A method for transmembrane transport of sucrose by escherichia coli using secy protein translocation channel mutants

Abstract

This invention provides a method for transmembrane sucrose transport in *E. coli* using a mutant of the SecY protein translocation channel. Firstly, the SecY protein is overexpressed in *E. coli*. Klebsiella pneumoniae Source scrB Genes are used to construct the intracellular sucrose hydrolysis pathway; then, the SecY(ΔP) protein translocation channel gene is overexpressed. secY ( ΔP ) ,secE,secG and the extracellular membrane porin gene derived from the SARS virus. sCVE A preliminary transmembrane transport pathway for sucrose was established; finally, using iterative saturation mutagenesis, site-directed random mutagenesis was employed to modify the gene. secY ( ΔP Modifications were made to adjust key gene elements of the SecY(ΔP) protein translocation channel. secY (ΔP) The degree of amino acid pore ring opening was measured to obtain various mutant strains; these mutant strains were then screened using fermentation to obtain a dominant strain that metabolizes sucrose. E.coli B3.

Description

Technical Field

[0001] This invention belongs to the field of metabolic engineering, specifically relating to a method for Escherichia coli to transport sucrose across the membrane using a mutant of the SecY protein translocation channel, thereby realizing a complete system for sucrose transport and utilization. Background Technology

[0002] Sucrose is a common sugar compound found in nature. It is a well-known disaccharide, primarily formed by the condensation and dehydration of the hemiacetal hydroxyl groups of one molecule of glucose and one molecule of fructose. Sucrose is solid at room temperature and has a certain degree of sweetness, making it widely used as a sweetener. It is readily soluble in water and glycerol, and is frequently added to food and beverages. While its structural composition indicates optical activity, structural analysis reveals that it does not exhibit mutarotation. As a common chemical, sucrose is ubiquitous in nature. Many plants on Earth contain varying amounts of sucrose in their leaves, flowers, stems, seeds, and fruits, with high concentrations found in sugarcane, sugar beets, and maple sap. As a widely distributed disaccharide in nature, sucrose, through refining, blending, and the addition of other substances, has given rise to many other sugars, such as white sugar, brown sugar, granulated sugar, rock sugar, and raw sugar (yellow sugar).

[0003] Sucrose metabolism plays a vital role in development, stress response, and normal life activities. The main function of sucrose is to break it down into a variety of compounds through a series of reactions. These compounds can promote the normal growth of many organisms and synthesize compounds necessary for the survival of organisms. They can also act as signals to regulate the expression of transcription factors and other genes, and can interact with hormones, oxidation, and defense signals.

[0004] Escherichia coli is one of the most commonly used bacterial strains in academic and industrial experiments and is widely applied in the field of metabolic engineering. Sucrose, a common disaccharide, could be a valuable substrate for E. coli metabolism, saving significant resources in this field. In this context, we first overexpress the *scrB* gene derived from *Klebsiella pneumoniae* in *E. coli* to establish an intracellular sucrose hydrolysis pathway. Then, we modify the pore-loop structure of the *SecY* (ΔP) channel protein in *E. coli*, enabling sucrose to diffuse freely into the cell, thus achieving efficient transmembrane sucrose transport in recombinant *E. coli*. Summary of the Invention

[0005] This invention aims to address the aforementioned problems by providing a method for transmembrane sucrose transport in *E. coli* using a mutant of the SecY protein translocation channel. First, the exogenous gene *scrB* from *Klebsiella pneumoniae* is introduced to enable *E. coli* BL21 (DE3) strain to metabolize sucrose in vivo. Then, recombinant plasmids *pETDuet-secY(ΔP)-secE* and *pRSFDuet-secG-sCVE* are transfected into the strain. By expressing the SecY(ΔP) protein translocation channel genes *secY(ΔP)*, *secE*, *secG*, and the extracellular membrane porogen gene *sCVE* derived from SARS virus in vivo, a preliminary sucrose transmembrane transport channel is established. Site-directed random mutations are performed on key sites of the pore-loop structure of the original gene *secY(ΔP)* to obtain a site-modified gene. After four rounds of random mutations at six sites, a *secY(ΔP)* mutant gene library is obtained. Then, the relevant *E. coli* BL21... (DE3) transport and metabolism genes were transfected into cells in the form of plasmids. After obtaining a defined strain library, M9 medium with sucrose as the substrate was used to screen for dominant strains that metabolize sucrose. After fermentation screening, the dominant strain E. coli B3 was obtained, and finally, recombinant E. coli was able to efficiently metabolize sucrose using the SecY channel protein mutant.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: A method for transmembrane sucrose transport in *E. coli* using a mutant of the SecY protein translocation channel was first proposed. First, the *scrB* gene, derived from *Klebsiella pneumoniae*, was overexpressed in *E. coli* BL21 (DE3). This gene helps establish an intracellular sucrose hydrolysis pathway. To enable efficient sucrose transport via the SecY(ΔP) transport channel, the secY(ΔP), secE, secG, and sCVE genes were introduced to establish the sucrose transport pathway. Then, the pore-loop structure of the secY(ΔP) gene was modified by altering the base sequence at the gene loci involved in the pore-loop structure. After site-directed random mutation modification at six sites related to the pore-loop structure in the secY(ΔP) gene, a modified secY(ΔP) gene library was obtained. Through fermentation screening, a dominant sucrose transport strain, *E. coli* B3, was obtained. The plasmid of this dominant strain was then extracted to obtain the secY(ΔP*) gene, thus enabling efficient transmembrane sucrose transport in recombinant *E. coli*.

[0007] Furthermore, the above-mentioned method for transmembrane sucrose transport in Escherichia coli using a SecY protein translocation channel mutant specifically includes the following steps: (1) To construct an intracellular sucrose hydrolysis pathway, genomic DNA of Klebsiella pneumoniae was extracted, and then scrB in the genome was amplified using primers scrB-F and scrB-R. The scrB amplification product and the empty plasmid pACYCDuet-1 were double-digested using Nde I and KpnI enzymes. The amplified product and the plasmid were then ligated using T4 DNA ligase to obtain the recombinant plasmid pACYCDuet-scrB. pACYCDuet-scrB was then transfected into Escherichia coli BL21 (DE3) by electrochemical transformation, successfully constructing an intracellular sucrose hydrolysis pathway and obtaining the recombinant strain E. coli (scrB). The sequences of the primers scrB-F and scrB-R are shown in Table 1. (2) To preliminarily establish a transmembrane sucrose transport pathway, plasmids pETDuet-secY(ΔP)-secE and pRSFDuet-secG-sCVE were extracted from strain E. coli (ΔPtsG) with SecY (ΔP) and SCVE; (3) To adjust the opening degree of the amino acid pore loop of the key gene element secY(ΔP) in the SecY(ΔP) protein translocation channel, the base sequence of six sites of the secY(ΔP) gene was randomly mutated using site-directed random mutagenesis through iterative saturation mutagenesis. Site-directed random mutagenesis adjusted the opening degree of the amino acid pore loop of the key gene element secY(ΔP) in the SecY(ΔP) channel. Based on the gene mutation principle of error-prone PCR, since the two groups of amino acid base sites at positions 82 and 86 and positions 187 and 191 are too close, positions 82 and 86 were combined for mutation when designing primers, and positions 187 and 191 were combined for mutation; therefore, a total of four rounds of mutation were performed; four pairs of primers containing degenerate codons were used: secY-1, 2-F and secY-1, 2-R, secY-3, 4-F and secY-3, Four rounds of amplification were performed using 4-R, secY-5-F, secY-5-R, secY-6-F, and secY-6-R. Using pETDuet-secY(ΔP)-secE as a mutation template, iterative saturation gene mutations were performed on six sites corresponding to the amino acid pore loop to establish a gene mutation library for secY(ΔP). The six mutation sites of the amino acid pore loop of secY(ΔP) are isoleucine residues at positions 82, 86, 187, 191, 278, and 408 on the secY(ΔP) gene fragment. The mutated recombinant plasmid, combined with pRSFDuet-secG-sCVE and pACYCDuet-scrB, was transfected into E. coli BL21 (DE3) to obtain mutant strains. Then, multiple mutant strains were screened by fermentation using M9 medium with sucrose as the sole carbon source to determine the dominant sucrose transport strain, E. coli B3, ultimately yielding secY(ΔP) mutants. (ΔP*) channels are used to enable efficient transmembrane transport of sucrose in recombinant E. coli; the sequences of the primers secY-1,2-F, secY-12-R, secY-3,4-F, secY-3,4-R, secY-5-F, secY-5-R, secY-6-F, and secY-6-R are shown in Table 1.

[0008] Furthermore, the site-directed mutagenesis method in step (3) mainly involves designing four pairs of recombinant primers and using iterative saturation mutagenesis to perform site-directed random mutagenesis on secY(ΔP), thereby optimizing the transport protein's sucrose transport by adjusting the pore-loop structure of the secY(ΔP) transport channel.

[0009] Furthermore, in step (3), when screening using a sucrose-containing culture medium, it is necessary to first activate and culture the various mutant strains after mutation in LB test tubes and add IPTG to induce the expression of multiple genes in the cells to synthesize the required proteins. Then, the strains are inoculated at a rate of 1 / 1000 in M9 culture medium with a substrate of 4 g / L sucrose for fermentation. By comparing the different growth densities of various mutant cells, the dominant strains are screened, and finally, strain E. coli B3 is obtained.

[0010] Compared with the prior art, the beneficial effects of the present invention are as follows: In this invention, the exogenous gene scrB is expressed in wild-type Escherichia coli to help E. coli BL21(DE3) establish an intracellular sucrose hydrolysis pathway. The translocation channel genes secY(ΔP), secE, and secG, as well as the extracellular membrane porogen gene sCVE derived from SARS virus, are introduced and induced to synthesize their corresponding proteins in the cell, thereby helping the cell to initially establish a sucrose transmembrane transport channel. Then, the known gene secY(ΔP) is subjected to site-directed random mutation using iterative saturation mutagenesis to obtain a mutant gene library. This mutant gene library, along with pRSFDuet-secG-sCVE and pACYCDuet-scrB, is transfected into E. coli BL21(DE3) to obtain various mutant strains. Through fermentation screening, the dominant sucrose strain E. coli B3 is identified, and the secY(ΔP*) channel gene is obtained. Attached Figure Description

[0011] Figure 1 Schematic diagram of sucrose metabolism process.

[0012] Figure 2 A: From left to right: PCR amplification of the scrB gene, plasmid and gene image after double enzyme digestion, and colony PCR gel image; B: SDS-PAGE images: Lane 1 (blank) is the SDS-PAGE image of E. coli BL21 (DE3), and Lane 2 (lane 1) is the SDS-PAGE image of E. coli (scrB).

[0013] Figure 3 Site-directed random mutation mapping of the secY(ΔP) gene. A: Gel map of mutations at positions 82 and 86; B: Gel map of mutations at positions 187 and 191; C: Gel map of mutation at position 278; D: Gel map of mutation at position 408. Lanes 1 and 2 in each image represent parallel samples from the same round of mutations. Figure 4The mutant plasmids, combined with pRSFDuet-secG-sCVE and pACYCDuet-scrB, were transfected into E. coli BL21 (DE3) to obtain various mutant strains. These strains were then activated by inoculating them into LB broth tubes, and subsequently fermented in M9 medium containing sucrose. The final growth states of each strain were plotted. Figure 4 .

[0014] Figure 5 Image A shows the fermentation of recombinant strains E. coli (scrB) and E. coli (scrB, secY [ΔP]), as well as the mutant-selected E. coli B3 strain, in M9 medium containing 4 g / L sucrose; Image B shows the fermentation of recombinant strains E. coli (scrB) and E. coli (scrB, secY [ΔP]), as well as the mutant-selected E. coli B3 strain, in LB medium containing 4 g / L sucrose. (Dashed line: sucrose consumption; solid line: cell growth) Detailed Implementation

[0015] To make the content of this invention easier to understand, the technical solution of this invention will be further described below with reference to specific embodiments, but this invention is not limited thereto.

[0016] The primers involved in this invention are shown in the table below.

[0017] Table 1

[0018] Example 1 The pACYCDuet-scrB plasmid was constructed and transfected into E. coli BL21(DE3) to obtain strain E. coli (scrB). The scrB gene derived from Klebsiella pneumoniae was overexpressed in strain E. coli (scrB), and the scrB protein was characterized using SDS-PAGE. Klebsiella pneumoniae (this strain is published in the reference: Sekar R, Shin HD, Chen R. Engineering Escherichia coli cells for cellobiose assimilation through a phosphorolytic mechanism.[J]. Applied & Environmental Microbiology,2012, 78(5):1611-1614.) was inoculated into LB agar plates using an inoculation loop and cultured at 37°C for 12-16 h. Then, it was transferred to 4 mL LB tubes for further culture for 12-16 h. Genomic DNA of the Klebsiella pneumoniae strain was extracted using a genomic DNA extraction kit. Following the kit's instructions, the extracted genomic DNA was collected in centrifuge tubes. A pair of primers, scrB-F and scrB-R, with Nde I and Kpn I restriction sites at the F and R ends, respectively, were designed. The scrB gene (sequence shown in SEQ ID NO. 1 of the sequence listing) was amplified using genomic DNA extracted from Klebsiella pneumoniae as a template. The amplified gene was then separated using agarose gel electrophoresis, and the gel containing the scrB gene was subsequently recovered. Figure 2 A). Then, plasmid pACYCDuet-1 (Novagen, catalog number 71147) and the gel recovery product were simultaneously digested with Nde I and Kpn I restriction endonucleases. After processing, two linear DNA fragments were obtained. These two DNA fragments were ligated together using T4 DNA ligase to obtain the recombinant plasmid pACYCDuet-scrB. The recombinant plasmid pACYCDuet-scrB was transfected into *E. coli* BL21(DE3) using electrochemical transformation to obtain the recombinant strain *E. coli* (scrB). *E. coli* (scrB) was inoculated into LB tubes and cultured at 37°C for 16 h. Then, 1 mL of the bacterial culture was transferred from the tubes to an Erlenmeyer flask containing 50 mL of LB liquid medium and cultured. The cell growth density (OD) of the bacterial culture was measured using a microplate reader. 600 IPTG to a final concentration of 100 mM was added to an Erlenmeyer flask at approximately 1.6°C. Induction was performed at 37°C for 15 h. The bacterial cells were collected by centrifugation and lysed to obtain cell lysate. The cell lysate was then centrifuged again to obtain the supernatant and precipitate. The supernatant was analyzed by SDS-PAGE. Figure 2 B).

[0019] Example 2 Recombinant plasmids pRSFDuet-secG-sCVE and pETDuet-secY(ΔP)-secE were extracted from the strain E. coli (ΔPtsG) with SecY(ΔP) and SCVE (this strain is published in the reference: Reprogramming of sugar transport pathways in Escherichia coli using apermeabilized SecY protein-translocation channel[J]. Biotechnology and Bioengineering, 2020, 117(6): 1738-1746.). The recombinant plasmid pETDuet-secY(ΔP)-secE contained in the E. coli (ΔPtsG) with SecY(ΔP) and SCVE strain was created using restriction endonucleases Nco I and Bam HI, Nde I and Kpn I to bind the secY(ΔP) gene (sequence shown in SEQ ID NO.2) and the secE gene (sequence shown in SEQ ID NO.2). NO.3) was obtained by ligating the pETDDuet-1 plasmid; the recombinant plasmid pRSFDuet-secG-sCVE contained in the E. coli (ΔPtsG) with SecY (ΔP) and SCVE strain was obtained by ligating the secG gene (sequence shown in SEQ ID NO.4) and the sCVE gene (sequence shown in SEQ ID NO.5) to the pRSFDuet-1 plasmid using restriction endonucleases Nco I and Bam HI, Nde I and Kpn I. The recombinant plasmids pETDuet-secY (ΔP)-secE and pRSFDuet-secG-sCVE were transfected into E. coli (scrB) using electrochemical transformation to obtain the recombinant strain E. coli (scrB, secY [ΔP]) containing the secY (ΔP) gene.

[0020] Site-directed random mutagenesis was performed on the secY (ΔP) gene, and the results were screened. The mutation sites were isoleucine residues at positions 82, 86, 187, 191, 278, and 408 of the secY (ΔP) gene fragment in the pETDuet-secY (ΔP)-secE plasmid. E. coli (scrB, secY[ΔP]) strain containing the secY (ΔP) gene was inoculated at a rate of 1 / 1000 into LB tubes containing 60 μg / mL of Kanamycin and 100 μg / mL of ampicillin. The tubes were then incubated in a shaker at 220 rpm for 12-16 h at 37°C. The plasmid pETDuet-secY(ΔP)-secE was extracted from the E. coli (scrB, secY[ΔP]) strain using the EZNA® Plasmid Mini Kit I. Using the extracted pETDuet-secY(ΔP)-secE plasmid as a template, four rounds of error-prone PCR were performed sequentially using four pairs of designed random mutant primers, as detailed below. Figure 3First, using the extracted plasmid pETDuet-secY (ΔP)-secE as a template, and secY-1,2-F and secY-1,2-R as primers, a first round of PCR was performed. The products of the first round of PCR were separated and purified by agarose gel electrophoresis. Then, the gel containing the PCR products was recovered using gel extraction. The gel-recovered products were then chemically transformed into competent E. coli Trans10 strains, resulting in the formation of circular DNA from the mutant PCR products within the cells, thus obtaining the recombinant strain E. coli Trans10 (secY [ΔP, 12]). The recombinant strain E. coli Trans10 (secY [ΔP, 12]) was inoculated into LB tubes for amplification culture. Plasmids were extracted from mixed cultures and used as templates, with secY-3, 4-F and secY-3, A second round of PCR was performed using primers 4-R. The products of the second round of PCR were separated and purified by agarose gel electrophoresis. The gel containing the target DNA was recovered using gel extraction. The recovered product was then chemically transformed into competent E. coli Trans10 strains, resulting in intracellular circularization of the mutated PCR product and obtaining the recombinant strain E. coli Trans10 (secY [ΔP, 12, 34]). The recombinant strain E. coli Trans10 (secY [ΔP, 12, 34]) was inoculated into LB tubes for amplification culture. Plasmids were extracted from mixed cultures and used as templates. A third round of PCR was performed using primers secY-5-F and secY-5-R. The products of the third round of PCR were separated and purified by agarose gel electrophoresis. The gel containing the target DNA was recovered using gel extraction. The recovered product was then chemically transformed into E. coli Trans10 strains. The PCR product from the mutant strain Trans10 was circularized within the cells to obtain the recombinant strain E. coli Trans10 (secY [ΔP, 12, 34, 5]). The recombinant strain E. coli Trans10 (secY [ΔP, 12, 34, 5]) was inoculated into LB broth tubes for amplification. Plasmids were extracted from mixed cultures and used as templates. A fourth round of PCR was performed using secY-6-F and secY-6-R primers. The products from the fourth round of PCR were separated and purified by agarose gel electrophoresis. The gel containing the target DNA was recovered using gel extraction. The recovered gel product was then chemically transformed into competent E. coli Trans10 strains, resulting in the intracellular circularization of the mutant PCR product and obtaining the recombinant strain E.E. coli Trans10 (secY [ΔP, 12, 34, 5, 6]). After expanding the recombinant strain E. coli Trans10 (secY [ΔP, 12, 34, 5, 6]), plasmids were extracted from the mixed cultures to obtain a library of randomly mutant plasmids. The library of randomly mutant plasmids, along with pRSFDuet-secG-sCVE and pACYCDuet-scrB, was transfected into E. coli BL21 (DE3) using electrochemical transformation to obtain multiple mutant strains. These mutant strains were inoculated into M9 medium containing 4 g / L sucrose for fermentation screening at a constant temperature of 37°C. Cell density (OD) was measured at the same time intervals. 600 Screening for those with the best growth (OD) 600 The strain with the highest value Figure 4 This was named B3. After expanding the culture of B3, the bacterial cells were collected and the mutated pETDuet-secY (ΔP)-secE plasmid was extracted. The extracted mutant plasmid was named pETDuet-secY (ΔP*)-secE. Sequencing revealed that the isoleucine residues at positions 187, 191, and 278 of the secY (ΔP) gene fragment in pETDuet-secY (ΔP*)-secE were mutated to phenylalanine, tyrosine, and tyrosine, respectively. This mutated secY (ΔP) gene was named secY (ΔP*).

[0021] Example 3 The selected strains were then validated through fermentation. The mutant plasmid pETDuet-secY (ΔP*)-secE, the recombinant plasmid pRSFDuet-secG-sCVE, and the recombinant plasmid pACYCDuet-scrB were transfected into E. coli BL21 (DE3) using electrochemical transformation to obtain the recombinant strain E. coli B3. The recombinant plasmid pACYCDuet-scrB was transfected into E. coli BL21 (DE3) using electrochemical transformation to obtain the recombinant strain E. coli (scrB). The recombinant plasmids pETDuet-secY (ΔP)-secE, pRSFDuet-secG-sCVE, and pACYCDuet-scrB were transfected into E. coli BL21 (DE3) using electrochemical transformation to obtain the recombinant strain E. coli (scrB, secY [ΔP]).

[0022] E. coli B3 was fermented, with E. coli (scrB) and E. coli (scrB, secY [ΔP]) as controls. The culture medium formulation was: M9 medium + 4 g / L sucrose + 60 μg / mL kanamycin + 30 μg / mL chloramphenicol + 100 μg / mL ampicillin. 0.2 mM IPTG was added at the initial inoculation fermentation. The final fermentation time was 144 h, and the culture temperature was kept constant at 37℃. The final cell growth density was OD. 600 like Figure 5 As shown in Figure A, this is consistent with expectations. Experimental results show that, compared to E. coli (scrB) and E. coli (scrB, secY [ΔP]), the cell growth density of E. coli B3 showed a more significant increase. Analysis of the fermentation broth after every 12 hours revealed that the substrate sucrose in the E. coli B3 group continuously decreased, while the substrate in the control group showed no significant change.

[0023] E. coli B3 was fermented, with E. coli (scrB) and E. coli (scrB, secY [ΔP]) as controls. The culture medium formulation was: LB liquid medium + 4 g / L sucrose + 60 μg / mL kanamycin + 30 μg / mL chloramphenicol + 100 μg / mL ampicillin. 0.2 mM IPTG was added at the initial inoculation of cells for fermentation. The final fermentation time was 72 h, and the culture temperature was kept constant at 37℃. The final cell density was OD. 600 Curves that change over time, such as Figure 5 As shown in B, this is consistent with expectations. The experimental results show that, compared to the two control groups, E. coli B3 showed significant growth and substantial consumption of the substrate sucrose in the first 12 hours of fermentation, while the control group, although showing a gradual increase in cell growth density, did not exhibit significant changes in substrate content.

[0024] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made within the scope of the claims of the present invention should be included in the scope of the present invention.

Claims

1. A method for transmembrane sucrose transport in *Escherichia coli* using a mutant of the SecY protein translocation channel, characterized in that: Escherichia coli ( Escherichia coli Klebsiella pneumoniae overexpressed in ) Klebsiella pneumoniae (Source) scrB Genes were used to establish an intracellular sucrose hydrolysis pathway; then, the SecY(ΔP) protein translocation channel gene was overexpressed. secY(Δ P), secE, secG and the extracellular membrane porin gene derived from the SARS virus. sCVE A preliminary transmembrane transport pathway for sucrose was established; then, key gene elements of the SecY(ΔP) protein translocation channel were adjusted using gene mutation techniques. secY(ΔP) The degree of openness of the amino acid pore ring ultimately enables E. coli to efficiently transport sucrose across the membrane using the SecY protein translocation channel mutant; Among them, the scrB The gene sequence is shown in SEQ ID NO.1 of the sequence listing. secY(ΔP) The gene sequence is shown in SEQ ID NO.2 of the sequence listing. secE The gene sequence is shown in SEQ ID NO.3 of the sequence listing. secG The gene sequence is shown in sequence listing SEQ ID NO.

4. sCVE The gene sequence is shown in the sequence listing SEQ ID NO.5; The key gene elements of the SeCY(ΔP) protein translocation channel are adjusted through gene mutation. secY(ΔP) The degree of openness of amino acid pore rings specifically refers to... secY(ΔP) The isoleucine residues at positions 187, 191, and 278 on the gene fragment were mutated to phenylalanine, tyrosine, and tyrosine, respectively.

2. The method according to claim 1, characterized in that: comprising the following steps: 1) scrB The gene recombines with the pACYCDuet-1 plasmid to form the recombinant plasmid pACYCDuet- scrB ; 2) secY(ΔP) Gene, secE The gene recombines with the pETDDuet-1 plasmid to form the recombinant plasmid pETDuet- secY(Δ P)-secE ,Will secG Gene, sCVE The gene recombines with the pRSFDuet-1 plasmid to form the recombinant plasmid pRSFDuet- secG- sCVE ; 3) The recombinant plasmid pETDuet- secY(ΔP)-secE of secY(ΔP) The isoleucine residues at positions 187, 191, and 278 of the gene fragment were mutated to phenylalanine, tyrosine, and tyrosine, respectively, to obtain the mutant plasmid pETDuet- secY (ΔP*)-secE The mutant plasmid pETDuet -secY (ΔP*)-secE Recombinant plasmid pRSFDuet -secG-sCVE and recombinant plasmid pACYCDuet- scrB Transfection to E. coli BL21 (DE3) yielded a recombinant strain. E. coli B3, the recombinant strain E. coli B3 enables efficient transmembrane transport of sucrose.

3. The method according to claim 2, characterized in that: The recombinant plasmid pACYCDuet -scrB The construction process involved: extracting genomic DNA from Klebsiella pneumoniae and using it as a template, then employing primers... scrB -F and scrB -R pairs scrB Genes were amplified by PCR using Nde I and Kpn I restriction endonuclease scrB The gene PCR product and pACYCDuet-1 plasmid were double-digested and ligated using T4 DNA ligase to obtain the recombinant plasmid pACYCDuet-1. scrB ; wherein, the primer scrB -F and scrB -R sequences are listed in SEQ ID NO. 6~7.

4. The method according to claim 2, characterized in that: The recombinant plasmid pETDuet- secY(ΔP)-secE and recombinant plasmid pRSFDuet- secG-sCVE From strains E. coli (ΔPtsG) was obtained from SecY (ΔP) and SCVE.

5. The method according to claim 2, characterized in that: Step 3) is as follows: Based on the gene mutation principle of error-prone PCR, four pairs of primers containing degenerate codons are used. secY -1,2-F and secY -12-R、 secY -3,4-F and secY -3,4-R、 secY -5-F and secY -5-R、 secY -6-F and secY -6-R, respectively with pETDuet- secY(ΔP)-secE plasmid as a template for mutation secY(ΔP) Iterative saturation gene mutations were performed on the six sites corresponding to the amino acid pore loop. The six mutation sites are pETDuet -secY(ΔP)-secE plasmid secY (ΔP) Isoleucine residues at positions 82, 86, 187, 191, 278, and 408 on the gene fragment were used to obtain a random mutation plasmid library. This library, along with the recombinant plasmid pRSFDuet- secG-sCVE and recombinant plasmid pACYCDuet- scrB Transfected using electrochemical conversion method E. coli BL21 (DE3) was used to obtain a mutant strain, which was then screened using M9 medium with sucrose as the sole carbon source. secY(ΔP) Mutants were created by mutating isoleucine at positions 187, 191, and 278 of the gene fragment to phenylalanine, tyrosine, and tyrosine, respectively. The mutated pETDuet- was then extracted from these mutant strains. secY(ΔP)-secE The plasmid was named pETDuet- secY (ΔP*)-secE The mutant plasmid pETDuet- secY (ΔP*)-secE Recombinant plasmid pRSFDuet- secG-sCVE and recombinant plasmid pACYCDuet -scrB Transfected using electrochemical conversion method E. coli BL21(DE3) was used to obtain a recombinant strain. E. coli B3, the recombinant strain E. coli B3 enables efficient transmembrane transport of sucrose; wherein, the primers... secY -1,2-F and secY -12-R、 secY -3,4-F and secY -3,4-R、 secY -5-F and secY -5-R、 secY -6-F and secY The -6-R sequence is listed in SEQ ID NO. 8~15.

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