国成; 陈坚
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-11-12
Filing date: 2020-11-12
Publication date: 2021-02-12
Anticipated expiration: 2040-11-12
Also published as: CN112359005B

Abstract

The invention discloses an escherichia coli engineering bacterium with improved acid stress capability and application thereof, and belongs to the technical field of microbial engineering. The invention takes the gene of the nickel ion ABC transporter periplasmic binding protein NikA as a target gene and takes escherichia coli as an expression host, and successfully constructs a class of escherichia coli engineering bacteria which can be widely applied to the preparation of foods, medicines, feeds and chemicals; the acid stress capability of the strain is obviously improved, and the resistance of the strain to the itaconic acid is 6.3 times that of a control strain; resistance to D-lactic acid was 3.3 times that of the control strain; resistance to succinic acid was 1.6 times that of the control strain.

Description

Escherichia coli engineering bacterium with improved acid stress capability and application thereof

Technical Field

The invention relates to an escherichia coli engineering bacterium with improved acid stress capability and application thereof, and belongs to the technical field of microbial engineering.

Background

Escherichia coli is an important host bacterium in prokaryotes. The bacteria are widely distributed in nature and have abundant species diversity. They are ideal materials for the research of biochemistry, genetics, molecular biology and genetic engineering, have important academic values in theory, and have extremely high application values in important fields closely related to human life, such as industry, agriculture and animal husbandry, food and medicine. At present, various high-value organic acid biological fermentation methods are successfully applied, and some people try to express the organic acid biological fermentation method by taking escherichia coli as a host, but the problem of acid stress often exists.

The itaconic acid is known as methylene succinic acid and itaconic acid, is unsaturated binary organic acid, contains unsaturated double bond, has active chemical property, can carry out polymerization among themselves, can also be polymerized with other monomers such as acrylonitrile and the like, is an important raw material of chemical synthesis industry, and is an important raw material of chemical production. When Escherichia coli was used as a natural host, since Escherichia coli itself had no itaconic acid metabolizing ability, it was originally reported that the yield was very low, less than 1 g.L^-1Later, the concentration of the compound was 4.3 g.L^-1. Until recently, the highest production of itaconic acid was only reported to be 47 g.L^-1The synthesis path is mainly generated by aconitic acid oxidation, and from the final yield, the yield is still different from the yield when aspergillus is used as a host, and the yield is 0.86 g.h^-1·L^-1Although there are some deficiencies in productivity as compared with the conventional Aspergillus producing strains, the use of E.coli as a host for the production of itaconic acid is still considered to be of potential.

Succinic acid, as a precursor substance with great potential, can be used for producing various derivatives such as tetrahydrofuran, and is evaluated as the compound or technology in 2010Having drawn great attention in the literature, the scale of products or technologies with powerful platform potential is being escalated to pilot, demo or full-scale promotion, the market potential of succinic acid and its direct derivatives is expected to be up to 245 x 103 tons per year, whereas the market scale of succinic acid-derived polymers is estimated to be up to 25 x 106 tons per year. In the traditional fermentation process of engineering strains, succinic acid is produced by fermenting the Mannheimia succiniciproducens and the like, and the yield is about 68.41 g.L^-1. The strains are naturally-occurring strains which can metabolize to produce succinic acid and have high yield, but a common problem of the strains is that the metabolic modification of the strains is relatively complex and difficult, and generally, auxotrophic strains need more nutrient additives during fermentation, so that large-scale industrial application cannot be realized, so researchers aim at escherichia coli.

D-lactic acid with high optical purity (more than 97 percent) is used as a chiral center, is a precursor of a plurality of chiral substances, is an important chiral intermediate and an organic synthesis raw material, is widely applied to chiral synthesis in the fields of pharmacy, high-efficiency low-toxicity pesticides, herbicides, cosmetics and the like, and is also a raw material of bioplastic polylactic acid. The existing fermentation method for preparing lactic acid has wide application prospect in industry because of green, high efficiency and environmental protection, although the existing lactic acid generation way in escherichia coli is available, pyruvic acid can be converted into lactic acid through lactate dehydrogenase (LdhA), and heterologous LdhA is not required to be additionally introduced to produce lactic acid, the yield of lactic acid is limited to be improved because the escherichia coli system has the problem of acid stress.

However, the escherichia coli has a limited development factor with low tolerance to organic acid, the growth environment of the escherichia coli is neutral, and the escherichia coli is about 50 g.L^-1The organic acid(s) will reduce the environmental pH to about pH 2.0, which is a challenge to the growth of E.coli.

In order to maintain the stability of the objective protein produced by fermentation of Escherichia coli and to improve the production efficiency under acid stress, in the past, it has been common in industry to maintain the pH in a stable range by adding an exogenous neutralizing agent during the fermentation of Escherichia coli, for example, by adding an alkaline substance (calcium carbonate) to control the pH of the fermentation environment. However, the addition of alkaline substances often results in the accumulation of byproducts, and the salts formed in the byproducts can cause the cells to be in a hypertonic environment again, thereby causing osmotic stress and influencing the growth and metabolism of the bacteria again. At present, the methods for improving the acid stress resistance of escherichia coli mainly comprise: (1) mutation breeding, the method has the characteristics of simplicity, convenience, various types and the like, but the method has the main defects of large workload and low efficiency, and the mutagenized strains are easy to degenerate; (2) the existing method for improving the environmental stress of escherichia coli by utilizing the metabolic engineering strategy mainly comprises the steps of constructing a new metabolic pathway, expanding an existing metabolic pathway and weakening the existing metabolic pathway, but the method has the problems of high cost and low success rate.

Therefore, a new method which has low cost, high success rate, outstanding effect, good genetic stability and simple operation and can improve the acid stress of the escherichia coli is urgently needed to be found.

Disclosure of Invention

In order to solve the above technical problems, the present invention provides a recombinant escherichia coli with improved acid stress resistance, comprising a recombinant plasmid and an expression host; the recombinant plasmid is an expression vector connected with a target gene; the target gene is a gene encoding the periplasmic binding protein NikA (Ni) of the nickel ion ABC transporter²⁺ABC transporter plasmid binding protein).

In one embodiment of the invention, the expression host is e.coli K12 MG 1655.

In one embodiment of the invention, the nickel ion ABC transporter periplasmic binding protein NikA is derived from escherichia coli e.coli K12 MG 1655.

In one embodiment of the invention, the amino acid sequence of the periplasmic binding protein NikA of the nickel ion ABC transporter is shown in SEQ ID NO. 6.

In one embodiment of the invention, the nucleotide sequence of the NikA gene encoding the periplasmic binding protein of the nickel ion ABC transporter is shown as SEQ ID NO. 1.

In one embodiment of the invention, the expression vector is pTrc99 a.

The invention provides a method for improving acid stress resistance of escherichia coli, which is used for over-expressing a periplasmic binding protein NikA of a nickel ion ABC transporter in the escherichia coli.

In one embodiment of the invention, the overexpression is to construct a recombinant plasmid containing a gene encoding the nickel ion ABC transporter periplasmic binding protein NikA through the gene encoding the nickel ion ABC transporter periplasmic binding protein NikA and an expression vector, and then introduce the recombinant plasmid into Escherichia coli.

In one embodiment of the invention, the acid stress is itaconic acid stress.

In one embodiment of the present invention, the acid stress is D-lactic acid stress.

In one embodiment of the present invention, the acid stress is succinic acid stress.

The invention also provides application of the recombinant escherichia coli with improved acid stress resistance in fermentation production of metabolites, wherein the metabolites are substances participating in organic acid metabolism.

In one embodiment of the invention, the metabolite is formic acid, acetic acid, lactic acid.

The invention also provides an acid stress resistant component which is an expression vector carrying the nickel ion ABC transport protein periplasm binding protein NikA gene with the nucleotide sequence shown as SEQ ID No. 1.

Has the advantages that:

(1) the overexpression of the NikA protein in the escherichia coli obviously improves the acid stress resistance of the escherichia coli, and the method is simple to operate and can be widely applied to industrial production.

(2) According to the invention, the recombinant Escherichia coli K12 MG1655/pTrc99a-NikA with remarkably improved acid stress resistance is obtained by over-expressing NikA protein in Escherichia coli; the resistance of the strain to itaconic acid is 6.3 times that of a control strain; resistance to D-lactic acid was 3.3 times that of the control strain; resistance to succinic acid was 1.6 times that of the control strain.

Drawings

FIG. 1: growth profiles of recombinant strain E.coli K12 MG1655/pTrc99a-NikA and control strain E.coli K12 MG1655/pTrc99a under normal conditions.

FIG. 2: survival plots for the recombinant strain E.coli K12 MG1655/pTrc99a-NikA and the control strain E.coli K12 MG1655/pTrc99a in stress tolerance tests under itaconic acid stress (pH 4.2).

FIG. 3: survival plots for the recombinant strain E.coli K12 MG1655/pTrc99a-NikA and the control strain E.coli K12 MG1655/pTrc99a in the D-lactate stress (pH 4.0) stress tolerance assay.

FIG. 4: survival plots for the recombinant strain E.coli K12 MG1655/pTrc99a-NikA and the control strain E.coli K12 MG1655/pTrc99a in succinic acid stress (pH4.3) stress tolerance experiments.

Detailed Description

Coli K12 MG1655 strain referred to in the examples below was obtained from baiopa bio-technologies ltd, beijing, and pTrc99a vector was obtained from vast ling bio-technologies ltd, wuhan.

The media involved in the following examples are as follows:

LB solid medium: peptone (Oxoid, UK) 10 g.L^-15 g.L of yeast powder (Oxoid)^-1Sodium chloride 10 g. L^-1And agar powder 20 g.L^-1。

LB liquid medium: peptone (Oxoid, UK) 10 g.L^-15 g.L of yeast powder (Oxoid)^-1Chlorine, chlorineSodium chloride 10 g.L^-1。

Itaconic acid LB liquid medium: peptone (Oxoid, UK) 10 g.L^-15 g.L of yeast powder (Oxoid)^-1Sodium chloride 10 g. L^-1pH 4.2 (itaconic acid adjustment).

D-lactic acid LB liquid medium: peptone (Oxoid, UK) 10 g.L^-15 g.L of yeast powder (Oxoid)^-1Sodium chloride 10 g. L^-1pH 4.0 (D-lactic acid adjustment).

Succinic acid LB liquid medium: peptone (Oxoid, UK) 10 g.L^-15 g.L of yeast powder (Oxoid)^-1Sodium chloride 10 g. L^-1pH4.3 (succinic acid adjustment).

Example 1: construction of recombinant Strain E.coli K12 MG1655/pTrc99a-NikA

The method comprises the following specific steps:

(1) designing primers pTrc99a/NikA-F and pTrc99a/NikA-R which are respectively shown as SEQ ID NO.2 and SEQ ID NO.3 based on nikA gene sequences (the NikA gene of the periplasm binding protein of the nickel ion ABC transporter is coded, participates in the ABC transporter metabolic pathway of nickel ions and regulates the combination of substrate proteins in a nickel transporter system) in an NCBI database;

(2) designing primers shown as SEQ ID NO.4 and SEQ ID NO.5 respectively, namely, a loop p-pTrc99a-F and a loop p-pTrc99 a-R;

(3) using E.coli K12 MG1655 genome as template, p-pTrc99a/NikA-F, p-pTrc99a/NikA-R as primer to obtain gene fragment shown in SEQ ID NO.1 by PCR amplification, and obtaining PCR product;

(4) obtaining a long fragment of the linearized vector by PCR amplification by taking the vector pTrc99a as a template, and taking the loop p-pTrc99a-F and the loop p-pTrc99a-R as primers to obtain a PCR product;

(5) and (3) connecting the PCR products obtained in the steps (3) and (4) to obtain a connecting product, then transforming the connecting product into escherichia coli E.coli K12 MG1655 competence to obtain a transformation product, inoculating the transformation product to an LB solid culture medium containing ampicillin to screen positive clones, verifying the correct size of the fragment through colony PCR, and then carrying out sequencing identification to finally obtain a recombinant strain E.coli K12 MG1655/pTrc99a-NikA containing the recombinant plasmid pTrc99a-NikA with the correct sequence.

(6) Construction of control bacteria E.coli K12 MG1655/pTrc99a-NikB, E.coli K12 MG1655/pTrc99a-NikC and E.coli K12 MG1655/pTrc99a

Based on the same method, the difference lies in that the NikA protein gene is replaced by NikB (Ni) with the nucleotide sequence shown as SEQ ID NO.7²⁺ABC transporter membrane subnikB) protein gene and NikC (Ni) with nucleotide sequence shown in SEQ ID No.8²⁺ABC transporter membrane disruption NikC) protein gene, NikB protein and NikC protein are other genes in the same metabolic pathway of NikA protein, and strains E.coli K12 MG1655/pTrc99a-NikB and E.coli K12 MG1655/pTrc99a-NikC are constructed.

Based on the same method as above, e.coli K12 MG1655/pTrc99a was constructed as a control strain.

Example 2: growth of recombinant and control strains under Normal conditions

The method comprises the following specific steps:

(1) the strains E.coli K12 MG1655/pTrc99a-NikA, E.coli K12 MG1655/pTrc99a-NikB, E.coli K12 MG1655/pTrc99a-NikC and the control strain E.coli K12 MG1655/pTrc99a obtained in example 1 were respectively inoculated into LB liquid medium for activation, and cultured in a shaker at 37 ℃ at 220rpm for 12 hours to obtain a seed solution;

(2) transferring the seed liquid obtained in the step (1) into an LB liquid culture medium with the inoculation amount of 2% (v/v), and culturing in a shaker at 37 ℃ at 220 rpm; samples were taken every 2 hours, and the OD at 600nm was measured to plot a growth curve (the resulting growth curve is shown in FIG. 1).

The results are shown in fig. 1, and through the analysis of growth performance tests, after 12h of culture, the growth of the recombinant strain over-expressing the nikA gene is slightly delayed compared with the growth cycle of the control strain, but the final bacterial quantities can be kept similar, which indicates that the overexpression of nikA protein in the e.coli K12 MG1655 has no influence on the growth performance of the strain, but the overexpression of NikB and NikC protein in the e.coli K12 MG1655 obviously increases the growth retardation of the recombinant strain, so that the growth of the strain is inhibited.

It can be seen that the genes of other NikA identical metabolic pathways are overexpressed in e.coli K12 MG1655, and the growth of the recombinant strain is inhibited.

Example 3: tolerance test of recombinant strain E.coli K12 MG1655/pTrc99a-NikA to itaconic acid stress (pH 4.2)

The method comprises the following specific steps:

(1) the control strain E.coli K12 MG1655/pTrc99a and the recombinant strain E.coli K12 MG1655/pTrc99a-NikA obtained in example 1 were respectively inoculated into LB liquid medium for activation, and cultured in a shaker at 37 ℃ for 12 hours at 220rpm to obtain a seed solution;

(2) transferring the seed solution obtained in the step (1) into a fresh LB liquid culture medium respectively with the inoculation amount of 2% (v/v), culturing for 4.5h in a shaking table at 37 ℃ at 220rpm until the logarithmic growth middle period, wherein the OD600 is 1.4-1.5, and obtaining a culture solution;

(3) centrifuging the culture solution obtained in the step (2) at 6000rpm for 5min, collecting thalli, washing the obtained thalli twice by using 0.85% PBS buffer solution, and suspending the thalli in an equal volume of fresh itaconic acid LB liquid medium (pH 4.2) for stressing for different times;

respectively washing the bacterial suspension after being stressed for 0h, 1h, 2h, 3h and 4h twice, then re-suspending the bacterial suspension in physiological saline with the same volume, taking 10 mu L of the re-suspension, diluting different gradient points, and inoculating the re-suspension on an LB solid culture medium to determine the viable count and the survival rate.

Survival rate ═ N/N₀)×100％；

Wherein N is₀Is the number of viable colonies of the bacterial suspension on the plate which is not subjected to acid stress treatment; n is the number of viable colonies growing on the plate after stress.

As shown in fig. 2 and table 1, after 4h of stress, the survival rate of the strain e.coli K12 MG1655/pTrc99a-NikA was 6.3 times that of the control strain e.coli K12 MG1655/pTrc99a after stress analysis by the tolerance test, and it can be seen that the tolerance of the recombinant strain to itaconic acid stress was improved by overexpression of NikA protein in e.coli K12 MG 1655.

TABLE 1 survival rate of acclimatized and control strains in the itaconic acid stress (pH 4.2) tolerance test

Example 4: recombinant strain E.coli K12 MG1655/pTrc99a-NikA in D-lactic acid stress (pH 4.0) tolerance test

The method comprises the following specific steps:

(3) centrifuging the culture solution obtained in the step (2) at 6000rpm for 5min, collecting thalli, washing the obtained thalli twice by using 0.85% PBS buffer solution, and suspending the thalli in a fresh D-lactic acid LB liquid culture medium (pH 4.0) with the same volume, and stressing for different times;

Survival rate ═ N/N₀)×100％；

As shown in fig. 3 and table 2, after 4h of stress, the survival rate of e.coli K12 MG1655/pTrc99a-NikA was 3.3 times that of the control strain e.coli K12 MG1655/pTrc99a after the stress test analysis, and it was found that the tolerance of the recombinant strain to D-lactic acid stress was improved by overexpression of NikA protein in e.coli K12 MG 1655.

TABLE 2 survival rate of acclimatized and control strains in D-lactic acid stress (pH 4.0) tolerance test

Example 5: recombinant Strain E.coli K12 MG1655/pTrc99a-NikA stress tolerance test under succinic acid stress (pH4.3)

The method comprises the following specific steps:

(1) respectively inoculating a control strain E.coli K12 MG1655/pTrc99a and the strain E.coli K12 MG1655/pTrc99a-NikA obtained in example 1 into an LB liquid culture medium for activation, and culturing in a shaker at 37 ℃ at 220rpm for 12h to obtain a seed solution;

(3) centrifuging the culture solution obtained in the step (2) at 6000rpm for 5min, collecting thalli, washing the obtained thalli twice by using 0.85% PBS buffer solution, and suspending the thalli in an equal volume of fresh succinic acid LB liquid culture medium (pH4.3) for stressing for different times;

Survival rate ═ N/N₀)×100％；

As shown in fig. 4 and table 3, after 4h of stress, the survival rate of the strain e.coli K12 MG1655/pTrc99a-NikA was 1.6 times that of the control strain e.coli K12 MG1655/pTrc99a after the stress test analysis, and it was found that the tolerance of the recombinant strain to succinic acid stress was improved by overexpression of NikA protein in e.coli K12 MG 1655.

TABLE 3 survival of acclimatized and control strains in succinic acid stress (pH4.3) tolerance test

Example 6: acid resistance stability test of recombinant strains

1. The recombinant strain E.coli K12 MG1655/pTrc99a-NikA is continuously passaged for 30 times in LB liquid culture medium, the cell growth performance has no obvious change, and the strain obtained by the technical scheme provided by the invention is proved to have passage stability.

2. The specific embodiment is the same as that of examples 3-5, except that the recombinant strain E.coli K12 MG1655/pTrc99a-NikA was adjusted to the strain obtained in step 1 after 30 serial passages, and the control strain E.coli K12 MG1655/pTrc99a was processed in the following manner: continuously passaging for 30 times in an LB liquid culture medium; the results are as follows:

after stress of itaconic acid, tolerance experiment analysis shows that after stress for 4h, the survival rate of the strain E.coli K12 MG1655/pTrc99a-NikA after continuous passage for 30 times is 0.00412586, is basically consistent with that of the strain before passage, and is 7.9 times of that of a control strain E.coli K12 MG1655/pTrc99a 0.000521518 after continuous passage for 30 times;

after the stress of D-lactic acid and the analysis of tolerance experiments, after the stress of 4h, the survival rate of the strain E.coli K12 MG1655/pTrc99a-NikA after continuous passage for 30 times is 0.00431548, is improved compared with the survival rate of the strain before passage, and is 3.7 times of the survival rate 0.00113931 of a control strain E.coli K12 MG1655/pTrc99a after continuous passage for 30 times;

after succinic acid stress, tolerance experiment analysis shows that after stress for 4h, the survival rate of the strain E.coli K12 MG1655/pTrc99a-NikA after continuous passage for 30 times is 0.000372715, is basically consistent with that of the strain before passage, and is 1.7 times of that of a control strain E.coli K12 MG1655/pTrc99a 0.00207063 after continuous passage for 30 times.

The above examples demonstrate that overexpression of NikA protein in e.coli K12 MG1655, the recombinant strain has improved tolerance to organic acid stress, and has passaging stability.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

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gatcagccgc tgtacgtcca gtacggcacc tggttgtgga aggcgctgca tcttgacttt 240

ggtatctcat tcgccagcca acgcccggta ctggacgata tgctgaactt cctgcccgcc 300

acgctggaac ttgcaggtgc ggcgctggta ttaattctgc tcacttccgt accgctcggt 360

atctgggcgg cgcgccatcg cgaccgtctg ccggatttcg ccgtacgttt catcgcgttt 420

cttggcgtgt cgatgcctaa cttctggctg gcgtttttac tggtgatggc gttttcggtg 480

tatctgcaat ggctacccgc gatgggttac ggcggctggc agcacatcat tttgcctgcg 540

gtttccattg cctttatgtc gctggcgatt aacgcgcgtt tactgcgcgc cagtatgctg 600

gacgtcgccg gtcagcgtca cgtcacctgg gcgcgtctgc gcggcctgaa cgacaaacag 660

accgaacgtc gccacatcct gcgcaatgcc tcgctgccga tgatcaccgc cgtggggatg 720

catatcggcg aactgattgg cgggacgatg attatcgaaa acatctttgc ctggccgggc 780

gtcgggcgct atgcggtgtc ggcgattttt aaccgtgact atccggtgat ccagtgcttt 840

acgctgatga tggtggtggt ttttgtggtc tgtaatttga ttgtcgattt gctcaacgcc 900

gcgctggacc cgcgcattcg tcgtcatgaa ggagcgcacg cgtga 945

<210> 8

<211> 1668

<212> DNA

<213> Artificial sequence

<400> 8

gtgaactttt tcctctcttc ccgctggtcg gtacgcctgg cgctgatcat tatcgccctg 60

ctggcgctga ttgcgctcac cagccagtgg tggctgccgt atgacccaca ggcgattgat 120

ttgccgtcgc gcctgctttc gccggatgcg cagcactggc tgggcaccga tcacttaggt 180

cgcgatattt tctcgcggct gatggcagcg acccgcgtgt cgctcggttc ggtaatggcc 240

tgcctgctgc tggtgctgac attagggctg gttattggcg gcagcgccgg gttgattggc 300

gggcgcgttg atcaggccac catgcgcgtc gccgatatgt ttatgacctt cccgacctcg 360

attctgtcgt tctttatggt tggcgtgctc ggcaccgggc tgaccaacgt aattatcgcc 420

atcgccctgt cgcactgggc gtggtatgca cgcatggtgc gcagcctggt gatttcacta 480

cgccaacgcg agtttgtgct ggcgtcacgg ctttccggtg cgggccatgt gcgggtgttt 540

gtcgatcatc tggcaggcgc ggtgatccct tcgctgctgg tgctggcaac gctggatatc 600

ggccatatga tgctgcacgt cgcggggatg tctttccttg gcctcggtgt gaccgcgccg 660

accgccgaat ggggcgtgat gattaacgac gcgcgccagt atatctggac ccagccgctg 720

caaatgttct ggccggggct ggcgctgttt atcagcgtga tggcctttaa cctggtgggt 780

gacgcactgc gcgatcatct ggaccctcat ctggtgacgg agcacgcaca ctaagtgaac 840

tttttcctct cttcccgctg gtcggtacgc ctggcgctga tcattatcgc cctgctggcg 900

ctgattgcgc tcaccagcca gtggtggctg ccgtatgacc cacaggcgat tgatttgccg 960

tcgcgcctgc tttcgccgga tgcgcagcac tggctgggca ccgatcactt aggtcgcgat 1020

attttctcgc ggctgatggc agcgacccgc gtgtcgctcg gttcggtaat ggcctgcctg 1080

ctgctggtgc tgacattagg gctggttatt ggcggcagcg ccgggttgat tggcgggcgc 1140

gttgatcagg ccaccatgcg cgtcgccgat atgtttatga ccttcccgac ctcgattctg 1200

tcgttcttta tggttggcgt gctcggcacc gggctgacca acgtaattat cgccatcgcc 1260

ctgtcgcact gggcgtggta tgcacgcatg gtgcgcagcc tggtgatttc actacgccaa 1320

cgcgagtttg tgctggcgtc acggctttcc ggtgcgggcc atgtgcgggt gtttgtcgat 1380

catctggcag gcgcggtgat cccttcgctg ctggtgctgg caacgctgga tatcggccat 1440

atgatgctgc acgtcgcggg gatgtctttc cttggcctcg gtgtgaccgc gccgaccgcc 1500

gaatggggcg tgatgattaa cgacgcgcgc cagtatatct ggacccagcc gctgcaaatg 1560

ttctggccgg ggctggcgct gtttatcagc gtgatggcct ttaacctggt gggtgacgca 1620

ctgcgcgatc atctggaccc tcatctggtg acggagcacg cacactaa 1668

Claims

1. A recombinant Escherichia coli with improved acid stress resistance is characterized in that the recombinant Escherichia coli comprises a recombinant plasmid, wherein the recombinant plasmid is an expression vector connected with a target gene; the target gene is NikA gene of periplasm binding protein of nickel ion ABC transporter.

2. The recombinant Escherichia coli having an improved acid stress resistance according to claim 1, wherein the expression vector is pTrc99 a.

3. The recombinant escherichia coli with improved acid stress resistance of claim 1 or 2, wherein the nickel ion ABC transporter periplasmic binding protein NikA is derived from escherichia coli e.coli K12 MG 1655.

4. The recombinant Escherichia coli having an improved acid stress resistance capability of any one of claims 1 to 3, wherein the amino acid sequence of the periplasmic binding protein NikA of the nickel ion ABC transporter is shown in SEQ ID NO. 6.

5. The application of the overexpression of the periplasm binding protein NikA of the nickel ion ABC transporter in improving the acid stress resistance of escherichia coli.

6. The use of claim 5, wherein the nickel ion ABC transporter periplasmic binding protein NikA is derived from Escherichia coli E.coli K12 MG 1655.

7. The use of claim 6, wherein the amino acid sequence of the periplasmic binding protein NikA of the nickel ion ABC transporter is shown in SEQ ID No. 6.

8. The use according to any one of claims 5 to 7, wherein the overexpression is carried out by constructing a recombinant plasmid containing a gene encoding the periplasmic binding protein NikA of the nickel ion ABC transporter and introducing the recombinant plasmid into E.coli.

9. Use of the recombinant E.coli strain of any one of claims 1 to 4 for the fermentative production of a metabolite, wherein the metabolite is a substance involved in the metabolism of organic acids.

10. An acid stress resistant component is characterized in that the component is an expression vector carrying a nickel ion ABC transport protein periplasm binding protein NikA gene with a nucleotide sequence shown as SEQ ID No. 1.