CN110564659B

CN110564659B - Escherichia coli resistant to sodium acetate, sodium chloride and isobutanol and construction method thereof

Info

Publication number: CN110564659B
Application number: CN201910878309.3A
Authority: CN
Inventors: 王智文; 李书廷; 陈聪; 陈涛
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2019-09-17
Filing date: 2019-09-17
Publication date: 2022-03-11
Anticipated expiration: 2039-09-17
Also published as: CN110564659A

Abstract

The invention discloses escherichia coli with resistance to sodium acetate, sodium chloride and isobutanol and a construction method thereof, and the construction method comprises the following steps: introducing the screened gene of the mutation site, wherein the 142 rd amino acid G of the Escherichia coli global regulatory factor CRP is replaced by I, into wild Escherichia coli to obtain Escherichia coli CRP-G142I resistant to sodium acetate, sodium chloride and isobutanol; according to the invention, based on the Escherichia coli global regulation factor, saturated mutation is rationally designed, so that the characteristics of prokaryote regulation network scalability are effectively utilized, the characteristic of completing the disturbance of the expression level of a host bacterium genome large-range network under the micro-disturbance gene change operation is realized, different physiological metabolic activities are coordinated, the thallus responds to the change of the external environment, the trouble of complexity among a plurality of genotype phenotypes is avoided, and the problem that the optimal phenotype cannot be obtained due to experiment limitation is solved.

Description

Escherichia coli resistant to sodium acetate, sodium chloride and isobutanol and construction method thereof

Technical Field

The invention belongs to the field of escherichia coli global regulatory factor engineering and escherichia coli tolerance engineering, and particularly relates to escherichia coli resistant to sodium acetate, sodium chloride and isobutanol and a construction method thereof.

Background

In order to meet the global demand for energy and reduce the negative environmental impact of fossil fuels, biofuels have become a potential alternative to petroleum and have become a focus of attention. And isobutanol in higher alcohol is called as a new generation of biofuel due to the characteristics of high energy density, low vapor pressure and low humidity. However, industrial fermentation for producing biofuels using inexpensive lignocelluloses is a complex and multifaceted process, and in order to increase the concentration, production rate and yield of products, further improvements in the robustness of strains are required, most of which are improvements in the tolerance of strains to adverse conditions. Adverse factors in the fermentation process include toxicity to high-concentration inhibitory products (such as acetate and furfural) generated in the pretreatment of fermentation raw material lignocellulose, solvent-based chemicals and biofuel (such as isobutanol), and osmotic pressure of the external environment, extreme pH and the like, among which acetate is one of the escherichia coli growth inhibitors widely studied at present, is not only a derivative of lignocellulose, but also a byproduct of fermentation of many other microorganisms, has high concentration in escherichia coli fermentation liquor, and research proves that (chong et al, 2013) can inhibit cell growth when the concentration exceeds 5g/L, especially can limit the growth of high-density strains and the generation of recombinant proteins when excessive sugar exists, and is valuable for enhancing acetate tolerance engineering bacteria to biological processes; secondly, the toxicity of isobutanol which can damage cell growth at 8g/L to escherichia coli is similar to that of other higher long-chain alcohols, and the toxicity is reflected in that the phospholipid composition and the cell membrane fluidity are changed, the intracellular pH and the ATP concentration are reduced, so that the glucose uptake of thalli is inhibited, the yield of the higher alcohols is limited, and the isobutanol is also a hot spot concerned by industrial production; finally, the extracellular environment, represented by osmotic pressure, affects the optimal activity and metabolism of key enzymes of the microorganism, ultimately resulting in decreased productivity and bioactivity of the microorganism. These factors are bottlenecks that limit achieving optimal yields. Research proves that the adverse factors are always present at the same time and have a synergistic effect, so that the improvement of the multiple tolerance of the strain is one of the important strategies for improving the fermentation performance of the strain and finally obtaining the optimal yield.

The tolerance of Escherichia coli is a complex phenotype controlled by multiple genes, and the simultaneous multi-gene modification can greatly promote the acquisition of a high-tolerance phenotype, but the capacity of simultaneously introducing the multi-gene modification is very limited. Research shows that prokaryotes and eukaryotes both have pyramid gene expression level regulation networks, and the global regulation factor is positioned at the top of the pyramid and is the regulation factor with the highest regulation level. The cyclic adenosine monophosphate receptor protein (CRP) is an important global transcription regulatory factor of escherichia coli, can directly regulate and control the expression of over 490 genes (Ravcheev et al, 2013), and can regulate and control about half of the genes of the escherichia coli by directly or indirectly regulating and controlling other transcription regulatory factors, so that the global disturbance of an escherichia coli cell network can be realized by mutating the regulatory factor (CRP), the limitation of simultaneously modifying a plurality of genes is solved, and the complex trouble between a phenotype and a genotype is avoided, so that different physiological metabolic activities are coordinated, and the somatic cells respond to the change of an external environment (Gottesman et al, 2007).

Meanwhile, in the existing aspect of improving the tolerance of escherichia coli, research on the CRP gene is also multiple, including obtaining a strain with tolerance for improving acetate by mutation of amino acid 138, obtaining a strain with tolerance for improving isobutanol by mutation of amino acid 179 and amino acid 199, and obtaining a strain with tolerance for high osmotic pressure by mutation of amino acid 69, 130, 52, 119, 74, 87, 114, 43, 71 and the like, but at present, 3 kinds of tolerance for improving the tolerance of sodium acetate, sodium chloride and isobutanol by mutation of amino acid 142 of the global regulatory factor CRP and the engineered strain CRP-G142I are not reported.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the escherichia coli with resistance to sodium acetate, sodium chloride and isobutanol.

The second purpose of the invention is to provide a construction method of escherichia coli resistant to sodium acetate, sodium chloride and isobutanol.

The technical scheme of the invention is summarized as follows:

the construction method of the escherichia coli with resistance to sodium acetate, sodium chloride and isobutanol comprises the following steps: introducing the screened gene containing the mutation site of the 142 th amino acid G of the escherichia coli global regulatory factor CRP replaced by I into wild type escherichia coli to obtain escherichia coli CRP-G142I resistant to sodium acetate, sodium chloride and isobutanol;

the amino acid sequence of the colibacillus global regulatory factor CRP is shown in SEQ ID NO. 1;

the nucleotide sequence of the gene obtained from the mutation site of the 142 th amino acid G of the colibacillus global regulatory factor CRP replaced by I is shown as SEQ ID NO. 2.

Coli CRP-G142I resistant to sodium acetate, sodium chloride and isobutanol constructed by the method of claim 1.

THE ADVANTAGES OF THE PRESENT INVENTION

Compared with the biomass of 48h of a control under the condition of sodium acetate (30G/L), the constructed escherichia coli CRP-G142I resistant to sodium acetate, sodium chloride and isobutanol is improved by 1633.96%, and the maximum specific growth rate is improved by 313.82%; the biomass is increased by 35.12 percent in 48 hours in sodium chloride (0.6mol/L), and the maximum specific growth rate is increased by 75.89 percent; in isobutanol (8g/L), the biomass increased 73.78% for 36h, and the maximum specific growth rate increased 37.25%.

According to the invention, based on the Escherichia coli global regulation factor, saturated mutation is rationally designed, so that the characteristics of prokaryote regulation network scalability are effectively utilized, the characteristic of completing the disturbance of the expression level of a host bacterium genome large-range network under the micro-disturbance gene change operation is realized, different physiological metabolic activities are coordinated, the thallus responds to the change of the external environment, the trouble of complexity among a plurality of genotype phenotypes is avoided, and the problem that the optimal phenotype cannot be obtained due to experiment limitation is solved.

The gene mutation is carried out by combining the global regulatory factor of the escherichia coli with the emerging genetic engineering technology, so that an ideal phenotype can be quickly obtained, the aim of improving the multiple tolerance of the escherichia coli is fulfilled, and a basis is further provided for researching a tolerance mechanism.

Drawings

FIG. 1 shows the biomass (48h biomass) of recombinant bacteria of different mutant libraries at a concentration of 20g/L sodium acetate.

FIG. 2 is a graph showing the tolerance of CRP-G142I under 30G/L sodium acetate stress.

FIG. 3 shows the tolerance of CRP-G142I under 0.6mol/L sodium chloride stress.

FIG. 4 is a graph of CRP-G142I tolerance under 8G/L isobutanol stress conditions.

Detailed Description

The present invention is further illustrated by the following examples, which are provided to enable those skilled in the art to better understand the present invention and are not intended to limit the present invention in any way.

Escherichia coli str.K-12MG1655, the original strain used in the present invention, is a typical strain purchased from ATCC deposit (S.) (

700926). Hereinafter abbreviated as MG1655

The invention comprises two culture mediums, LB culture medium and M9 basic salt culture medium. The formula is respectively (the solvent is water):

LB culture medium: 10g/L of tryptone, 5g/L of yeast extract and 10g/L of sodium chloride.

M9 minimal salts medium:

macroelements: 4% carbon source (xylose), NaH₂PO₄·7H₂O 12.8g/L、KH₂PO₄3g/L, 0.5g/L, NH g of sodium chloride₄Cl1g/L、MgSO₄0.24g/L、CaCl₂0.011g/L。

Trace element FeCl₃·6H₂O 3.2mg/L、CoCl₂·6H₂O 0.4mg/L、CuCl₂·2H₂O 0.2mg/L、ZnCl₂0.4mg/L、Na₂MoO₄·2H₂O 0.4mg/L、MnCl₂·4H₂O 0.4mg/L H₃BO₃0.1mg/L。

Stress-conditioned drugs such as sodium acetate, isobutanol and sodium chloride were purchased from sigma.

(http://www.sigmaaldrich.com/sigma-aldrich)

Example 1 construction of E.coli Global regulatory factor mutant library

The construction of the global regulatory factor mutation library is divided into two parts: design and synthesis of global regulatory factors and a diversity library flora containing global regulatory factor mutations.

Design and Synthesis of Global regulatory factors

CRISPR-based traceable genome engineering system (CREATE), comprising 3 sets of plasmids:

X2-Cas9 plasmid for expressing Cas9 protein induced by arabinose,

Temperature-sensitive pSIM5 plasmid for expressing lambda-red recombination system through heat shock induction

And gRNA and donor DNA expression global regulatory factor library plasmids (Garst et al, 2016).

Wherein the global regulatory factor library plasmids are mixed plasmids and are divided into 5 groups, and the 5 groups of library plasmids are composed of saturation mutations of corresponding sites of corresponding global regulatory factors and contain 34340 site saturation mutations in total. The process of designing and synthesizing the library plasmid is as follows:

firstly, according to literature research and analysis of the databases of regulatory factors, RegulonDB and EcoCyc, the 20 Escherichia coli global regulatory factors determined to be mutated specifically include: HNS, CspA, ArcA, Fur, NarL, Lrp, Mlc, IHF, CspE, ArgP, CRP, PhoB, CytR, DnaA, Rpos, Rpon, RpoE, FNR, SoxR, and FIS. In conjunction with databases such as: NCBI, Uniprot, etc. and protein modeling prediction information software such as: I-TASSER (http:// zhangglab. ccmb. med. umich. edu/I-TASSER) collects and extracts the information of the key action sites of 20 global regulatory factors, such as active sites, DNA binding sites, ligand binding sites, metal binding sites and dimer action sites, and the like, and uses the information as saturation mutation sites. Then, a global regulatory factor mutant library automated design tool Bioverse (http:// www.thebioverse.org /) was used to design the mutant library. Next, the designed library sequences were synthesized by Agilent Technologies, Inc. USA, the CREATE library fragments synthesized by the company were first subjected to 20 PCR extension reactions, and the extension products were separated and purified using 6% PAGE gel. The purified product is amplified by sub-libraries by using designed primers carrying different barcodes (the primer sequence table of the amplified library is shown in table 1), and finally is connected to the vector plasmid of the global regulatory factor mutation library by using a Gibson assembly method, so that plasmid sub-libraries of 5 different global regulatory factors containing 34340 mutations of 20 global regulatory factors are obtained, and the sub-libraries are divided into 5 global regulatory factor libraries according to the functions of mutation sites, wherein the 5 global regulatory factor libraries respectively comprise: g1 with a functional site being a live site, G2 and G3 with a functional site being a DNA binding site, G4 with a functional site being a dimer, and G5 with a predicted site.

TABLE 1 amplification library primer sequence Listing

Diverse library flora containing global regulator mutations

In addition to the above plasmids, a blank plasmid (nrg) (garstel., 2016) was subsequently used as a blank.

Firstly, X2-cas9 and pSIM5 plasmids are sequentially introduced into MG1655 through electrotransfer (2.5kv) to obtain MGZC (MG1655, harboring pSIM5, harboring cas9) strains, then 5 mutant library plasmids and a control plasmid (nrg) are respectively introduced into MGZC through electrotransfer, recovered at 30 ℃, 3h and LB culture medium is recovered, then inoculated into 50ml LB culture medium at 37 ℃, 220rpm and LB culture is carried out overnight, and then bacteria are preserved to obtain 5 global mutant library (G1, G2, G3, G4 and G5) floras (hereinafter, G1, G2, G3, G4 and G5) and a control strain (nrg), and the global mutant library is preserved at-80 ℃ for standby.

Example 2 enrichment screening of different mutant library recombinant bacteria under sodium acetate stress conditions

Based on the Escherichia coli global regulatory factor mutant library flora constructed in the embodiment 1, enrichment of the mutant flora of the forward library is carried out by gradually increasing the screening pressure under the condition of 10g/L-30g/L sodium acetate stress. The activated culture medium in the screening process is LB culture medium, and the screening culture medium is M9 basic salt culture medium.

First, 1ml of the thawed bacterial liquid of 6 strains (G1, G2, G3, G4, G5 and ntg) stored at-80 ℃ is inoculated into 5ml of LB test tube (15ml standard test tube) and cultured overnight at 37 ℃ and 220rpm, and then 6ml of the bacterial liquid is inoculated into 50ml of LB culture medium (250ml standard conical flask) and cultured to OD₆₀₀At 5-6, the initial cell concentration OD₆₀₀The initial OD was transferred to M9 minimal salt medium with 10g/L sodium acetate final concentration for screening at 0.1 to enrich the population of forward mutant library. Waiting for advantages in the culture processWhen the flora grows to the middle logarithmic growth stage, the initial thallus concentration OD is also used₆₀₀The initial OD (0.1) is transferred to the next M9 basic salt culture medium containing high-concentration sodium acetate, tolerance screening is carried out, the experiment is continuously transferred for 3 times, and the concentration of the selected sodium acetate is 10g/L, 20g/L and 30g/L in sequence. After each stress condition is screened, the strains are preserved, placed at minus 80 ℃ and preserved for later use. The thallus growth conditions of different mutant library recombinant bacteria under different sodium acetate concentrations are obtained by measuring the biomass of the different mutant library recombinant bacteria under different sodium acetate concentrations. FIG. 1 shows the biomass (48h biomass) of recombinant bacteria of different mutant libraries at a sodium acetate concentration of 20 g/L.

The forward mutant flora is enriched by the tolerance screening, and a library recombinant flora capable of tolerating 20g/L of sodium acetate is obtained, and comprises the following steps: g1, G2, G3, G4 and G5.

Because of the mutant flora constructed by means of the CREATE technique, the E.coli global regulator mutations in each cell after the tolerance screening were consistent with those of the CREATE plasmid library carried by it. Therefore, plasmids are extracted from the 5 dominant recombinant floras and used for deep sequencing and fitness fraction calculation, and the mutant escherichia coli global regulatory factor with high fitness is obtained.

Deep sequencing is carried out by designing Illumina compatible primers by using reported Golaybarcodes (Hamady et al, 2008), and amplifying CREATE library by 20 rounds of PCR reaction by using mixed plasmids as templates. The PCR reaction product was detected by agarose gel electrophoresis and purified using a kit. And distributing an experimental sequencing sequence according to the Illumina sequencing requirement, and carrying out Illumina deep sequencing. The fitness score is also called an enrichment score, and the formula is as follows:

fx, f is the frequency of CREATE box X after screening enrichment, Fx, i is the frequency of CREATE box X before screening, W indicates the absolute fitness of each mutation, and the frequency of mutations refers to the proportion of each mutation in the sequence to all read counts in the sequence. The fitness contribution of each mutation under sodium acetate screening stress conditions was evaluated by evaluation of fitness scores. The experimental data were analyzed and the data enriched for each tolerability screen reached a 95% confidence interval estimate, concluding that the fitness score of the mutation was statistically significant as a potential tolerability forward mutation.

In summary, in this example, 1 potential forward mutation associated with sodium acetate tolerance was obtained by replacing amino acid G (glycine) at position 142 of the E.coli global regulator CRP with I (isoleucine) and the enrichment score was 5.0946.

The nucleotide sequence of the gene obtained from the mutation site of the Escherichia coli global regulatory factor CRP in which the 142 rd amino acid G is replaced by I is shown as SEQ ID NO. 2.

Example 3 construction of mutant Strain CRP-G142I

The CRP potential tolerance forward mutation obtained in example 2 was subjected to non-nick editing of the genome of wild-type E.coli MG1655 using an I-Scel-based DNA double strand break gene recombination technique, starting from wild-type E.coli MG1655, to introduce the mutation of interest into the genome, to obtain a reconstructed strain. And then, the obtained mutant restructured strains are respectively cultured under the tolerance condition of 30g/L sodium acetate, and the effect of the escherichia coli global regulatory factor mutation obtained by screening is further verified. The specific operation is as follows:

construction of mutant reconstitution strains:

(1) preparation of recombinant fragments for crp Gene editing

The preparation of the recombinant fragment is divided into 3 steps

First, using wild type Escherichia coli MG1655 genome DNA as a template, PCR amplification is carried out by using high Fidelity enzyme Phata Max Super-Fidelity DNA polymerase (hereinafter referred to as Phata DNA polymerase) and primer CRP-F1(SEQ ID NO.8)/CRP-142I-L1(SEQ ID NO.9) and CRP-142I-F2(SEQ ID NO.10)/CRP-L2(SEQ ID NO.11) to obtain upstream (580bp) and downstream (554bp) homology arms for introducing mutation, which are respectively called fragment I (580bp) and fragment IV (554 bp).

And secondly, using pTKS/CS plasmid ((Lin et al, 2014) DNA as a template, and using high fidelity enzyme PhataDNA polymerase and primers CRP142I-Tet-F1(SEQ ID NO.12)/Tet-R (SEQ ID NO.15), Tet-F (SEQ ID NO.14)/CRP142I-Tet-L2(SEQ ID NO.13) (carrying out PCR amplification respectively to obtain two fragments (called fragments II and III) of the tetracycline resistance gene tag, wherein the sizes of the gene fragments are respectively about 900bp and 800bp, and the two fragments comprise an I-SceI recognition site sequence and a tetracycline gene promoter sequence.

And thirdly, performing fusion PCR amplification on the fragments I, II, III and IV obtained in the first two steps in stages to obtain an I + II fusion fragment (using a primer CRP-F1(SEQ ID NO.8)/Tet-R (SEQ ID NO.15)), and a III + IV fusion fragment (using a primer Tet-F (SEQ ID NO.14)/CRP-L2(SEQ ID NO.11)), and further performing PCR amplification by using a primer CRP-F1(SEQ ID NO.8)/CRP-L2(SEQ ID NO.11) to obtain an I + II + III + IV fusion fragment, wherein the amplification enzyme is high-fidelity Phata DNA polymerase. The finally obtained fusion fragment (I + II + III + IV) is a recombination fragment containing the upstream and downstream homology arms of the mutated crp gene, the I-SceI recognition site sequence and the tetracycline gene.

(2) Electrotransformation, positive clone screening and validation of recombinant fragments for traceless genome editing

The fusion fragment (I + II + III + IV) obtained in the third step of (1) was introduced into wild-type MG1655 digested with helper plasmid pTKRED by electroporation (1.8kv) as a starting bacterium at 30 ℃ for 3 hours at 220rpm, 100. mu.L of the bacterial solution was applied to an LB plate containing IPTG (2mM), spectinomycin (final concentration of 100ug/L) and tetracycline (final concentration of 20ug/L), after overnight culture at 30 ℃, 5 single colonies were selected for PCR-confirmation, sequencing-confirmation was performed using primer CRP-F1(SEQ ID NO. 8)/L2 (SEQ ID NO.11), correct mutation-introduced single colonies were selected in 15ml of CRP-containing LB medium (final concentration of 2mMIP, 0.4% arabinose, 100ul/ml of CRP), culturing at 220rpm and 30 ℃ for 24h, then taking the bacterial liquid and coating the bacterial liquid on an LB plate containing the spectinomycin with the final concentration of 2mMIPTG, 0.4% arabinose and 100ul/ml, then picking out the single colony which grows out and carrying out colony PCR and sequencing verification by using a primer CRP-F1(SEQ ID NO.8)/CRP-L2(SEQ ID NO.11) to obtain a recombinant strain which is successfully introduced with mutation and is named as CRP-G142I: and the strain was stored at-80 ℃ until use. The primers used are shown in Table 2.

TABLE 2 primer sequences used for the construction of the strains

Example 4 evaluation of sodium acetate tolerance of mutant Strain CRP-G142I

The mutant strain and the wild strain MG1655 (control strain) constructed in the above example 3 were inoculated into a 15ml test tube of LB medium for 12 hours, and then 1ml of the strain was inoculated into a 250ml Erlenmeyer flask of LB medium for 12 hours to activate the seeds. Acetate tolerance was verified by inoculating the above activated seeds at an initial OD of 0.1 in M9 minimal salt medium containing sodium acetate at a final concentration of 30g/L, and measuring the OD once at 12h, resulting in tolerance of the mutant dominant recombinant strain as well as the wild type control strain as shown in FIG. 2.

In conclusion, according to the growth situation, the global regulatory factor CRP mutation which is favorable for tolerating acetate is obtained:

the G142 position of the amino acid sequence shown in SEQ ID No.1 coded by the crp gene is replaced by I;

and CRP-G142I increased 1633.96% over the 48h biomass of the control under sodium acetate (30G/L) and 313.82% over that rate of growth.

Example 5 evaluation of sodium chloride tolerance of mutant Strain CRP-G142I

Similarly, the mutant strain CRP-G142I and the wild type strain MG1655 (control) constructed in example 3 were subjected to tolerance evaluation in 0.6mol/L NaCl in a manner similar to that in example 4, and as shown in FIG. 3, the mutant strain CRP-G142I had an advantage in growth over the wild type strain MG1655 (control) in 48h, and the biomass increased by 35.12%; and the maximum specific growth rate is improved by 75.89%.

Example 6 evaluation of Isobutanol tolerance of mutant Strain CRP-G142I

Also taking the mutant strain CRP-G142I and the wild type strain MG1655 (control) constructed in the above example 3 and carrying out the tolerance evaluation in 8G/L isobutanol similarly to the method in example 4 as shown in FIG. 4, it was found that the mutant strain CRP-G142I had a significant growth advantage after 12 hours compared with the wild type strain MG1655 (control) and the biomass at 36 hours was increased by 73.78% compared with the control and the maximum specific growth rate was increased by 37.25%.

In conclusion, the mutant strain CRP-G142I was obtained which was highly resistant to sodium acetate, osmotic pressure-related sodium chloride and isobutanol.

Reference documents:

[1]Chong H,Yeow J,Wang I,et al.Improving Acetate Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein(CRP)[J].PLOS ONE,2013,8.

[2]Ravcheev D A,Best A A,Sernova N V,et al.Genomic reconstruction of transcriptional regulatory networks in lactic acid bacteria[J].BMC Genomics,2013,14(1):94-94.

[3]Gottesman,Susan.Bacterial Regulation:Global Regulatory Networks[J].Annual Review of Genetics,1984,18(1):415-441.

[4]Garst A D,Bassalo M C,Pines G,et al.Genome-wide mapping of mutations at single-nucleotide resolution for protein,metabolic and genome engineering[J].Nature Biotechnology,2016,35(1):48-55.

[5]Hamady M,Walker J J,Harris J K,et al.Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex[J].NATURE METHODS,2008,5(3):235-237.

[6]Lin Z,Xu Z,Li Y,et al.Metabolic engineering of Escherichia coli for the production of riboflavin[J].Microbial Cell Factories,2014,13(1).

[7]Kuhlman T E,Cox E C.Site-specific chromosomal integration of large synthetic constructs[J].Nucleic Acids Research,2010,38(6):e92-e92.

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gctattcttc ctgccacaa 19

Claims

1. The construction method of the escherichia coli with resistance to sodium acetate, sodium chloride and isobutanol is characterized by comprising the following steps: introducing the screened gene of the mutation site, wherein the 142 rd amino acid G of the Escherichia coli global regulatory factor CRP is replaced by I, into wild Escherichia coli to obtain Escherichia coli CRP-G142I resistant to sodium acetate, sodium chloride and isobutanol; the amino acid sequence of the colibacillus global regulatory factor CRP is shown in SEQ ID NO. 1; the nucleotide sequence of the gene obtained from the mutation site of the 142 th amino acid G of the colibacillus global regulatory factor CRP replaced by I is shown as SEQ ID NO. 2.

2. Coli CRP-G142I resistant to sodium acetate, sodium chloride and isobutanol constructed by the method of claim 1.