CN112391331A - Recombinant escherichia coli for overexpression of GatA gene and application thereof - Google Patents
Recombinant escherichia coli for overexpression of GatA gene and application thereof Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/54—Acetic acid
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
- C12P7/56—Lactic acid
Abstract
The invention discloses recombinant escherichia coli for over-expressing a GatA gene and application thereof, belonging to the technical field of genetic engineering and microbial engineering. The invention takes the gatA gene of the special IIA component of the galactitol as a target gene and takes the escherichia coli as an expression host, successfully constructs a class of escherichia coli engineering bacteria which can be widely applied to the preparation of medicines, feeds and chemicals; the itaconic acid stress resistance of the escherichia coli engineering bacteria is obviously improved, and is improved by 29.4 times compared with that of a wild strain, and the D-lactic acid stress resistance of the escherichia coli engineering bacteria is obviously improved and is improved by 101 times compared with that of the wild strain; the succinic acid stress resistance of the engineering bacteria of the escherichia coli is obviously improved and is improved by 1.6 times compared with the wild strain.
Description
Technical Field
The invention relates to recombinant escherichia coli for over-expressing a GatA gene and application thereof, belonging 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.
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.
Succinic acid, as a precursor substance with great potential, can be used to produce various derivatives such as tetrahydrofuran, which was evaluated in 2010 as "this compound or technology has attracted great attention in the literature, has a strong platform potential, and the scale of products or technologies is being gradually enlarged for pilot trials, demonstrations or general promotion", the market potential of succinic acid and its direct derivatives is expected to be up to 245 x 103 tons per year, while 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.
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 for improving the acid stress of escherichia coli, which has the advantages of excellent effect, high success rate, good genetic stability, low cost and simple operation, is urgently needed to be found.
Disclosure of Invention
In order to solve the above problems, the present invention provides an engineered escherichia coli having improved acid stress resistance, the engineered escherichia coli comprising a recombinant plasmid; the recombinant plasmid is an expression vector connected with a target gene; the target gene is the IIA component GatA gene specific to the coding galactitol.
In one embodiment of the invention, the expression host is e.coli K12 MG 1655.
In one embodiment of the invention, the IIA component GatA specific for galactitol is derived from escherichia coli e.coli K12 MG 1655.
In one embodiment of the invention, the amino acid sequence of IIA component GatA, which is specific for galactitol, is shown in SEQ ID No. 6.
In one embodiment of the invention, the nucleotide sequence of the IIA component GatA gene specific for galactitol is shown in SEQ ID No. 1.
In one embodiment of the invention, the expression vector is pTrc99 a.
The invention also provides a method for improving the acid stress resistance of escherichia coli, and IIA component GatA specific to excessive epi-galactitol in the escherichia coli.
In one embodiment of the invention, the IIA component GatA specific for galactitol is derived from escherichia coli e.coli K12 MG 1655.
In one embodiment of the invention, the amino acid sequence of IIA component GatA, which is specific for galactitol, is shown in SEQ ID No. 6.
In one embodiment of the invention, the nucleotide sequence of the IIA component GatA gene specific for galactitol is shown in SEQ ID No. 1.
In one embodiment of the invention, the overexpression is to connect the gene encoding IIA component GatA specific to galactitol with an expression vector, construct a recombinant plasmid containing the IIA component GatA gene specific to galactitol, and introduce the recombinant plasmid into escherichia coli.
In one embodiment of the present invention, the acid stress is D-lactic acid stress.
In one embodiment of the invention, the acid stress is itaconic acid stress.
In one embodiment of the present invention, the acid stress is succinic acid stress.
The invention also provides application of the escherichia coli engineering bacteria 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 IIA component GatA gene specific to galactitol with a nucleotide sequence shown as SEQ ID No. 1.
Has the advantages that:
(1) according to the invention, the recombinant escherichia coli E.coli K12 MG1655/pTrc99a-GatA with remarkably improved acid stress resistance is obtained by over-expressing GatA protein in escherichia coli; the method is simple to operate and can be widely applied to industrial production.
(2) The resistance of the recombinant escherichia coli E.coli K12 MG1655/pTrc99a-GatA obtained by the method of the invention to acid stress is obviously improved compared with that of a control strain, and the resistance to itaconic acid is 29.4 times that of the control strain; it was 101 times more resistant to D-lactic acid than the control strain; it was 1.6 times more resistant to succinic acid than the control strain.
Drawings
FIG. 1: growth profiles of recombinant strain E.coli K12 MG1655/pTrc99a-GatA and control strain E.coli K12 MG1655/pTrc99a under normal conditions.
FIG. 2: viability plots for the recombinant strain E.coli K12 MG1655/pTrc99a-GatA and the control strain E.coli K12 MG1655/PTrc99a in the itaconic acid stress (pH 4.2) tolerance test.
FIG. 3: viability profiles of recombinant strain E.coli K12 MG1655/pTrc99a-GatA and control strain E.coli K12 MG1655/pTrc99a in D-lactate stress (pH 4.0) tolerance assays.
FIG. 4: viability profiles of the recombinant strain E.coli K12 MG1655/pTrc99a-GatA and the control strain E.coli K12 MG1655/pTrc99a in the succinic acid stress (pH 4.3) tolerance test.
Detailed Description
Coli K12 MG1655 strain referred to in the examples below was obtained from baiopa great biotechnology limited, beijing, and pTrc99a vector was obtained from vast ling biotechnology limited, 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)-1Sodium 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-1pH 4.3 (succinic acid adjustment).
Example 1: construction of recombinant Strain E.coli K12 MG1655/pTrc99a-GatA
The method comprises the following specific steps:
(1) primers pTrc99a/GatA-F, pTrc99a/GatA-R shown in SEQ ID NO.2 and SEQ ID NO.3 respectively are designed based on the gatA gene sequence (encoding the gene of IIA component GatA peculiar to galactitol, participating in galactose metabolic pathway, regulating galactitol metabolism) in 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) a gene fragment shown as SEQ ID NO.1 is obtained by PCR amplification by taking a genome of E.coli K12 MG1655 as a template and pTrc99a/GatA-F and pTrc99a/GatA-R as primers;
(4) carrying out PCR amplification by using a vector pTrc99a as a template and a loop p-pTrc99a-F and a loop p-pTrc99a-R as primers to obtain a long fragment with linearized vector;
(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-GatA containing a recombinant plasmid pTrc99a-GatA with a correct sequence.
(6) Coli e.coli K12 MG1655 was transformed with the empty plasmid based on the same method as above, and a control strain e.coli K12 MG1655/PTrc99a was constructed.
Example 2: growth of recombinant and control strains under Normal conditions
(1) The recombinant strain E.coli K12 MG1655/pTrc99a-GatA and the control strain E.coli K12 MG1655/pTrc99a obtained in example 1 were inoculated into LB liquid medium for activation, respectively, and cultured overnight at 220rpm in a shaker at 37 ℃;
(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 the growth conditions of the recombinant strain E.coli K12 MG1655/PTrc99a-GatA after 10h of culture are not obviously different from those of the control strain through growth performance test analysis, which shows that the GatA protein is over-expressed in E.coli K12 MG1655, and has no influence on the growth performance of the strain.
Example 3: the concrete steps of the tolerance test of the recombinant strain E.coli K12 MG1655/pTrc99a-GatA in itaconic acid stress (pH 4.2) are as follows:
(1) the control strain E.coli K12 MG1655/pTrc99a and the recombinant strain E.coli K12 MG1655/pTrc99a-GatA obtained in example 1 were respectively inoculated in 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-GatA was 29.4 times that of the control strain e.coli K12 MG1655/pTrc99a after stress, and it was found that the tolerance of the recombinant strain to itaconic acid stress was improved by overexpression of GatA 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-GatA tolerance test at D-lactic acid stress (pH 4.0)
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-GatA obtained in example 1 were respectively inoculated in 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 a fresh D-lactic acid LB liquid culture medium (pH 4.0) with the same volume, and 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. 3 and table 2, after 4h of stress, the survival rate of e.coli K12 MG1655/pTrc99a-GatA was 101 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 lactic acid stress was improved by overexpression of GatA 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-GatA stress tolerance test at succinic acid stress (pH 4.3)
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-GatA obtained in example 1 were respectively inoculated in 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 a fresh succinic acid LB liquid culture medium (pH 4.3) with the same volume for stressing for different time;
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. 4 and table 3, after 4h of stress, the survival rate of the strain e.coli K12 MG1655/pTrc99a-GatA 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 GatA protein in e.coli K12 MG 1655.
TABLE 3 survival of acclimatized and control strains in succinic acid stress (pH 4.3) tolerance test
Example 6: acid resistance stability test of recombinant strains
1. The recombinant strain E.coli K12 MG1655/pTrc99a-GatA is continuously passaged for 30 times in LB liquid culture medium, and the cell growth performance has no obvious change, thereby proving that the strain obtained by the technical scheme provided by the invention has passage stability.
2. The specific embodiment is the same as that of examples 3 to 5, except that the recombinant strain E.coli K12 MG1655/pTrc99a-GatA was adjusted to the strain obtained in step 1 after 30 serial passages, and the control strain E.coli K12 MG1655/pTrc99a was treated in the following manner: continuously passaging for 30 times in an LB liquid culture medium; the results were:
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-GatA after continuous passage for 30 times is 0.0246, is basically consistent with that of the strain before passage, and is 35.1 times of that of a control strain E.coli K12 MG1655/pTrc99a 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-GatA after continuous passage for 30 times is 0.05793, is improved compared with the survival rate of the strain before passage, and is 128.3 times of the survival rate 0.00045148 of the 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-GatA after 30 times of continuous passage is 0.00048471, is basically consistent with that of the strain before passage, and is 2.0 times of that of the control strain E.coli K12 MG1655/pTrc99a 0.00024232 after 30 times of continuous passage.
The above examples demonstrate that overexpression of the GatA 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
<110> university of south of the Yangtze river
<120> recombinant escherichia coli overexpressing GatA gene and application thereof
<130> BAA200779A
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<170> PatentIn version 3.3
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atgactaacc tgtttgttcg tagcggaatt tcttttgtcg atcgtagcga agttttaacc 60
catatcggta atgagatgct cgccaaaggt gtggttcatg atacctggcc acaggcatta 120
attgccagag aagcagaatt ccctaccggg ataatgcttg agcagcacgc tattgcaata 180
ccgcattgtg aggcgattca tgctaagtcg tcagccattt atctgttaag gccaacaaat 240
aaagttcatt ttcagcaagc ggatgatgat aacgacgtgg cggtatcgtt ggttattgcg 300
ttgattgtgg aaaatccgca gcagcaattg aaacttttac gctgtttatt tggcaagtta 360
caacagcccg atatcgtcga gacactaatc actcttcctg aaacccagtt aaaggaatac 420
ttcacaaagt atgttttaga ttcagacgaa taa 453
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gtcgactcta gaggatccgc gttattcgtc tgaatctaaa acatactttg tg 52
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ggatcctcta gagtcgac 18
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gaattccatg gtctgtttcc tg 22
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Met Thr Asn Leu Phe Val Arg Ser Gly Ile Ser Phe Val Asp Arg Ser
1 5 10 15
Glu Val Leu Thr His Ile Gly Asn Glu Met Leu Ala Lys Gly Val Val
20 25 30
His Asp Thr Trp Pro Gln Ala Leu Ile Ala Arg Glu Ala Glu Phe Pro
35 40 45
Thr Gly Ile Met Leu Glu Gln His Ala Ile Ala Ile Pro His Cys Glu
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Ala Ile His Ala Lys Ser Ser Ala Ile Tyr Leu Leu Arg Pro Thr Asn
65 70 75 80
Lys Val His Phe Gln Gln Ala Asp Asp Asp Asn Asp Val Ala Val Ser
85 90 95
Leu Val Ile Ala Leu Ile Val Glu Asn Pro Gln Gln Gln Leu Lys Leu
100 105 110
Leu Arg Cys Leu Phe Gly Lys Leu Gln Gln Pro Asp Ile Val Glu Thr
115 120 125
Leu Ile Thr Leu Pro Glu Thr Gln Leu Lys Glu Tyr Phe Thr Lys Tyr
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Val Leu Asp Ser Asp Glu
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Claims (10)
1. An engineering bacterium of escherichia coli with improved acid stress resistance is characterized in that the engineering bacterium comprises a recombinant plasmid, wherein the recombinant plasmid is an expression vector connected with a target gene; the target gene is the IIA component GatA gene specific to the coding galactitol.
2. The engineered escherichia coli having increased acid stress resistance of claim 1, wherein the IIA component GatA specific to galactitol is derived from escherichia coli e.coli K12 MG 1655.
3. The engineered escherichia coli having increased acid stress resistance as claimed in claim 1 or 2, wherein the amino acid sequence of IIA component GatA specific to galactitol is shown in SEQ ID No. 6.
4. The engineered Escherichia coli having improved acid stress resistance according to any one of claims 1 to 3, wherein the expression vector is pTrc99 a.
5. A method for increasing acid stress resistance of E.coli, characterized in that the IIA component, GatA, specific for galactitol is overexpressed in E.coli.
6. The method of claim 5, wherein the IIA component GatA specific to galactitol is derived from E.coli K12 MG 1655.
7. The method for improving acid stress resistance of Escherichia coli according to claim 6, wherein the amino acid sequence of IIA component GatA specific to galactitol is shown in SEQ ID No. 6.
8. The method for improving acid stress resistance of Escherichia coli according to any one of claims 5 to 7, wherein said overexpression comprises ligating a gene encoding IIA component GatA specific to galactitol with an expression vector to construct a recombinant plasmid containing the gene encoding IIA component GatA specific to galactitol, and introducing the recombinant plasmid into Escherichia coli.
9. Use of an engineered escherichia coli having increased acid stress resistance according to any one of claims 1 to 4 for producing a metabolite by fermentation, wherein the metabolite is a substance involved in the metabolism of an organic acid.
10. An acid stress resistant component is characterized in that the component is an expression vector carrying a IIA component GatA gene specific to galactitol with a nucleotide sequence shown as SEQ ID No. 1.
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