Application of gene pmtA in improving broad-spectrum tolerance of escherichia coli

Abstract

The invention relates to application of a gene pmtA in improving broad-spectrum tolerance of escherichia coli. According to the present invention, the research results show that the codon-optimized (derived from Rhodobacter sphaeroids) gene pmtA for coding phosphatidyl ethanolamine-N-methyltransferase EC 2.1. 1.17 is integrated to the escherichia coli genome ldhA site, such that phosphatidylcholine can be introduced to remodel the escherichia coli cell membrane phospholipid system so as to improve the broad-spectrum tolerance of escherichia coli; in addition, an artificial regulation element M1-12 and a gene pmtA are assembled to construct a tolerance module, and the tolerance module is integrated to a lactic dehydrogenase encoding gene ldhA site of an escherichia coli genome through a lambda Red homologous recombination method to obtain an engineering bacterium named + PC. According to the engineering strain provided by the invention, the broad-spectrum tolerance of the strain is improved, the methanol utilization capability of escherichia coli is remarkably enhanced while the tolerance of an intermediate toxic substrate formaldehyde is enhanced, and the TAL synthesis capability is remarkably enhanced while the tolerance to TAL is enhanced.

Description

Technical Field

[0001] The invention belongs to the field of microbial synthetic biology, and specifically relates to an application of a gene pmtA in improving the broad-spectrum tolerance of Escherichia coli. Background Art

[0002] Compared with the highly polluting and energy-intensive petroleum-based compound refining process, biomanufacturing technology has the advantages of being green and sustainable. The core of biomanufacturing is the microbial cell factory. As a model strain, Escherichia coli has excellent characteristics such as clear genetic background, short iteration time and mature gene editing technology. It is the preferred chassis cell for building microbial cell factories. Existing studies have shown that Escherichia coli can produce a variety of compounds, such as 1,3-succinic acid, TAL, citric acid, etc. However, studies have found that when using Escherichia coli to create a cell factory to produce the target product, the growth and product synthesis ability of the microbial cell factory are severely inhibited due to the toxic effect of the target product on the cell. There are many mechanisms by which synthetic products are toxic to cells, such as causing cell membrane damage, inducing reactive oxygen production, DNA damage, protein denaturation, etc. Recent studies have found that cell membrane damage is considered to be an important common mechanism for cell toxicity caused by various compounds.

[0003] The E. coli cell membrane is an elastic semipermeable membrane composed of a phospholipid bilayer and membrane proteins embedded therein, with a thickness of 8-10 nm. The phospholipid bilayer is the basic support of the cell membrane. The E. coli cell membrane has three types of phospholipid molecules, namely 70-75% phosphatidylethanolamine (PE), 15-20% phosphatidylglycerol (PG) and 5-10% cardiolipin (CL). The E. coli cell membrane plays an important role in maintaining the stability of the intracellular environment and exchanging material information and energy with the outside world, and damage to the cell membrane will seriously affect the function of the cell.

[0004] Building a robust cell membrane is an effective way to solve the problem of membrane damage. At present, the transformation of cell membrane mainly includes the following aspects: on the one hand, by regulating the configuration of unsaturated fatty acids at the tail of phospholipid molecules, regulating the length of fatty acids at the tail of phospholipid molecules, or regulating the integration of non-straight-chain fatty acids into the tail of phospholipid molecules, the fluidity of cell membrane is reduced; on the other hand, by regulating the fatty acid composition at the tail of phospholipid molecules, regulating the distribution of phospholipid head groups and regulating the abundance of membrane proteins, the integrity of cell membrane is improved.

[0005] At present, there are no reports in China on introducing phosphatidylcholine (PC) molecules into the cell membrane of Escherichia coli to enhance the tolerance of Escherichia coli to various compounds. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an application of a gene pmtA in improving the broad-spectrum tolerance of Escherichia coli, including the application of the gene pmtA and the protein expressed therein and a recombinant vector comprising a tolerance module M12-pmtA composed of an artificial regulatory element M1-12 and the gene pmtA in improving the broad-spectrum tolerance of Escherichia coli. In addition, the present invention also provides an engineering strain (named +PC) for introducing the gene pmtA or the tolerance module M12-pmtA into Escherichia coli and a construction method thereof, which is characterized in that phosphatidylcholine (PC) is introduced to reshape the phospholipid system of the cell membrane of Escherichia coli, and the cell membrane contains phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylcholine (PC) and cardiolipin (CL). An Escherichia coli strain introducing the gene pmtA or the tolerance module M12-pmtA and the triacetin synthesis module is provided, and the results show that the introduction of phosphatidylcholine significantly enhances the TAL synthesis ability while enhancing the tolerance to TAL. Provided is an Escherichia coli strain in which the gene pmtA or a tolerance module is integrated with a methanol utilization chassis strain, and the growth curve and substrate consumption are measured. The results show that the introduction of phosphatidylcholine significantly enhances the methanol utilization ability of Escherichia coli while enhancing the tolerance to the intermediate toxic substrate formaldehyde.

[0007] The method for introducing phosphatidylcholine into the cell membrane is as follows: the artificial regulatory element M1-12 is assembled with the codon-optimized pmtA gene (encoding phosphatidylethanolamine-N-methyltransferase EC2.1.1.17) from Rhodobacter sphaeroides to construct a tolerance module, and the tolerance module is integrated into the ldhA (encoding lactate dehydrogenase) site of the Escherichia coli genome by the λRed homologous recombination method to obtain an engineered strain (named +PC).

[0008] The tolerance analysis method is: in the presence of different concentrations of inhibitors and inhibitory conditions, a broad-spectrum tolerance analysis is performed by measuring the growth curve of the engineered strain (+PC), and inhibitors and inhibitory conditions with significantly improved tolerance are screened.

[0009] The first aspect of the present invention provides an application of a gene pmtA in improving the broad-spectrum tolerance of Escherichia coli.

[0010] The second aspect of the present invention provides an application of a protein expressed by a gene pmtA in improving the broad-spectrum tolerance of Escherichia coli.

[0011] The third aspect of the present invention provides an application of a recombinant vector in improving the broad-spectrum tolerance of Escherichia coli.

[0012] A fourth aspect of the present invention provides an Escherichia coli strain with improved broad-spectrum tolerance.

[0013] The fifth aspect of the present invention provides a method for constructing the Escherichia coli strain with improved broad-spectrum tolerance.

[0014] A sixth aspect of the present invention provides an Escherichia coli strain for synthesizing triacetic acid lactone.

[0015] A seventh aspect of the present invention provides an Escherichia coli strain with improved methanol utilization.

[0016] An eighth aspect of the present invention provides a method for constructing an Escherichia coli strain with improved methanol utilization.

[0017] The objective of the present invention is achieved through the following technical solutions:

[0018] In a first aspect, the present invention provides an application of a gene pmtA in improving the broad-spectrum tolerance of Escherichia coli. The nucleotide sequence of the gene pmtA is shown in SEQ ID NO.1.

[0019] The gene pmtA of the present invention is a codon-optimized gene pmtA (derived from Rhodobacter sphaeroides) encoding phosphatidylethanolamine-N-methyltransferase EC 2.1.1.17. The pmtA gene is capable of synthesizing phosphatidylcholine and remodeling the phospholipid system of the Escherichia coli cell membrane.

[0020] As an embodiment of the present invention, the broad-spectrum tolerant inhibitor includes any one of furfural, 5-methylfurfural, vanillin, formaldehyde, lactic acid, ethanol, n-butanol, isobutanol, triacetic acid lactone (TAL), limonene, and sodium chloride.

[0021] As an embodiment of the present invention, the Escherichia coli includes any one of K-12MG1655 and K-12MG1655(DE3).

[0022] In a second aspect, the present invention provides an application of a protein expressed by a gene pmtA in improving the broad-spectrum tolerance of Escherichia coli, wherein the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0023] In a third aspect, the present invention provides an application of a recombinant vector in improving the broad-spectrum tolerance of Escherichia coli, wherein the recombinant vector comprises a tolerance module M12-pmtA, and the M12-pmtA comprises an artificial regulatory element M1-12 and the gene pmtA.

[0024] As an embodiment of the present invention, the nucleotide sequence of the artificial regulatory element M1-12 is shown in SEQ ID NO.3.

[0025] In a fourth aspect, the present invention provides an Escherichia coli strain with improved broad-spectrum tolerance, which is obtained by taking Escherichia coli as a starting strain, knocking out the gene ldhA in the Escherichia coli genome, and integrating the gene pmtA or the tolerance module M12-pmtA in the recombinant vector into the ldhA site.

[0026] As one embodiment of the present invention, the Escherichia coli includes K-12MG1655 Escherichia coli.

[0027] As one embodiment of the present invention, the gene ldhA is the gene ldhA encoding lactate dehydrogenase.

[0028] Furthermore, the nucleotide sequence of the gene ldhA is shown in SEQ ID NO.4.

[0029] As an embodiment of the present invention, the knockout method comprises λRed homologous recombination; and the integration method comprises one-step homologous recombination.

[0030] The present invention integrates the gene pmtA into the ldhA site to obtain a strain, wherein the integration process includes: integrating the gene pmtA and its promoter sequence into the ldhA site together;

[0031] Alternatively, the tolerance module M12-pmtA is integrated into the ldhA locus.

[0032] In some preferred embodiments, the present invention utilizes the λRed homologous recombination method to replace the lactate dehydrogenase ldhA gene of Escherichia coli with the pmtA gene, and uses M1-12 as an artificial regulatory element of pmtA to obtain an engineered strain (+PC), denoted as MG1655ΔldhA::FKF-M12-pmtA.

[0033] In a fifth aspect, the present invention provides a method for constructing the Escherichia coli strain with improved broad-spectrum tolerance, comprising the following steps:

[0034] S1, using plasmid pKD4, strain M1-12, and plasmid pUC-pmtA as templates, PCR amplification was performed to obtain the FKF fragment, M1-12 fragment, and pmtA fragment;

[0035] S2, assembling the FKF fragment, the M1-12 fragment and the pmtA fragment;

[0036] or, assembling the FKF fragment and the pmtA fragment to obtain the FMP1 fragment and the FMP2 fragment, respectively;

[0037] S3. Transform the FMP1 fragment or the FMP2 fragment into competent cells of Escherichia coli MG1655 containing plasmid pKD46, eliminate the plasmid pKD46, and identify to obtain the Escherichia coli strain with improved broad-spectrum tolerance.

[0038] As an embodiment of the present invention, in step S1, the nucleotide sequence of the FKF fragment is shown as SEQ ID NO. 5. The nucleotide sequences of the M1-12 fragment and the pmtA fragment are shown as SEQ ID NO. 3 and SEQ ID NO. 1, respectively.

[0039] As an embodiment of the present invention, in step S2, the nucleotide sequence of the FMP1 fragment is as shown in SEQ ID NO.6.

[0040] As an embodiment of the present invention, in step S3, the Escherichia coli competent cells containing the pKD46 plasmid are obtained by transforming the pKD46 plasmid into Escherichia coli using a heat shock transformation method, and then electro-transformed into competent cells to obtain Escherichia coli competent cells containing the pKD46 plasmid, which are recorded as MG1655 (pKD46) electro-transformed competent cells.

[0041] In a sixth aspect, the present invention provides an Escherichia coli strain for synthesizing triacetic acid lactone, which is obtained by using the Escherichia coli strain with improved broad-spectrum tolerance as a starting strain and transferring the pTrc99A-bktB plasmid into the strain.

[0042] As one embodiment of the present invention, the nucleotide sequence of the pTrc99A-bktB plasmid is shown as SEQ ID NO.7.

[0043] As one embodiment of the present invention, the transfer method comprises electroporation.

[0044] In some preferred embodiments, the broad-spectrum tolerance-enhanced Escherichia coli is MG1655ΔldhA::FKF-M12-pmtA. The Escherichia coli strain synthesizing triacetic acid lactone of the present invention (denoted as MG1655ΔldhA::FKF-M12-pmtA (pTrc99A-bktB)) enhances tolerance to triacetic acid lactone and significantly enhances the ability to synthesize triacetic acid lactone.

[0045] In the seventh aspect, the present invention provides an Escherichia coli strain with improved methanol utilization. The strain is obtained by taking Escherichia coli as the starting strain, knocking out the genes frmA, rpiA, rpiB, ldhA and cyaA in the Escherichia coli genome, integrating the gene pmtA or the tolerance module M12-pmtA in the recombinant vector into the ldhA site, and transferring the plasmid pCDF-CT4-1mdh-hps-phi into the strain.

[0046] As one embodiment of the present invention, the Escherichia coli includes Escherichia coli K-12 MG1655 (DE3).

[0047] As an embodiment of the present invention, the nucleotide sequences of the gene frmA, gene rpiA, gene rpiB and gene cyaA are shown as SEQ ID NOs. 8-11, respectively.

[0048] In an eighth aspect, the present invention provides a method for constructing an Escherichia coli strain with improved methanol utilization, comprising the following steps:

[0049] A1, using pETduet-nudF-mdh2-hps-phi plasmid and pCDFduet-CT4-1 mdh plasmid as templates, PCR amplification was performed to obtain hps-phi fragment and pCDF-P2 fragment;

[0050] A2, assemble the hps-phi fragment and the pCDF-P2 fragment, and transfer them into DH5α chemically competent cells to screen and obtain the plasmid pCDF-CT4-1 mdh-hps-phi;

[0051] A3, knocking out the genes frmA, rpiA, rpiB, ldhA and cyaA of Escherichia coli, and integrating the gene pmtA or the tolerance module M12-pmtA into the ldhA site to obtain a strain;

[0052] A4. Transform the plasmid pCDF-CT4-1 mdh-hps-phi into the strain, and identify to obtain the Escherichia coli strain with improved methanol utilization.

[0053] As an embodiment of the present invention, in step A1, the nucleotide sequences of the hps-phi fragment and the pCDF-P2 fragment are shown as SEQ ID NO.12-13 respectively.

[0054] As an embodiment of the present invention, in step A2, the transfer method includes the induced transformation method.

[0055] As an embodiment of the present invention, in step A2, the nucleotide sequence of the plasmid pCDF-CT4-1 mdh-hps-phi is shown in SEQ ID NO.14.

[0056] In some preferred embodiments, in step A3 or A4, the strain is a strain obtained by integrating the tolerance module M12-pmtA into the ldhA site, which is recorded as MG1655(DE3)ΔfrmAΔrpiAΔrpiBΔldhA:M12-pmtA strain.

[0057] In some preferred embodiments, in step A4, the Escherichia coli strain with improved methanol utilization is MG1655(DE3)ΔfrmAΔrpiAΔrpiBΔldhA:M12-pmtAΔcyaA::FKF strain.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] 1. The present invention found that integrating the codon-optimized gene pmtA (derived from Rhodobacter sphaeroides encoding phosphatidylethanolamine-N-methyltransferase EC 2.1.1.17) into the ldhA site of the Escherichia coli genome can introduce phosphatidylcholine to reshape the phospholipid system of the Escherichia coli cell membrane, thereby improving the broad-spectrum tolerance of Escherichia coli.

[0060] 2. The present invention assembles the artificial regulatory element M1-12 and the gene pmtA to construct a tolerance module, and integrates it into the ldhA site of the gene encoding lactate dehydrogenase in the Escherichia coli genome to obtain an engineered strain +PC. The engineered strain +PC introduces phosphatidylcholine to reshape the phospholipid system of the Escherichia coli cell membrane, thereby improving the broad-spectrum tolerance of the strain.

[0061] 3. The engineered strain + PC provided by the present invention introduces phosphatidylcholine to reshape the phospholipid system of the Escherichia coli cell membrane, thereby enhancing the tolerance to the intermediate toxic substrate formaldehyde and significantly enhancing the methanol utilization ability of Escherichia coli.

[0062] 4. The engineered strain + PC provided by the present invention introduces phosphatidylcholine to reshape the phospholipid system of the Escherichia coli cell membrane, thereby enhancing tolerance to TAL and significantly enhancing TAL synthesis capacity. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

[0064] Figure 1 A conceptual diagram of the phosphatidylcholine synthesis pathway is introduced for the present invention;

[0065] Figure 2 The quantitative detection results of phosphatidylcholine in the cell membrane of +PC strain in Example 3;

[0066] Figure 3 The results of the broad-spectrum tolerance analysis of the engineered strain + PC compared to the control strain Control in Example 4;

[0067] Figure 4The results of the cell membrane integrity analysis of the engineered strain + PC compared to the control strain Control in Example 5;

[0068] Figure 5 The results of the analysis of the fatty acid composition of the cell membrane of the engineered strain + PC compared with the control strain Control in Example 6;

[0069] Figure 6 Concept diagram for the construction of a strain for the coordinated introduction of phosphatidylcholine into the methanol-utilizing chassis strain in Example 7;

[0070] Figure 7 The growth curves of the MK-1 strain and the MK-2 strain in the methanol growth medium in Example 7;

[0071] Figure 8 The results of xylose and methanol consumption of MK-1 strain and MK-2 strain in methanol growth medium in Example 7 are shown in the figure on the left; the results of xylose consumption and the results of methanol consumption are shown on the right;

[0072] Fig. 9 The growth curves of the MK-3 strain and the MK-4 strain in Example 8 in LB like MOPS medium;

[0073] Fig.10 These are the triacetic acid lactone (TAL) production results of the MK-3 strain and the MK-4 strain in Example 8. DETAILED DESCRIPTION

[0074] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples are implemented on the premise of the technical solution of the present invention, provide detailed implementation methods and specific operation processes, and will help those skilled in the art to further understand the present invention. It should be pointed out that the protection scope of the present invention is not limited to the following embodiments, and several adjustments and improvements made under the premise of the concept of the present invention all belong to the protection scope of the present invention.

[0075] The concentrations of inducers and antibiotics in the embodiments of the present invention are shown in the following table:

[0076] name Solvents Working concentration Sterilization method Storage conditions Kanamycin <![CDATA[ddH 2 The]]> 50 μg / mL 0.22μm filter membrane for sterilization -20℃ Ampicillin <![CDATA[ddH 2 The]]> 100 μg / mL 0.22μm filter membrane for sterilization -20℃ Spectinomycin <![CDATA[ddH 2 The]]> 50 μg / mL 0.22μm filter membrane for sterilization -20℃ L-Arabinose <![CDATA[ddH 2 The]]> 10mM 0.22μm filter membrane for sterilization 4℃ IPTG <![CDATA[ddH 2 The]]> 0.1mM 0.22μm filter membrane for sterilization -20℃

[0077] The kit in the embodiment of the present invention is shown in the following table:

[0078]

[0079]

[0080] It should be noted that the Escherichia coli MG1655 in the embodiment of the present invention is K-12 Escherichia coli MG1655, and the Escherichia coli MG1655 (DE3) is K-12MG1655 (DE3). The nucleotide sequence of the tolerance module M12-pmtA is shown in SEQ ID NO.93.

[0081] Example 1: Construction of control strain Control

[0082] The control strain Control is characterized by knocking out the lactate dehydrogenase gene ldhA (GenBank No: YP_001725238.1; GI: 170020284) site of Escherichia coli MG1655. The construction method is specifically divided into the following two steps:

[0083] 1.1 Obtaining DNA fragment KO-1

[0084] Using pKD4 plasmid (reference Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products [J]. Proceedings of the National Academy of Sciences, 2000, 97 (12): 6640-6645.) DNA as a template and using the primer pair ldhA-FKF-F / ldhA-KO-R (as shown in SEQ ID NO.15-16), a 1649 bp DNA fragment was amplified and named KO-1 (as shown in SEQ ID NO.17). The fragment includes a 59 bp DNA fragment upstream of the ldhA site of Escherichia coli MG1655, a DNA fragment encoding a kanamycin resistance gene required for the λ Red homologous recombination method, a DNA fragment encoding two FRT sites, and a 61 bp DNA fragment downstream of the ldhA site of Escherichia coli MG1655.

[0085] The amplification system is:

[0086] Component Volume(μL) 2×Phanta max buffer 25 dNTP (10mM each) 1 ldhA-FKF-F 2 ldhA-KO-R 2 Phanta max Super-Fidelity DNA polymerase 1 pKD4 plasmid 1 <![CDATA[ddH 2 The]]> up to 50

[0087] The reaction procedure is:

[0088]

[0089] 1 μL DpnI was added to the PCR product and digested at 37°C for 30 minutes to eliminate the pKD4 plasmid template. After detection on 1% agarose gel, the correct DNA fragment was purified using a DNA gel recovery kit.

[0090] 1.2 Construction of control strains

[0091] (1) The pKD46 plasmid (reference: Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products [J]. Proceedings of the National Academy of Sciences, 2000, 97(12): 6640-6645) was transformed into Escherichia coli MG1655 by heat shock transformation (42°C, 90 seconds) to obtain the MG1655 (pKD46) strain.

[0092] (2) Preparation of MG1655 (pKD46) electroporation competent cells: E. coli MG1655 (pKD46) was inoculated into LB liquid medium (the formula is shown in Table 1) and shaken at 37°C and 200 rpm until the OD 550 =0.3-0.4, collect the bacteria and place them in an ice bath for 20 minutes, then centrifuge at 4°C, 5400 rpm and wash the bacteria three times with ice-cold sterile 10% glycerol (wt:vol), and dispense them into 50 μL / tubes for electroporation of competent cells of MG1655 (pKD46).

[0093] (3) Electroporation: Place 50 μL of MG1655 (pKD46) electroporation competent cells on ice, add 100 ng of KO-1 DNA fragment, place on ice for 3 minutes, and transfer to a 0.1 cm Bio-Rad electroporation cup. Use a Micro-Pulser electroporator with a voltage parameter of 1.8 kV for electroporation. After electroporation, quickly add 1 mL of sterile LB medium to the electroporation cup, pipette to mix, and transfer to a 15 mL sterile centrifuge tube. Resume culture at 37°C, 200 rpm for 2 hours while the pKD46 plasmid is removed.

[0094] (4) Take an appropriate amount of bacterial solution and spread it on LB plate containing Kanamycin (50 mg / L) and culture it upside down in a 37°C incubator overnight.

[0095] (5) After the colonies grew, 8 monoclonal colonies were selected for transfer and colony PCR was performed using a 2× Rapid Taq Master Mix kit. The primer pair was verified to be pmtA-ZH-F / pmtA-ZH-R (as shown in SEQ ID NOs. 18-19), and a 1723 bp target DNA fragment was amplified.

[0096] The colony PCR reaction is:

[0097] Component Volume(μL) 2× Rapid Taq Master Mix 25 pmtA-ZH-F 2 pmtA-ZH-R 2 <![CDATA[ddH 2 The]]> 21

[0098] The reaction procedure is:

[0099]

[0100] The colonies with correct verification were selected for sequencing, and the sequencing primer pair was pmtA-CX-F / pmtA-CX-R (as shown in SEQ ID NO.20-21). The colonies with correct sequencing were inoculated in LB liquid medium containing 50 mg / L Kanamycin, and the culture was shaken at 37°C and 200 rpm for 12 hours, and maintained with 50% glycerol (wt:vol). It was named Control, and the genotype was MG1655ΔldhA::FKF.

[0101] Table 1 LB medium formula

[0102]

[0103] Example 2: Construction of recombinant engineering strain + PC

[0104] The engineered strain (named +PC) is characterized by knocking out the lactate dehydrogenase gene ldhA (GenBank No: YP_001725238.1; GI: 170020284) site of Escherichia coli MG1655, and inserting the artificial regulatory element M1-12 and the codon-optimized pmtA gene from Rhodobacter sphaeroides (synthesized by GENEWIZ) at the ldhA site. Figure 1 The introduction of the phosphatidylcholine synthesis pathway is shown.

[0105] 2.1 Obtaining DNA fragment FKF

[0106] Using pKD4 plasmid (purchased from addgene) DNA as a template and using primer pair ldhA-FKF-F / FKF-R (as shown in SEQ ID NO.22), a 1599 bp DNA fragment was amplified and named FKF (as shown in SEQ ID NO.5), which included a 50 bp DNA homologous fragment upstream of the ldhA site of Escherichia coli MG1655, a DNA fragment encoding a Kanamycin resistance gene required for the one-step homologous recombination method, and a DNA fragment encoding two FRT sites.

[0107] The amplification system is:

[0108] Component Volume(μL) 2×Phanta max buffer 25 dNTP (10mM each) 1 ldhA-FKF-F 2 FKF-R 2 Phanta max Super-Fidelity DNA polymerase 1 pKD4 plasmid 1 <![CDATA[ddH 2 The]]> up to 50

[0109] The reaction procedure is:

[0110]

[0111] Add 1 μL DpnI to the PCR product and digest it at 37°C for 30 minutes to eliminate the pKD4 plasmid template. After detection on agarose gel, use a DNA gel recovery kit to purify the correct DNA fragment.

[0112] 2.2 Obtaining DNA fragments of artificial regulatory element M1-12

[0113] Using the genomic DNA of strain M1-12 (reference Lu J, Tang J, Liu Y, et al. Combinatorial modulation of galP and glk gene expression for improved alternative glucose utilization [J]. Applied Microbiology and Biotechnology, 2012, 93 (6): 2455-2462.) as a template and primers M1-FKF-F / M1-pmtA-R (SEQ ID NO. 23-24), a 128 bp DNA fragment was amplified and named M1-12 fragment (SEQ ID NO. 3). The fragment included a 20 bp homologous fragment at the end of the DNA fragment FKF, an artificial regulatory element M1-12 fragment, and a 20 bp homologous fragment at the end of the pmtA gene.

[0114] The amplification system is:

[0115] Component Volume(μL) 2×Phanta max buffer 25 dNTP (10mM each) 1 M1-FKF-F 2 M1-pmtA-R 2 Phanta max Super-Fidelity DNA polymerase 1 M1-12 genomic DNA 1 <![CDATA[ddH 2 The]]> up to 50

[0116] The reaction procedure is:

[0117]

[0118] After detection on 1% agarose gel, the correct DNA fragment was purified using a DNA gel recovery kit.

[0119] 2.3 Obtaining DNA fragment pmtA

[0120] Using plasmid pUC-pmtA (synthesized by Jinweizhi Biotechnology Co., Ltd., plasmid pUC-pmtA is a pUC plasmid carrying a codon-optimized pmtA gene sequence) as a template, using primer pair pmtA-F / pmtA-ldhA-R (as shown in SEQ ID NO.25-26), a 672 bp DNA fragment was amplified and named pmtA fragment (as shown in SEQ ID NO.1), which includes a DNA fragment of the pmtA gene and a homologous DNA fragment 60 bp downstream of the ldhA site of the Escherichia coli MG1655 genome.

[0121] The amplification system is:

[0122]

[0123]

[0124] The reaction procedure is:

[0125]

[0126] 1 μL DpnI was added to the PCR product and digested at 37°C for 30 minutes to eliminate the pUC-pmtA plasmid template. After detection on 1% agarose gel, the correct DNA fragment was purified using a DNA gel recovery kit.

[0127] 2.4 Obtaining the linear DNA fragment FMP1

[0128] The DNA fragments FKF, M1-12, and pmtA were assembled using the Seamless Cloning Kit. The assembly system is:

[0129] Component Volume(μL) 2×Seamless Cloning Mix 5 FKF:M1-12:pmtA=1:5:1 (molar ratio) 5 Total volume 10

[0130] The assembly conditions were: 50° C., 30 minutes. Using the assembled product as a template and pmtA-ZH-F / pmtA-ZH-R as primers, a 2328 bp linear DNA fragment was amplified and named FMP1 (as shown in SEQ ID NO.6).

[0131] The amplification system is:

[0132] Component Volume(μL) 2×Phanta max buffer 25 dNTP (10mM each) 1 pmtA-ZH-F 2 pmtA-ZH-R 2 Phanta max Super-Fidelity DNA polymerase 1 Assembled products 1 <![CDATA[ddH 2 The]]> up to 50

[0133] The reaction procedure is:

[0134]

[0135] After detection on 1% agarose gel, the correct DNA fragment was purified using a DNA gel recovery kit.

[0136] Construction of 2.5+PC strain

[0137] (1) The pKD46 plasmid was transformed into Escherichia coli MG1655 by heat shock transformation (42°C, 90 seconds) to obtain the MG1655 (pKD46) strain.

[0138] (2) Preparation of MG1655 (pKD46) electroporation competent cells: E. coli MG1655 (pKD46) was inoculated into LB liquid medium and shaken at 37°C and 200 rpm until the OD 550 =0.3-0.4, collect the bacteria and place them in an ice bath for 20 minutes, then centrifuge at 4°C, 5400 rpm and wash the bacteria three times with ice-cold sterile 10% glycerol (wt:vol), and dispense them into 50 μL / tubes for electroporation of competent cells of MG1655 (pKD46).

[0139] (3) Electroporation: Place 50 μL of MG1655 (pKD46) electroporation competent cells on ice, add 100 ng of FMP1 fragment, place on ice for 3 minutes, and transfer to a 0.1 cm Bio-Rad electroporation cup. Use a Micro-Pulser electroporator with a voltage parameter of 1.8 kV for electroporation. After electroporation, quickly add 1 mL of sterile LB medium to the electroporation cup, pipette to mix, and transfer to a 15 mL sterile centrifuge tube. Resume culture at 37°C, 200 rpm for 2 hours while the pKD46 plasmid is removed.

[0140] (4) Take an appropriate amount of bacterial solution and spread it on LB plate containing Kanamycin (50 mg / L) and culture it upside down in a 37°C incubator overnight.

[0141] (5) After the colonies grew, eight monoclonal colonies were selected for transfer and colony PCR was performed using a 2× Rapid Taq Master Mix kit. The primer pair was verified to be pmtA-CX-F / pmtA-CX-R, and a 934 bp target DNA fragment was amplified.

[0142] The colony PCR reaction is:

[0143] Component Volume(μL) 2× Rapid Taq Master Mix 25 pmtA-CX-F 2 pmtA-CX-R 2 <![CDATA[ddH 2 The]]> 21

[0144] The reaction procedure is:

[0145]

[0146] The colonies with correct verification were selected for sequencing, and the sequencing primer pair was pmtA-CX-F / pmtA-CX-R. The colonies with correct sequencing were inoculated in LB liquid medium containing 50 mg / L Kanamycin, and the bacteria were shaken at 37°C, 200 rpm for 12 hours and maintained with 50% glycerol (wt:v). They were named +PC, and the genotype was MG1655ΔldhA::FKF-M12-pmtA.

[0147] Example 3: Detection of phosphatidylcholine on the membrane of recombinant engineering strain + PC cells

[0148] 3.1 Extraction of total cell membrane phospholipids

[0149] The total phospholipids of the cell membrane of the Control strain and the +PC strain were extracted using the method of Bligh and Dyer (reference Bligh EG, Dyer W JA RAPID METHOD OF TOTAL LIPID EXTRACTION AND PURIFICATION [J]. Canadian Journal of Biochemistry and Physiology, 1959, 37 (8): 911-917).

[0150] (1) The control strain and the +PC strain were inoculated into an appropriate volume of MOPS + 2% glucose medium (formula as shown in Table 2) containing 50 mg / L Kanamycin, and the seed solution was cultured overnight at 37°C and 200 rpm. 550 =0.1 was transferred to an appropriate volume of MOPS + 2% glucose medium containing 50 mg / L Kanamycin, cultured at 37°C, 200 rpm for 7 hours, centrifuged at 12000 rpm for 1 minute, and the supernatant was discarded.

[0151] (2) Resuspend the cell pellet in 1 mL of 1× PBS buffer (pH 7.0, the formula is shown in Table 3) and transfer to a 10 mL glass vial. Add 3.75 mL of chloroform / methanol / HCl (2 / 4 / 0.1, vol:vol) and vortex at 2000 rpm for 15 min to mix thoroughly.

[0152] (3) Add 1.25 mL of chloroform, vortex for 1 min, and then add 1.25 mL of ddH 2 O, vortex on a vortex shaker at 2000 rpm for 15 min to mix thoroughly.

[0153] (4) Centrifuge at 2000 × g for 10 min, remove the lower chloroform layer into a new glass vial, and blow dry with nitrogen.

[0154] Table 2MOPS+2% glucose medium formula

[0155]

[0156] Table 3 1×PBS buffer

[0157]

[0158] 3.2 Quantitative detection of phosphatidylcholine in total cell membrane phospholipids

[0159] The Phosphatidylcholine Assay Kit (Colorimetric / Fluorometric) (purchased from Abcam) was used to detect the phosphatidylcholine content in the total phospholipids of the cell membrane of the Control strain and the +PC strain.

[0160] Results Figure 2 No phosphatidylcholine was detected in the total phospholipids of the cell membrane of the Control strain, while phosphatidylcholine was successfully detected in the +PC strain and the content of phosphatidylcholine was 100.60±56.44mg / gDCW.

[0161] Example 4: Broad-spectrum tolerance analysis of recombinant engineering strains + PC

[0162] 4.1 Tolerance analysis conditions

[0163] The medium used for tolerance analysis was MOPS + 2% glucose medium;

[0164] The types and concentrations of inhibitors used in the tolerance analysis were: 10.4 mM furfural, 20 mM 5-methylfurfural, 6.6 mM vanillin, 0.5 mM formaldehyde, 500 mM lactic acid, 600 mM ethanol, 66 mM n-butanol, 66 mM isobutanol, 3.47 mM triacetic acid lactone (TAL), 3.67 mM limonene, and 600 mM sodium chloride.

[0165] 4.2 Tolerance analysis steps

[0166] (1) Take out the Control strain and +PC strain from the -80°C freezer, streak them on LB solid plates containing 50 mg / L Kanamycin to activate them, and culture them upside down in a 37°C incubator overnight.

[0167] (2) Pick 4-5 single clones of the control strain and the +PC strain and inoculate them into MOPS + 2% glucose medium containing 50 mg / L Kanamycin. Shake the medium at 37°C and 200 rpm until the OD 550 =1.5-2.0.

[0168] (3) Prepare MOPS + 2% glucose medium containing 50 mg / L Kanamycin and divide it into two equal parts. One part is used to culture the control strain at OD 550 = 0.1, and the other was used to transfer the +PC strain at OD 550 = 0.1 transfer and mix thoroughly.

[0169] (4) Prepare a MOPS + 2% glucose medium containing 50 mg / L Kanamycin and a certain concentration of inhibitor, and divide it into two equal parts. One part is used to grow the control strain at OD 550 = 0.1, and the other was used to transfer the +PC strain at OD 550 = 0.1 transfer and mix thoroughly.

[0170] (5) Add the bacterial solution in "step (3)" and "step (4)" to a 96-well transparent ELISA plate, 200 μL of bacterial solution per well, make 4 replicate wells for each bacterial solution and each treatment, and cover with sealing film.

[0171] (6) Place the 96-well plate in a shaker at 37°C and 200 rpm, and take it out every hour to measure the OD using an ELISA reader. 550 Or use Biotek SynergyⅡ microplate reader to run continuous kinetic program for tolerance analysis experiment, and the continuous kinetic program is set as follows:

[0172]

[0173] (7) After exporting the tolerance analysis experimental data, the OD of each treatment for each strain was calculated. 550 Perform exponential curve fitting, and the exponential formula obtained is OD 550,t =OD 550,0 e μt (R 2 ≥0.95), where μ is the specific growth rate (hr -1 ).

[0174] The results of the tolerance analysis are attached. Figure 3Compared with the Control strain, the +PC strain showed a certain degree of increased growth rate for the inhibitors furfural, 5-methylfurfural, vanillin, formaldehyde, lactate, ethanol, butanol, iso-butanol, TAL, limonene, and sodium chloride, which means that the +PC strain has a broad spectrum of tolerance to multiple inhibitors.

[0175] Example 5: Analysis of cell membrane integrity of the recombinant engineered strain + PC relative to the control strain Control

[0176] Cell membrane integrity was measured using a BD Cytoflex flow cytometer and SYTOX Green dye. The specific steps are:

[0177] (1) The control strain and the +PC strain were inoculated on LB solid plates containing 50 mg / L Kanamycin for streak activation and incubated in an inverted incubator at 37°C for 12 hours.

[0178] (2) 4-5 single clones of the control strain and the +PC strain were selected and inoculated into MOPS + 2% glucose medium containing 50 mg / L Kanamycin, and cultured overnight at 37°C and 200 rpm to obtain seed solution.

[0179] (3) Prepare MOPS + 2% glucose medium containing 50 mg / L Kanamycin and divide it into two equal parts. One part is used to transfer the Control strain at OD550 = 0.1, and the other part is used to transfer the +PC strain at OD550 = 0.1. Incubate at 37°C and 200 rpm until the strain grows to the logarithmic phase.

[0180] (4) Centrifuge at 6000 rpm for 5 min, wash the bacterial pellet twice with sterile 1× PBS buffer (pH 7.0), and then resuspend the bacterial pellet with sterile 1× PBS buffer (pH 7.0) at a final OD550 of 0.1, add formaldehyde at a final concentration of 0.5 mM, mix thoroughly, and treat at 37°C, 200 rpm for 3 h.

[0181] (5) Centrifuge at 6000 rpm for 5 minutes, resuspend the bacterial pellet in sterile 1× PBS buffer (pH 7.0), add SYTOX Green dye at a final concentration of 5 μM, mix thoroughly, and stain at room temperature in the dark for 15 minutes.

[0182] (5) 10,000 cells were collected using a BD Cytoflex flow cytometer, and the fluorescence intensity of the FITC channel was detected. The negative control sample of each bacteria (bacterial solution without SYTOX Green) was first loaded to set the acquisition parameters, and then the treated sample was loaded to obtain the ratio of live cells to dead cells in the cell population.

[0183] The results of cell membrane integrity are shown in the attached Figure 4 The cell membrane integrity of the recombinant engineering strain + PC strain was improved by 50% compared with the cell membrane integrity of the control strain Control.

[0184] Example 6: Analysis of fatty acid composition of cell membrane of recombinant engineering strain + PC relative to control strain Control The steps of extraction, esterification and content analysis of fatty acids in cell membrane phospholipids are as follows:

[0185] (1) Escherichia coli cells grown to the exponential growth phase were collected by centrifugation, washed twice with ice-cold 1× PBS buffer (pH 7.0), then resuspended in 1.4 mL of methanol and transferred to a glass tube.

[0186] (2) Add 10 μL of 1 mg / L C15:0 (solvent: ethanol) as an internal standard. Then, place the sample in an ultrasonic cleaner and perform ultrasonic cleaning three times, each time for 20 seconds.

[0187] (3) The samples were incubated at 70°C for 15 minutes and centrifuged at 5000 g for 5 minutes.

[0188] (4) Transfer the supernatant to a new glass tube and add 0.75 mL of chloroform to resuspend the cell debris at the bottom of the glass tube. Shake at 37°C and 150 rpm for 15 minutes.

[0189] (5) Mix the transferred supernatant and cell debris suspension together, vortex for 1 minute, and centrifuge at 5000g for 2 minutes. The system will be separated into layers. Transfer the lower layer of liquid to a new glass tube and blow dry with nitrogen.

[0190] (6) Add 2 mL of a mixture of methanol and sulfuric acid (98:2, v / v), vortex mix, and react at 80° C. for 30 minutes to perform an esterification reaction.

[0191] (7) Add 2 mL of 0.9% (wt / v) NaCl and 1 mL of n-hexane, vortex thoroughly, and centrifuge at 2000 g for 2 minutes to extract the fatty acid ethyl esters formed in the reaction.

[0192] (8) Gas chromatography-mass spectrometry (GC-MS) analysis was performed. The analysis procedure was as follows: 50°C for 2 minutes, then heated to 200°C at a rate of 25°C / min and maintained for 1 minute, then heated to 300°C at a rate of 25°C / min and maintained for 2 minutes. Helium was used as the carrier gas at a flow rate of 1 mL / min, and the chromatographic column was HP-5.

[0193] (9) The ratio of saturated fatty acids to unsaturated fatty acids (saturated / unsaturated ratio, S / U) and the average fatty acid chain length (length, L) of the extracted cell membrane were calculated according to the formula, and L / nL was used to characterize the changes in the fluidity of the cell membrane.

[0194]

[0195] The results of cell membrane fatty acid composition are shown in the attached Figure 5 , the ratio of saturated fatty acids to unsaturated fatty acids in the cell membrane of the +PC strain was significantly increased ( Figure 5 Left), without changing the average fatty acid chain length ( Figure 5 right).

[0196] Example 7: Introduction of phosphatidylcholine to enhance substrate utilization

[0197] 7.1 Construction of a carbon utilization module

[0198] 7.1.1 Obtaining HPS-Phi fragments

[0199] Using pETduet-nudF-mdh2-hps-phi (reference Wang X, Wang X, Lu X, et al. Methanolfermentation increases the production of NAD(P)H-dependent chemicals insynthetic methylotrophic Escherichia coli [J]. Biotechnology for Biofuels, 2019, 12 (1): 17.) plasmid DNA as a template, using primer pair C1-HPS-F / C1-PHI-R (as shown in SEQ ID NO.27-28), a 1237 bp DNA fragment was amplified and named hps-phi fragment (as shown in SEQ ID NO.12). The fragment includes the hps-phi gene and the homology arm fragments of 20 bp upstream and downstream of the pCDF-P2 site.

[0200] The amplification system is:

[0201] Component Volume(μL) 2×Phanta max buffer 25 dNTP (10mM each) 1 C1-HPS-F 2 C1-PHI-R 2 Phanta max Super-Fidelity DNA polymerase 1 pETduet-nudF-mdh2-hps-phi plasmid 1 <![CDATA[ddH 2 The]]> up to 50

[0202] The reaction procedure is:

[0203]

[0204] After 1% agarose gel detection, the correct DNA fragment was purified using a DNA gel recovery kit. 1 μL DpnI was added to the PCR product and digested at 37°C for 30 minutes to digest the pETduet-nudF-mdh2-hps-phi plasmid template. After 1% agarose gel detection, the correct hps-phi fragment was purified using a DNA gel recovery kit.

[0205] 7.1.2 Obtaining pCDF-P2 fragment

[0206] Using pCDFduet-CT4-1 mdh plasmid (synthesized by Jinweizhi Biotechnology Co., Ltd., the pCDFduet plasmid carries a codon-optimized CT4-1 mdh gene sequence; the pCDFduet plasmid contains T7 promoter, 6 His tags, and the CT4-1 mdh gene sequence is shown in SEQ ID NO.94) DNA as a template, using primer pair C1-P2-F / C1-P2-R (SEQ ID NO.29-30), a 4956 bp DNA fragment was amplified and named pCDF-P2 fragment (as shown in SEQ ID NO.13).

[0207] The amplification system is:

[0208] Component Volume(μL) 2×Phanta max buffer 25 dNTP(10mM each) 1 C1-P2-F 2 C1-P2-R 2 Phanta max Super-Fidelity DNA polymerase 1 pETduet-nudF-mdh2-hps-phi plasmid 1 <![CDATA[ddH 2 The]]> up to 50

[0209] The reaction procedure is:

[0210]

[0211] Add 1 μL DpnI to the PCR product and digest at 37°C for 30 minutes to digest the pCDFduet-CT4-1 mdh plasmid template. After detection on a 1% agarose gel, purify the correct pCDF-P2 fragment using a DNA gel recovery kit.

[0212] 7.1.3 Obtaining pCDF-CT4-1 mdh-hps-phi plasmid

[0213] Seamless Cloning was performed with a ratio of hps-phi fragment: pCDF-P2 fragment = 3:1 (molar ratio). The assembly reaction system was:

[0214] Component Volume(μL) 2×Seamless Cloning Mix 5 hps-phi:pCDF-P2 = 3:1 (molar ratio) 5 Total volume 10

[0215] The assembly conditions were: 50°C, 30 minutes. Take 5 μL of the assembly product and transfer it into DH5α chemically competent cells by heat shock transformation at 42°C, 90 seconds. After 1 hour of recovery culture at 37°C, 200 rpm, take an appropriate amount of bacterial solution and spread it on LB solid plates containing 50 mg / L Spectinomycin, and culture it inverted in a 37°C incubator for 12 hours.

[0216] After the colonies grow, 8 monoclonal colonies are selected for colony PCR. The primers are CT4-1-F / P2-YZ-KR (as shown in SEQ ID NO.31-32), and the colony PCR reaction is:

[0217] Component Volume(μL) 2×Rapid Taq Master Mix 25 CT4-1-F 2 P2-YZ-KR 2 <![CDATA[ddH 2 The]]> 21

[0218] The reaction procedure is:

[0219]

[0220] The correct colony was verified to be plasmid-extracted and sent for sequencing. The sequencing primer pair was P1-YZ-KF / P2-YZ-KR (the sequence of P1-YZ-KF is shown in SEQ ID NO.33). The colony with correct sequencing was inoculated in LB liquid culture medium containing 50 mg / L Spectinomycin, and the culture was shaken at 37°C and 200 rpm for 12 hours to extract the plasmid to obtain the plasmid pCDF-CT4-1mdh-hps-phi (shown in SEQ ID NO.14).

[0221] 7.2 Construction of Tolerance Module and Methanol Utilization Chassis Strains

[0222] 7.2.1 Knockout of frmA gene

[0223] 7.2.1.1 Construction of pEcgRNA-ΔfrmA plasmid

[0224] (1) Using the pEcgRNA plasmid (reference Li QI, Sun B, Chen J, et al. A modified pCas / pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli [J]. Acta Biochimica et Biophysica Sinica, 2021, 53(5): 620-627.) as a template, the primer pair pEcgRNA-frmA-F / pEcgRNA-frmA-R (as shown in SEQ ID NO.34-35) was used to amplify a 2118 bp DNA fragment named pEcgRNA-ΔfrmA fragment (as shown in SEQ ID NO.36), which contains the N20 sequence fragment for knocking out the frmA site.

[0225] The amplification system is:

[0226] Component Volume(μL) 2×Phanta max buffer 25 dNTP(10mM each) 1 pEcgRNA-frmA-F 2 pEcgRNA-frmA-R 2 Phanta max Super-Fidelity DNA polymerase 1 pEcgRNA plasmid 1 <![CDATA[ddH 2 The]]> up to 50

[0227] The reaction procedure is:

[0228]

[0229] Add 1 μL DpnI to the PCR product and digest at 37°C for 30 minutes to digest the pEcgRNA plasmid template. After detection on a 1% agarose gel, use a DNA gel recovery kit to purify the correct pEcgRNA-ΔfrmA fragment.

[0230] (2) The obtained linear pEcgRNA-ΔfrmA fragment was self-circularized, and the seamless assembly reaction system was

[0231] Component Volume(μL) 2×Seamless Cloning Mix 5 pEcgRNA-ΔfrmA 5 Total volume 10

[0232] Take 5 μL of the assembled product and transfer it into DH5α (Vazyme) chemically competent cells by heat shock transformation at 42°C for 90 seconds. After recovery culture at 37°C and 200 rpm for 1 hour, take an appropriate amount of bacterial solution and spread it on LB solid plates containing 50 mg / L Spectinomycin, and culture it upside down in a 37°C incubator for 12 hours.

[0233] After the colonies grew out, 8 monoclonal colonies were selected and transferred to LB liquid culture medium containing 50 mg / L Spectinomycin. The bacteria were shaken at 37°C and 200 rpm for 12 hours and the plasmid was extracted. The plasmid was sent for sequencing. The sequencing primer was N20-YZ (SEQ ID NO. 77). The correctly sequenced plasmid was named pEcgRNA-ΔfrmA.

[0234] 7.2.1.2 Obtaining ΔfrmA donor DNA

[0235] (1) Obtaining the ΔfrmA-L DNA fragment. Using MG1655 genomic DNA as a template and the primer pair frmA-LF / frmA-LR (as shown in SEQ ID NOs. 37-38), a 487 bp DNA fragment was amplified and named the ΔfrmA-L fragment (as shown in SEQ ID NO. 39). The fragment contained a 460 bp homology arm DNA fragment upstream of the frmA site and a 20 bp homology arm DNA fragment downstream of the frmA site.

[0236] The amplification system is:

[0237] Component Volume(μL) 2×Phanta max buffer 25 dNTP(10mM each) 1 frmA-L-F 2 frmA-L-R 2 Phanta max Super-Fidelity DNA polymerase 1 MG1655 genomic DNA 1 <![CDATA[ddH 2 The]]> up to 50

[0238] The reaction procedure is:

[0239]

[0240] After detection on a 1% agarose gel, the correct ΔfrmA-L fragment was purified using a DNA gel recovery kit.

[0241] (2) Obtaining the ΔfrmA-R DNA fragment. Using MG1655 genomic DNA as a template and the primer pair frmA-RL / frmA-RR (as shown in SEQ ID NOs. 40-41), a 444 bp DNA fragment was amplified and named the ΔfrmA-R fragment (as shown in SEQ ID NO. 42). The fragment contained a 444 bp homology arm DNA fragment downstream of the frmA site.

[0242] The amplification system is:

[0243] Component Volume(μL) 2×Phanta max buffer 25 dNTP(10mM each) 1 frmA-R-L 2 frmA-R-R 2 Phanta max Super-Fidelity DNA polymerase 1 MG1655 genomic DNA 1 <![CDATA[ddH 2 The]]> up to 50

[0244] The reaction procedure is:

[0245]

[0246] After detection on a 1% agarose gel, the correct ΔfrmA-R fragment was purified using a DNA gel recovery kit.

[0247] (3) Obtaining ΔfrmA donor DNA. Seamless cloning was performed with a ratio of ΔfrmA-L fragment: ΔfrmA-R fragment = 1:1 (molar ratio). The seamless assembly reaction system is:

[0248]

[0249]

[0250] Using the assembled product as a template and the primer pair frmA-LF / frmA-RR, a 911 bp DNA fragment was amplified and named ΔfrmA donor DNA fragment (as shown in SEQ ID NO.43).

[0251] The amplification system is:

[0252] Component Volume(μL) 2×Phanta max buffer 25 dNTP(10mM each) 1 frmA-L-F 2 frmA-R-R 2 Phanta max Super-Fidelity DNA polymerase 1 MG1655 genomic DNA 1 <![CDATA[ddH 2 The]]> up to 50

[0253] The reaction procedure is:

[0254]

[0255] After checking on a 1% agarose gel, purify the correct ΔfrmA donor DNA fragment using a DNA gel recovery kit.

[0256] 7.2.1.3 Construction of MG1655(DE3)ΔfrmA(pEcCas) strain

[0257] (1) The pEcCas plasmid (reference Li QI, Sun B, Chen J, et al. A modified pCas / pTargetF system for CRISPR-Cas9-assisted genome editing in Escherichia coli [J]. Acta Biochimica et Biophysica Sinica, 2021, 53(5): 620-627.) was transformed into Escherichia coli MG1655 (DE3) by 42°C, 90 sec heat shock transformation method to obtain MG1655 (DE3) (pEcCas) strain.

[0258] (2) MG1655(DE3)(pEcCas) was inoculated into LB liquid medium containing 50 mg / L Kanamycin and 10 mM L-arabinose and shaken at 37°C and 200 rpm until OD 550 When the pH reaches 0.3-0.4, wash the bacteria three times with 10% glycerol at 4°C to prepare MG1655(DE3)(pEcCas) electroporation competent cells, and dispense the competent cells into 50 μL / tubes.

[0259] (3) Add 100ng pEcgRNA-ΔfrmA plasmid and 400ng ΔfrmA donor DNA fragment to 50μL MG1655(DE3)(pEcCas) electroporation competent cells, incubate on ice for 3 minutes, and transfer to a 0.1cm Bio-Rad electroporation cuvette. Use a MicroPulser electroporator with a voltage parameter of 1.8kV. After electroporation, quickly add 1mL of sterile LB medium to the electroporation cuvette, pipette to mix, and transfer to a 15mL sterile centrifuge tube. Resume culture at 37°C, 200rpm for 1 hour.

[0260] (4) Take an appropriate amount of bacterial solution and spread it on a LB solid plate containing 50 mg / L Kanamycin and 50 mg / L Spectinomycin, and invert and culture at 37°C for 12 hours. After the colonies grow, select 8 monoclonal colonies for transfer and perform colony PCR. The primers are frmA-YZ-F / frmA-YZ-R (as shown in SEQ ID NO.44-45).

[0261] The colony PCR reaction is:

[0262] Component Volume(μL) 2×Rapid Taq Master Mix 25 frmA-YZ-F 2 frmA-YZ-R 2 <![CDATA[ddH 2 The]]> 21

[0263] The reaction procedure is:

[0264]

[0265] (5) Colony PCR verification: The correct colonies were inoculated into LB liquid medium containing 50 mg / L Kanamycin and 10 mM rhamnose to induce Cas protein expression and cut the pEcgRNA-ΔfrmA plasmid. The culture was shaken at 37°C and 200 rpm for 12 h. An appropriate amount of bacterial liquid was spread on an LB solid plate containing 50 mg / L Kanamycin and incubated inverted at 37°C for 12 h.

[0266] (6) After the colonies grow, single clones are picked for transfer, first streaked onto an LB solid plate containing 50 mg / L Spectinomycin, and then streaked onto an LB solid plate containing 50 mg / L Kanamycin. The single clone that is only on the LB solid plate containing 50 mg / L Kanamycin is the MG1655(DE3)ΔfrmA(pEcCas) strain.

[0267] 7.2.2 Knockout of rpiA gene

[0268] The rpiA gene was knocked out based on the MG1655(DE3)ΔfrmA(pEcCas) strain to obtain the MG1655(DE3)ΔfrmAΔrpiA(pEcCas) strain. The method for knocking out the rpiA gene is described in 7.2.1.

[0269] The primer pair for constructing pEcgRNA-ΔrpiA was pEcgRNA-rpiA-F / pEcgRNA-rpiA-R (SEQ ID NO.46-47), with an annealing temperature of 52°C, an extension time of 2 minutes, and a target DNA fragment size of 2138 bp.

[0270] The primer pair for constructing the ΔrpiA-L fragment (SEQ ID NO.78) was rpiA-LF / rpiA-LR (SEQ ID NO.48-49), the annealing temperature was 56°C, the extension time was 30 seconds, and the target DNA fragment size was 571 bp. The primer pair for constructing the ΔrpiA-R fragment (SEQ ID NO.79) was rpiA-RF / rpiA-RR (SEQ ID NO.50-51), the annealing temperature was 59°C, the extension time was 30 seconds, and the target DNA fragment size was 729 bp. The primer pair for constructing the ΔrpiA donor DNA (SEQ ID NO.80) was rpiA-LF / rpiA-RR, the annealing temperature was 57°C, the extension time was 1 minute, and the target DNA fragment size was 1300 bp.

[0271] The primer pair for colony PCR verification of rpiA gene knockout was rpiA-YZ-F / rpiA-RR (the sequence of rpiA-YZ-F is shown in SEQ ID NO.52), with an annealing temperature of 55°C and an extension time of 45 seconds.

[0272] 7.2.3 Knockout of rpiB gene

[0273] The rpiB gene was knocked out based on the MG1655(DE3)ΔfrmAΔrpiA(pEcCas) strain to obtain the MG1655(DE3)ΔfrmAΔrpiAΔrpiB(pEcCas) strain. The method for knocking out the rpiB gene is described in 7.2.1.

[0274] The primer pair pEcgRNA-ΔrpiB-F / pEcgRNA-rpiB-R (SEQ ID NO.53-54) of pEcgRNA-ΔrpiB (SEQ ID NO.81) was constructed, with an annealing temperature of 52°C, an extension time of 2 minutes, and a target DNA fragment size of 2118 bp.

[0275] The primer pair for constructing the ΔrpiB-L (SEQ ID NO.82) fragment was rpiB-LF / rpiB-LR (SEQ ID NO.55-56), the annealing temperature was 58°C, the extension time was 30 seconds, and the target DNA fragment size was 493 bp. The primer pair for constructing the ΔrpiB-R fragment (SEQ ID NO.83) was rpiB-RF / rpiB-RR (SEQ ID NO.57-58), the annealing temperature was 55°C, the extension time was 30 seconds, and the target DNA fragment size was 537 bp. The primer pair for constructing the ΔrpiB donor DNA (SEQ ID NO.84) was rpiB-LF / rpiB-RR, the annealing temperature was 57°C, the extension time was 1 minute, and the target DNA fragment size was 1015 bp.

[0276] The primer pair for colony PCR verification of rpiB gene knockout was rpiB-LF / rpiB-YZ-R (SEQ ID NO.59), with an annealing temperature of 55°C and an extension time of 30s.

[0277] 7.2.4 Knockout of ldhA gene

[0278] The ldhA gene was knocked out based on the MG1655(DE3)ΔfrmAΔrpiAΔrpiB(pEcCas) strain to obtain the MG1655(DE3)ΔfrmAΔrpiAΔrpiBΔldhA(pEcCas) strain. The method for knocking out the ldhA gene is described in 7.2.1.

[0279] The primer pair pEcgRNA-ΔldhA (SEQ ID NO.85) was constructed as pEcgRNA-ldhA-F / pEcgRNA-ldhA-R (SEQ ID NO.60-61), with an annealing temperature of 53°C, an extension time of 2 minutes, and a target DNA fragment size of 2118 bp.

[0280] The primer pair for constructing the ΔldhA-L fragment (SEQ ID NO.86) was ldhA-LF / ldhA-LR (SEQ ID NO.62-63), the annealing temperature was 58°C, the extension time was 30 seconds, and the target DNA fragment size was 519 bp. The primer pair for constructing the ΔldhA-R fragment (SEQ ID NO.87) was ldhA-RF / ldhA-RR (SEQ ID NO.64-65), the annealing temperature was 55°C, the extension time was 30 seconds, and the target DNA fragment size was 516 bp. The primer pair for constructing the ΔldhA donor DNA (SEQ ID NO.88) was ldhA-LF / ldhA-RR, the annealing temperature was 55°C, the extension time was 1 minute, and the target DNA fragment size was 1035 bp.

[0281] Colony PCR verified that the primer pair for ldhA gene knockout was ldhA-YZ-F / ldhA-YZ-R (SEQ ID NO.66-67), with an annealing temperature of 56°C and an extension time of 30 seconds.

[0282] 7.2.5 Integration of the tolerance module at the ldhA locus

[0283] Integration tolerance module at the ldhA site based on the MG1655 (DE3) ΔfrmA ΔrpiA ΔrpiB (pEcCas) strain: The artificial regulatory element M1-12 was assembled with the codon-optimized pmtA gene (encoding phosphatidylethanolamine-N-methyltransferase EC 2.1.1.17) from Rhodobacter sphaeroides to obtain the MG1655 (DE3) ΔfrmA ΔrpiA ΔrpiB ΔldhA::M12-pmtA (pEcCas) strain. The integration method of the tolerance module is the same as the knockout method of the ldhA gene, see 7.2.1.

[0284] The primer pair for constructing pEcgRNA-ΔldhA was pEcgRNA-ldhA-F / pEcgRNA-ldhA-R, with an annealing temperature of 53°C, an extension time of 2 minutes, and a target DNA fragment size of 2138 bp.

[0285] The donor DNA construction method of the tolerance module is as follows:

[0286] (1) Obtaining the DNA fragment of ldhA-PC-L (SEQ ID NO.89). Using MG1655 genomic DNA as a template and the primer pair ldhA-LF / ldhA-PC-LR (SEQ ID NO.68), a 540 bp DNA fragment was amplified and named the ldhA-PC-L fragment, which contained a 518 bp homology arm DNA fragment upstream of the ldhA site and a 22 bp pmtA gene homology arm DNA fragment.

[0287] The amplification system is:

[0288] Component Volume(μL) 2×Phanta max buffer 25 dNTP (10 mM each) 1 ldhA-L-F 2 ldhA-PC-L-R 2 Phanta max Super-Fidelity DNA polymerase 1 MG1655 genomic DNA 1 <![CDATA[ddH 2 The]]> up to 50

[0289] The reaction procedure is:

[0290]

[0291] After detection on 1% agarose gel, the correct ldhA-PC-L fragment was purified using a DNA gel recovery kit.

[0292] (2) Obtaining the DNA fragment of M12-PC. Using the genomic DNA of the +PC strain as a template and the primer pair PC-F / M12-R (SEQ ID NO.69-70), a 720 bp DNA fragment was amplified and named M12-PC fragment (SEQ ID NO.90). This fragment contained the constitutive artificial promoter M12, the pmtA gene, and a 20 bp homology arm DNA fragment downstream of the ldhA site.

[0293] The amplification system is:

[0294]

[0295]

[0296] The reaction procedure is:

[0297]

[0298] After detection on a 1% agarose gel, the correct M12-PC fragment was purified using a DNA gel recovery kit.

[0299] (3) Obtaining the DNA fragment of ldhA-R. Using MG1655 genomic DNA as a template and the primer pair ldhA-PC-RF / ldhA-RR (the nucleotide sequence of ldhA-PC-RF is shown in SEQ ID NO.71), a 516 bp DNA fragment was amplified and named ldhA-R fragment, which contained a 516 bp homology arm DNA fragment downstream of the ldhA site.

[0300] The amplification system is:

[0301] Component Volume (μL) 2×Phanta max buffer 25 dNTP (10 mM each) 1 ldhA-PC-R-F 2 ldhA-R-R 2 Phanta max Super-Fidelity DNA polymerase 1 MG1655 genomic DNA 1 <![CDATA[ddH 2 The]]> up to 50

[0302] The reaction procedure is:

[0303]

[0304] After detection on 1% agarose gel, the correct ldhA-R fragment was purified using a DNA gel recovery kit.

[0305] (4) Seamless cloning was performed at a ratio of ldhA-PC-L:M12-PC:ldhA-R=1:1:1 (molar ratio). The seamless assembly reaction system was:

[0306]

[0307] Using the assembled product as a template and the primer pair ldhA-LF / ldhA-RR, a 1735 bp DNA fragment was amplified. The amplification system was:

[0308]

[0309]

[0310] The reaction procedure is:

[0311]

[0312] After detection on 1% agarose gel, the correct M12-pmtA donor DNA fragment (SEQ ID NO. 91) was purified using a DNA gel recovery kit.

[0313] The primer pair for successful integration of the tolerance module verified by colony PCR was PC-F / ldhA-YZ-R, with an annealing temperature of 55°C and an extension time of 30 seconds. The colonies that were verified correctly were sent for sequencing, and the sequencing primers were ldhA-YZ-F / ldhA-YZ-R. The colonies that were sequenced correctly were inoculated in LB liquid culture medium, shaken at 37°C, 200 rpm for 12 hours, and maintained with 50% glycerol (wt:v).

[0314] 7.2.6 Elimination of pEcCas plasmid

[0315] The MG1655(DE3)ΔfrmAΔrpiAΔrpiBΔldhA(pEcCas) strain and the MG1655(DE3)ΔfrmAΔrpiAΔrpiBΔldhA::M12-pmtA(pEcCas) strain were inoculated into LB antibiotic-free medium, shaken at 37°C, 200 rpm for 6 hours to eliminate the pEcCas plasmid, streaked onto LB solid plates containing 10 g / L sucrose, and inverted cultured at 37°C for 12 hours.

[0316] After the colonies grew out, single clones were picked for transfer, first streaked on an LB solid plate containing 50 mg / L Kanamycin, then on an LB solid plate containing 50 mg / L Kanamycin, and finally on an LB solid plate containing 10 g / L sucrose, and cultured upside down at 37°C for 12 hours. Single clones only on the LB solid plate containing 10 g / L sucrose were picked, namely the MG1655(DE3)ΔfrmAΔrpiAΔrpiBΔldhA strain and the MG1655(DE3)ΔfrmAΔrpiAΔrpiBΔldhA:M12-pmtA strain.

[0317] 7.2.7 Knockout of cyaA gene

[0318] Using MG1655(DE3)ΔfrmAΔrpiAΔrpiBΔldhA strain and MG1655(DE3)ΔfrmAΔrpiAΔrpiBΔldhA:M12-pmtA strain as starting strains, the cyaA gene was knocked out by λRed homologous recombination method. The knockout method was the same as in Example 1, and the methanol utilization chassis Control strain MG1655(DE3)ΔfrmAΔrpiAΔrpiBΔldhAΔcyaA::FKF and the methanol utilization chassis + PC strain MG1655(DE3)ΔfrmAΔrpiAΔrpiBΔldhA::M12-pmtAΔcyaA::FKF were obtained.

[0319] The DNA fragment ΔcyaA::FKF (SEQ ID NO.92) was constructed using the pKD4 plasmid as a template and the primer pair cyaA-F / cyaA-R (SEQ ID NO.72-73) to amplify a 1659 bp DNA fragment, which includes a 63 bp DNA fragment upstream of the cyaA site of Escherichia coli MG1655, a 66 bp DNA fragment downstream of the cyaA site, a DNA fragment encoding the Kanamycin resistance gene required for the λRed homologous recombination method, and a DNA fragment encoding two FRT sites. The PCR amplification system is:

[0320] Component Volume (μL) 2×Phanta max buffer 25 dNTP (10 mM each) 1 cyaA-F 2 cyaA-R 2 Phanta max Super-Fidelity DNA polymerase 1 pKD4 plasmid 1 <![CDATA[ddH 2 The]]> up to 50

[0321] The reaction procedure is:

[0322]

[0323] After detection on a 1% agarose gel, the correct ΔcyaA::FKF fragment was purified using a DNA gel recovery kit.

[0324] The primer pair for colony PCR verification of cyaA gene knockout was cyaA-F / cyaA-YZ-R (SEQ ID NO.74). The colony PCR reaction was:

[0325] Component Volume (μL) 2×Rapid Taq Master Mix 25 cyaA-F 2 cyaA-YZ-R 2 <![CDATA[ddH 2 The]]> 21

[0326] The reaction procedure is:

[0327]

[0328] 7.3 Construction of MK-1 and MK-2 strains

[0329] The methanol efficient utilization plasmid pCDF-CT4-1 mdh-hps-phi was transformed into the methanol utilization chassis strain MG1655(DE3)ΔfrmAΔrpiAΔrpiBΔldhAΔcyaA::FKF strain and MG1655(DE3)ΔfrmAΔrpiAΔrpiBΔldhA::M12-pmtAΔcyaA::FKF strain to obtain the methanol utilization Control strain MK-1: MG1655(DE3)ΔfrmAΔrpiAΔrpiBΔldhAΔcyaA::FKF (pCDF-CT4-1 mdh-hps-phi) and the methanol utilization +PC strain MK-2: MG1655(DE3)ΔfrmAΔrpiAΔrpiBΔldhA::M12-pmtAΔcyaA::FKF (pCDF-CT4-1mdh-hps-phi). The strain construction of methanol utilization + PC strain MK-2 with the introduction of phosphatidylcholine in the methanol utilization chassis strain is as follows Figure 6 shown.

[0330] 7.4 Methanol Utilization Growth Experiment

[0331] (1) The methanol utilization control strain MK-1: MG1655 (DE3) ΔfrmA ΔrpiA ΔrpiB ΔldhA ΔcyaA:: FKF (pCDF-CT4-1 mdh-hps-phi) and the methanol utilization + PC strain MK-2: MG1655 (DE3) ΔfrmA ΔrpiA ΔrpiB ΔldhA:M12-pmtA ΔcyaA:: FKF (pCDF-CT4-1 mdh-hps-phi) were inoculated into an appropriate volume of TB medium containing 50 mg / L Kanamycin and 50 mg / L Spectinomycin (the formula is shown in Table 4) and incubated at 37°C at 250 rpm until the OD 550 Reach 1.6-2.0.

[0332] (2) Wash the bacteria twice with MOPS salt medium (without glucose) and resuspend in an appropriate volume. 550 The strain was transferred to 0.1 in 5 mL methanol growth medium (the formula is shown in Table 5) containing 50 mg / L Kanamycin, 50 mg / L Spectinomycin and 0.1 mM IPTG. The treatment group without methanol was used as a control to verify whether the strain depends on methanol for growth. The bacteria were shaken at 37°C and 250 rpm. Samples were taken every 48 hours with a sampling volume of 380 μL. The samples were stored at -20°C.

[0333] For growth curves, see the attached Figure 7 MK-1 basically did not grow within 13 days (final OD550 0.04±0.05), while MK-2 grew better (final OD 550 The growth trend of MK-2 strain was as follows: 0-4 days was the growth lag phase, 4 to 10 days was the logarithmic phase, and 10 to 13 days was the stable phase.

[0334] Table 4 TB medium formula

[0335]

[0336] Table 5 Methanol utilization growth medium formulation

[0337]

[0338]

[0339] 7.5 Substrate detection

[0340] The sample was centrifuged at 12000rpm for 1 minute, the supernatant was taken out and filtered through 0.22μm into a liquid chromatography detection bottle, and the content of xylose and methanol was detected by high performance liquid chromatography. The chromatographic column was Aminex HPX-87H, 5mM sulfuric acid was used as the mobile phase, the column temperature was 65℃, the flow rate was 0.6mL / min, each sample was 30 minutes, and the RID detector was used.

[0341] See attached for the results Figure 8 After the 13th day of growth, the MK-1 strain consumed 1.43±0.28mM xylose and 6.30±0.87mM methanol, and the MK-2 strain consumed 42.79±0.94mM xylose and 146.25±0.55mM methanol. The introduction of phosphatidylcholine significantly improved the ability of E. coli to utilize xylose and methanol.

[0342] Example 8: Introduction of phosphatidylcholine to enhance product synthesis ability

[0343] 8.1 Construction of triacetyl lactone (TAL) synthesis strain

[0344] The pTrc99A-bktB plasmid (as shown in SEQ ID NO.7) was transformed into the Control strain and the +PC strain by electroporation to obtain the triacetic acid lactone (TAL) Control strain MK-3: MG1655ΔldhA::FKF (pTrc99A-bktB) and the triacetic acid lactone (TAL) +PC strain MK-4: MG1655ΔldhA::FKF-M12-pmtA (pTrc99A-bktB). Colony PCR was performed on the pTrc99A-bktB plasmid in the strain, and the primer pair was verified to be 99A-YZ-F / 99A-YZ-R (SEQ ID NO.75-76).

[0345] The colony PCR reaction is:

[0346] Component Volume (μL) 2×Rapid Taq Master Mix 25 99A-YZ-F 2 99A-YZ-R 2 <![CDATA[ddH 2 The]]> 21

[0347] The reaction procedure is:

[0348]

[0349] 8.2 Triacetic acid lactone (TAL) fermentation and metabolite detection

[0350] (1) The strains MK-3 and MK-4 were inoculated into LB medium containing 50 mg / L Kanamycin and 50 mg / L Spectinomycin, and the seed solution was cultured at 37°C and 200 rpm.

[0351] (2) The seed solution was heated to an initial OD 550 The culture medium was transferred to LB like MOPS medium (formula shown in Table 6) containing 50 mg / L Kanamycin, 50 mg / L Spectinomycin and 0.1 mM IPTG at 0.1, and fermented at 37°C, 200 rpm for 48 hours.

[0352] (3) Draw a standard curve for triacetin (TAL): Dissolve the triacetin (TAL) standard in LB like MOPS medium and prepare a concentration gradient of triacetin standard solution. After thorough mixing, take 2.5 μL of the supernatant and detect the ultraviolet absorbance at 282 nm to draw a standard curve.

[0353] (4) After 48 hours of fermentation, samples were taken every 24 hours to measure OD 550 , then sample 1 mL, centrifuge at 12000 rpm for 3 minutes, take 2.5 μL of supernatant to detect the ultraviolet absorbance at 282 nm, and calculate the yield of triacetic acid lactone in the fermentation broth according to the standard curve.

[0354] See attached for the results Figure 9 and attachedFigure 10 .

[0355] At the 48th hour of fermentation, the MK-3 strain produced 43.91±1.28 mg / L triacetic acid lactone, and the MK-4 strain produced 61.74±0.46 mg / L triacetic acid lactone, with a yield increase of 40.69±4.77%.

[0356] At the 72nd hour of fermentation, MK-3 strain produced 49.82±2.90 mg / L triacetate lactone, and MK-4 strain produced 69.27±0.52 mg / L triacetate lactone, with a yield increase of 39.36±8.25%.

[0357] At the 96th hour of fermentation, MK-3 strain produced 59.75±3.15 mg / L triacetic acid lactone, and MK-4 strain produced 89.58±0.28 mg / L triacetic acid lactone, with a yield increase of 50.19±7.89%.

[0358] Table 6 LB like MOPS medium formula

[0359]

[0360] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. Use of gene pmtA or a protein expressed by gene pmtA in improving the broad-spectrum tolerance of Escherichia coli, characterized in that: The nucleotide sequence of the gene pmtA is shown in SEQ ID NO.1, and the amino acid sequence of the protein is shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The broad-spectrum tolerant inhibitor includes any one of furfural, 5-methylfurfural, vanillin, formaldehyde, lactic acid, ethanol, n-butanol, isobutanol, triacetic acid lactone, limonene, and sodium chloride.

3. Use of a recombinant vector in improving the broad-spectrum tolerance of Escherichia coli, characterized in that: The recombinant vector comprises a tolerance module M12-pmtA, wherein the M12-pmtA comprises an artificial regulatory element M1-12 and the gene pmtA according to claim 1, and the nucleotide sequence of the artificial regulatory element M1-12 is shown in SEQ ID NO.

3.

4. An Escherichia coli strain with improved broad-spectrum tolerance, characterized in that Using Escherichia coli as the starting strain, knocking out the gene ldhA in the Escherichia coli genome, and integrating the gene pmtA or the tolerance module M12-pmtA in the recombinant vector of claim 3 into the ldhA site to obtain a strain; the nucleotide sequence of the gene ldhA is shown in SEQ ID NO.

4.

5. A method for constructing an Escherichia coli strain with improved broad-spectrum tolerance as claimed in claim 4, characterized in that: The following steps are involved: S1, using plasmid pKD4, strain M1-12, and plasmid pUC-pmtA as templates, PCR amplification was performed to obtain the FKF fragment, M1-12 fragment, and pmtA fragment; S2, assembling the FKF fragment, the M1-12 fragment and the pmtA fragment; or, assembling the FKF fragment and the pmtA fragment to obtain the FMP1 fragment and the FMP2 fragment, respectively; S3. Transform the FMP1 fragment or the FMP2 fragment into competent cells of Escherichia coli MG1655 containing plasmid pKD46, eliminate the plasmid pKD46, and identify to obtain the Escherichia coli strain with improved broad-spectrum tolerance.

6. The construction method according to claim 5, characterized in that: The nucleotide sequence of the FKF fragment is shown in SEQ ID NO.5; the nucleotide sequence of the FMP1 fragment is shown in SEQ ID NO.

6.

7. An Escherichia coli strain for synthesizing triacetic acid lactone, characterized in that: The strain is obtained by using the Escherichia coli strain with improved broad-spectrum tolerance as claimed in claim 4 as the starting strain and transferring the pTrc99A-bktB plasmid into it; the nucleotide sequence of the pTrc99A-bktB plasmid is shown in SEQ ID NO.

7.

8. An Escherichia coli strain with improved methanol utilization, characterized in that Using Escherichia coli as the starting strain, knocking out the genes frmA, rpiA, rpiB, ldhA and cyaA in the Escherichia coli genome, integrating the gene pmtA or the tolerance module M12-pmtA in the recombinant vector described in claim 3 into the ldhA site, and transferring the resultant strain into the plasmid pCDF-CT4-1 mdh-hps-phi; the nucleotide sequences of the genes frmA, rpiA, rpiB and cyaA are shown in SEQ ID NOs.8-11, respectively.

9. A method for constructing an Escherichia coli strain with improved methanol utilization as claimed in any one of claims 7 to 8, characterized in that: The following steps are involved: A1, using pETduet-nudF-mdh2-hps-phi plasmid and pCDFduet-CT4-1 mdh plasmid as templates, PCR amplification was performed to obtain hps-phi fragment and pCDF-P2 fragment; A2, assemble the hps-phi fragment and the pCDF-P2 fragment, and transfer them into DH5α chemically competent cells to screen and obtain the plasmid pCDF-CT4-1 mdh-hps-phi; A3, knocking out the genes frmA, rpiA, rpiB, ldhA and cyaA of Escherichia coli, and integrating the gene pmtA or the tolerance module M12-pmtA into the ldhA site to obtain a strain; A4. The plasmid pCDF-CT4-1 mdh-hps-phi is transferred into the strain, and the Escherichia coli strain with improved methanol utilization is obtained through identification.

10. The construction method according to claim 9, characterized in that: The nucleotide sequences of the hps-phi fragment and the pCDF-P2 fragment are shown in SEQ ID NO.12-13 respectively.

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