Recombinant escherichia coli of galactose and galactitol as well as preparation method and application of recombinant escherichia coli

Abstract

The invention relates to recombinant escherichia coli of galactose and galactitol as well as a preparation method and application thereof, and belongs to the field of gene engineering. The method comprises the following steps: by taking escherichia coli MG1655 as an original strain, knocking out a gene ptsG, a gene glk, a gene ugd and a gene galK on a genome; the gene galE is subjected to overexpression; the recombinant Escherichia coli capable of producing galactose at high yield is obtained by taking recombinant escherichia coli as a template and carrying out free expression on an exogenous gene glf and a gene NmLgtB, and a gene GRE3 is integrated on the basis of the recombinant Escherichia coli capable of producing galactose, so that the recombinant Escherichia coli capable of producing galactitol at high yield is obtained. In the shake flask fermentation experiment process, the yield of galactose can reach 6.18 g / L, the yield of galactitol can reach 3.98 g / L, and the method has a good industrialization prospect.

Description

Technical Field

[0001] The present invention relates to a recombinant Escherichia coli of galactose and galactitol, a preparation method and an application thereof, and belongs to the field of genetic engineering. Background Art

[0002] Galactose is a reducing monosaccharide composed of six carbon atoms and an aldehyde group. It is a diastereoisomer of glucose and serves as a bioenergy source and a fundamental building block of glycolipids and glycoproteins. D-galactose is an aldose naturally present in lactose, cerebrosides, gangliosides, and mucins. It can be metabolized into glucose or stored as a polysaccharide. As a monosaccharide precursor, galactose can be synthesized into functional substances through various pathways: in amination reactions, galactosamine-6-sulfatase catalyzes the synthesis of N-acetylgalactosamine (GalNAc); GDP-galactose is converted to GDP-fucose through diastereoisomerization, ultimately forming fucose; and in industrial applications, galactose can also be used to produce tagatose through the catalysis of L-arabinose isomerase.

[0003] Galactitol has advantages such as suitable sweetness, low calories, strong hygroscopicity, good stability, and sustained-release properties, making it suitable as a sweetener substitute, humectant, and drug stabilizer / sustained-release agent for diabetic patients. Therefore, galactose and its derivatives have shown significant commercial value in the pharmaceutical, cosmetic, and food industries.

[0004] Traditional production methods rely primarily on natural resource extraction or chemical synthesis, but these methods face limitations such as complex processes, low yields, poor stereoselectivity, and high environmental costs. While β-galactosidase is commonly used industrially to hydrolyze lactose, the enzyme is expensive. While galactose reductase-catalyzed production of galactitol offers excellent product purity, it relies on a coenzyme regeneration system. Although some yeasts can naturally accumulate the relevant products, the yields are insufficient to meet industrial production requirements. Metabolic engineering-based cell factory strategies, which utilize inexpensive substrates such as glycerol and glucose for de novo synthesis, offer the advantage of easy scalability and are a promising and efficient production solution. Escherichia coli, with its clear genetic background, high efficiency in expressing exogenous genes, and short culture cycles, is a promising microbial platform for engineered chemical production. Therefore, constructing galactose and galactitol biosynthesis pathways suitable for E. coli and optimizing the expression levels of key enzymes is an effective strategy for improving production efficiency. Summary of the Invention

[0005] The recombinant Escherichia coli constructed by the present invention uses Escherichia coli MG1655 as a starting strain, and knocks out the competitive pathway glucose-specific PTS enzyme IIBC component gene ptsG, the glucokinase gene glk, the UDP-glucose 6-dehydrogenase gene ugd, and the galactokinase gene galK in the genome; homologously overexpresses the UDP-glucose 4-epimerase gene galE; and freely expresses the exogenous UDP-galactopyranose mutase gene glf and the β-1,4-galactosyltransferase gene NmLgtB, thereby obtaining a recombinant Escherichia coli with high galactose production. The aldose reductase gene GRE3 is integrated into the galactose-producing recombinant Escherichia coli, thereby obtaining a recombinant Escherichia coli with high galactitol production.

[0006] The first object of the present invention is to provide a recombinant Escherichia coli that can efficiently produce galactose, characterized in that the recombinant Escherichia coli knocks out the glucose-specific PTS enzyme IIBC component gene ptsG (Gene ID: 945651), the glucokinase gene glk (Gene ID: 946858), the UDP-glucose 6-dehydrogenase gene ugd (Gene ID: 946571), the galactokinase gene galK (Gene ID: 945358), the gene ilvG (Gene ID: 2847699), and the gene rph (Gene ID: 948156) on the Escherichia coli genome; and overexpresses the endogenous gene UDP-glucose-4-epimerase gene galE (Gene ID: 945354); and integrates and overexpresses the exogenous gene UDP-galactopyranose mutase gene glf (Gene ID: 79904430) and the β-1,4-galactosyltransferase gene NmlgtB.

[0007] The second object of the present invention is to provide a recombinant Escherichia coli that can efficiently produce galactitol, characterized in that the recombinant Escherichia coli knocks out the glucose-specific PTS enzyme IIBC component gene ptsG, the glucokinase gene glk, the UDP-glucose 6-dehydrogenase gene ugd, the galactokinase gene galK, the gene ilvG, and the xylulose kinase xylB (Gene ID: 948133) on the Escherichia coli genome; and overexpresses the endogenous gene UDP-glucose-4-epimerase gene galE; and integrates and overexpresses the exogenous genes UDP-galactopyranose mutase gene glf, the β-1,4-galactosyltransferase gene NmlgtB, and the aldose reductase gene GRE3 (Gene ID: 856504).

[0008] In one embodiment of the present invention, the genetically engineered bacterium in a recombinant Escherichia coli that can efficiently produce galactose is Escherichia coli MG1655 in which galK is knocked out, the glf gene is integrated at the ptsG site on the Escherichia coli genome, the NmlgtB gene is integrated at the glk site on the Escherichia coli genome, the galE gene is integrated at the ugd site on the Escherichia coli genome, the NmlgtB gene is integrated at the ilvG site on the Escherichia coli genome, and the galE gene is integrated at the rph site on the Escherichia coli genome.

[0009] In one embodiment of the present invention, the genetically engineered bacterium in a recombinant Escherichia coli that can efficiently produce galactitol is Escherichia coli MG1655 in which galK is knocked out, the gene glf is integrated at the ptsG site on the Escherichia coli genome, the gene NmlgtB is integrated at the glk site on the Escherichia coli genome, the gene galE is integrated at the ugd site on the Escherichia coli genome, the gene NmlgtB is integrated at the ilvG site on the Escherichia coli genome, and the gene GRE3 is integrated at the xylB site on the Escherichia coli genome.

[0010] In one embodiment, the β-1,4-galactosyltransferase gene NmlgtB is derived from Neisseria meningitidis with the NCBI sequence number of AAC44085.1.

[0011] In one embodiment, the glf gene is derived from Zymomonas mobilis subsp. mobilisstr. CP4.

[0012] In one embodiment, the GRE3 gene is derived from Saccharomyces cerevisiae, and the nucleotide sequence is shown in SEQ ID NO.3.

[0013] The third object of the present invention is to provide a method for producing galactose and galactitol, wherein the method comprises using the recombinant Escherichia coli to ferment and produce galactose and galactitol.

[0014] In one embodiment, the method comprises fermenting the recombinant Escherichia coli in a culture medium containing 30 g / L glycerol at a shaking speed of 200 rpm at 37°C until the OD 600 After 0.6-0.8, induce culture at 30℃ for at least 72h.

[0015] In one embodiment of the present invention, the added amount of the substrate glucose is: 5 g / L.

[0016] In one embodiment, the culture medium further contains 4.0 g / L diammonium hydrogen phosphate, 13.5 g / L potassium dihydrogen phosphate, 1.4 g / L magnesium sulfate heptahydrate, 1.7 g / L citric acid and 10 ml / L trace metal elements; the trace metal elements include: 0.35 g / L manganese sulfate monohydrate, 10 g / L ferrous sulfate, 1.0 g / L anhydrous copper sulfate, 2.25 g / L zinc sulfate heptahydrate, 2.0 g / L calcium chloride dihydrate, 0.23 g / L sodium borate decahydrate and 0.11 g / L ammonium molybdate.

[0017] The fourth object of the present invention is to provide the use of the above-mentioned recombinant E. coli in the fields of medicine, food and chemical industry. Preferably, the use of the above-mentioned recombinant E. coli in the production of products containing galactose.

[0018] The fifth object of the present invention is to provide the use of the above-mentioned recombinant Escherichia coli in the fields of medicine, food and chemical industry. Preferably, the use of the above-mentioned recombinant Escherichia coli in the production of products containing galactitol.

[0019] Beneficial effects:

[0020] The recombinant E. coli constructed in the present invention uses E. coli MG1655 as the starting strain. By knocking out the ptsG gene, a component of the competing pathway glucose-specific PTS enzyme IIBC, the glucokinase gene glk, the UDP-glucose 6-dehydrogenase gene ugd, and the galactokinase gene galK from the genome, and homologously overexpressing the UDP-glucose 4-epimerase gene galE, and expressing exogenous β-1,4-galactosyltransferase gene NmLgtB and UDP-galactopyranose mutase gene glf, the resulting recombinant E. coli can produce 6.18 g / L of galactose under shake flask fermentation conditions. By integrating the aldose reductase gene GRE3 into the chassis strain, the resulting recombinant E. coli can produce 3.98 g / L of galactitol under shake flask fermentation conditions. These two strains do not require the addition of additional inducers, reducing production costs and operational procedures. The chassis strain has good genetic stability and industrial application prospects.

[0021] The innovation of the present invention is:

[0022] (1) NmLgtB from Neisseria meningitidis is a β-1,4-galactosyltransferase that can transfer the galactosyl group of UDP-galactose (UDP-Gal) to N-acetylglucosamine (GlcNAc) or glucose (Glc), forming Galβ1-4GlcNAc (lactosamine structure). Existing technology shows that humanized glycosylation modification can be achieved by expressing the NmLgtB gene in yeast, but it has not been applied in Escherichia coli. It was also found that the increase of NmLgtB at different sites helps to increase galactose production.

[0023] (2) Homologous overexpression of the UDP-glucose 4-epimerase gene galE, and the increase of galE at different sites also contributes to the improvement of galactose production.

[0024] (3) The present invention discovered for the first time the Gly-DNAP-Fru-1,6-P-Fru-p-Glc-1-p-UDP-Glc-UDP-Gal signaling pathway, which has not been reported in the literature. It can increase the supply of UDP-Gal and help improve galactose production.

[0025] (4) The present invention creatively integrates the aldose reductase gene GRE3 on the basis of the chassis strain, and the resulting recombinant Escherichia coli achieves high production of galactitol. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Metabolic pathways for galactose and galactitol biosynthesis in recombinant Escherichia coli.

[0027] Figure 2 Comparison of production before and after construction of the recombinant E. coli galactose pathway.

[0028] Figure 3 Comparison of galactose production by fermentation with different integrated strains. DETAILED DESCRIPTION

[0029] The PCR amplification enzymes, plasmids, DNA gel recovery kits, and column-based plasmid extraction kits used in the examples were commercial products, and all procedures were performed according to the kit instructions. Conventional procedures such as colony PCR, agarose gel electrophoresis, heat shock transformation, electroporation, preparation of competent cells, and extraction and storage of bacterial genomes were performed according to Molecular Cloning: A Laboratory Manual (Fourth Edition). Sequencing of plasmids and DNA products was performed by GeneWeiZhi (Suzhou).

[0030] (1) Culture medium

[0031] (1) LB liquid medium: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.

[0032] (2) LB solid medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, and 15 g / L agar powder.

[0033] (3) Fermentation medium (DM): 30 g / L glycerol, 13.5 g / L potassium dihydrogen phosphate, 4.0 g / L diammonium hydrogen phosphate, 1.7 g / L citric acid, 1.4 g / L magnesium sulfate heptahydrate, and 10 ml / L trace metal elements; trace metal elements include: 10 g / L ferrous sulfate, 2.25 g / L zinc sulfate heptahydrate, 1.0 g / L anhydrous copper sulfate, 0.35 g / L manganese sulfate monohydrate, 0.23 g / L sodium borate decahydrate, 0.11 g / L ammonium molybdate, and 2.0 g / L calcium chloride dihydrate.

[0034] (2) Fermentation production of galactose and galactitol

[0035] (1) Shake flask fermentation process of galactose and galactitol: During the fermentation process, a single colony with normal colony morphology on the corresponding LB solid plate was taken and transferred to a 4 mL test tube LB culture liquid. After culturing for 10-12 h, the seed liquid in the test tube was transferred to a 20 mL shake flask DM medium at a 2% (v / v) inoculation rate. The culture conditions were 37°C and 200 rpm. When the bacterial growth density reached 0.6-0.8, glucose was added at a final concentration of 5 g / L, and the fermentation temperature was adjusted to 30°C. After the fermentation time reached 72 h, the fermentation liquid was taken and samples were prepared for product detection.

[0036] (3) Galactose and galactitol:

[0037] Take 1 mL of fermentation broth, centrifuge at 12,000 rpm for 10 min, take the supernatant, dilute it a certain number of times, and then pass it through a 0.22 μm water-based syringe filter for HPLC determination.

[0038] HPLC detection conditions: high performance liquid chromatography (HPLC) system (Waters e2695); chromatographic column: RezexROA organic acid column; detector: Waters 2414RIDetector differential detector; mobile phase: 5 mM sulfuric acid; flow rate: 0.6 mL / min; column temperature: 60°C; injection volume: 10 μL.

[0039] (IV) Plasmids and strains

[0040] Table 1 Strains involved in the following examples

[0041]

[0042]

[0043] Example 1: Construction of recombinant plasmid vector

[0044] The primers used for plasmid construction are listed in Table 2.

[0045] Table 2 Primers for the construction of recombinant plasmid vectors

[0046]

[0047] The PCR reaction systems involved in the following examples are shown in Table 3.

[0048] Table 3: PCR system

[0049]

[0050] PCR reaction system conditions: 95°C pre-denaturation for 3 min; 95°C denaturation for 30 s, 56°C annealing for 30 s, 72°C extension at 2 kb / min; 32 cycles, storage at 4°C, and PCR product recovery by gel.

[0051] The specific steps are as follows:

[0052] The nucleotide sequence of NmLgt B (NCBI reference sequence: AAC44085.1) was custom synthesized by GENEWIZ Biotech (Suzhou, China) and inserted into the first multiple cloning site of the pCDFDuet-1 plasmid vector. The NmlgtB gene fragment was amplified using primers pCD-NmlgtB-F / R. The promoter on the plasmid vector was replaced with a pair of primers pCD-J23119-F / R using the vector pCDFDuet-1 as a template. The two fragments were gel-recovered separately and ligated using a Gibson kit (NEB Reagents, USA) to obtain the plasmid pCD-J23119-NmlgtB (P J23119 The nucleotide sequence of the promoter is shown in SEQ ID NO. 2).

[0053] Example 2: Knockout of host bypass genes and promoter enhancement of key enzymes

[0054] Table 4 Construction primers used in host transformation examples

[0055]

[0056] The specific steps are as follows:

[0057] The pEcCpf1 / pcrEG method was used to knock out the glK gene in Escherichia coli and replace the original promoter sequence of the NmlgtB gene.

[0058] (1) pEcCpf1 plasmid was transformed into MG1655ΔptsG::glfΔglK::NmlgtBΔugd::galE (EG03). After a single colony grew on a plate, it was inoculated into a test tube containing 4 mL of LB medium containing kanamycin resistance and cultured for 12 h to prepare electrocompetent cells.

[0059] (2) The long primers galK-F and galK-R used for homologous recombination were synthesized by GENEWIZ Biotech (Suzhou, China) with a final concentration of 100 μM.

[0060] (3) The pcrEG plasmid was PCR amplified using primers galK-N23-F / R, and the resulting linear DNA was circularized into the pcrEG-ΔgalK plasmid targeting galK by single-fragment one-step cloning.

[0061] (4) Place the prepared electroporation competent medium containing the pEcCpf1 plasmid on ice, thaw, add the pcrEG-ΔgalK plasmid and the long primers galK-F or galK-R, and immediately add 1 mL of fully pre-chilled LB medium after electroporation. Gently pipette to mix and transfer to a sterile 1.5 mL EP tube. Incubate at 37°C and 200 rpm for 2 h. Then, spread all the cells on a double-antibody plate (final concentration of 50 μg / L kanamycin and 50 μg / L spectinomycin) and incubate inverted in a 37°C incubator for 12 to 15 h.

[0062] (5) Use colony PCR to verify the single colonies that have grown. If the external verification band is shorter than the negative control band, the gene knockout may be successful. Use the external verification primers ΔgalK-YZ-F / R to perform bacterial P verification. Send the samples with shorter external verification bands to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The colonies that match the theoretical sequence are the colonies with successful galK knockout.

[0063] (6) The successfully verified bacteria were inoculated in a 4 mL test tube and cultured for 12 h (50 μg / L kanamycin and a final concentration of 10 mM rhamnose were added during inoculation) to remove the pcrEG-ΔgalK plasmid. The target bacteria were further inoculated into liquid LB medium containing 5 g / L glucose and the bacterial liquid was further streaked onto a plate containing 5 g / L glucose and 15 g / L sucrose to remove the pEcCpf1 plasmid. The final strain obtained was:

[0064] MG1655ΔptsG::glfΔglk::NmlgtBΔugd::galEΔgalK was designated (EG04).

[0065] (7) In addition to knocking out galK, the host bacteria were also modified to strengthen the promoter of the NmlgtB gene. J23119 To replace the original promoter of NmlgtB gene. The construction method is basically the same as that of knocking out galK, except that the promoter fragment used for homologous recombination is obtained by overlap extension PCR. The final P J23119 The enhanced host was named EG05.

[0066] Example 3: Construction of genetically engineered bacteria

[0067] Table 5 Construction primers used in host transformation examples

[0068]

[0069] The specific steps are as follows:

[0070] As above, using the E01 strain as an example, the pEcCpf1 / pcrEG method was used to knock out the ptsG gene in E. coli and integrate it into the glf gene. The glf nucleotide sequence (NCBI Reference Sequence: AAA27691.1) was custom synthesized by GENEWIZ Biotech (Suzhou, China) and inserted into the first multiple cloning site of the pCDFDuet-1 plasmid vector. The glf gene fragment was amplified using primers pCD-glf-F / R.

[0071] (1) The pEcCpf1 plasmid was transformed into the original MG1655 strain. After a single colony grew on a plate, it was inoculated into a test tube containing 4 mL of LB medium containing kanamycin resistance and cultured for 12 h to prepare electrocompetent cells.

[0072] (2) The long primers ptsG-F and ptsG-R used for homologous recombination were synthesized by GENEWIZ Biotech (Suzhou, China) with a final concentration of 100 μM.

[0073] (3) The pcrEG plasmid was PCR amplified using primers ptsG-N23-F / R, and the resulting linear DNA was circularized into the pcrEG-ΔptsG plasmid targeting ptsG by single-fragment one-step cloning.

[0074] (4) Take the prepared electroporation competent medium containing pEcCpf1 plasmid on ice, thaw it, add pcrEG-ΔptsG plasmid and long primers ptsG-F, ptsG-R and glf gene combination fragment, and immediately add 1 mL of fully pre-cooled LB medium after electroporation. Gently pipette to mix and transfer to a sterile 1.5 mL EP tube, and culture at 37°C and 200 rpm for 2 hours. Then, spread all the bacteria on a double-antibody plate (final concentration of 50 μg / L kanamycin and 50 μg / L spectinomycin) and culture in an incubator at 37°C for 12 to 15 hours.

[0075] (5) Use colony PCR to verify the single colonies that have grown. If there is a band in the internal verification, the gene knockout and integration may be successful. Use the internal verification primers glf-YZ-F / glf–NB-R to verify the bacterial P. The colonies whose band length matches the theoretical value are then verified with the external verification primers glf-YZ-F / R. The samples with the external verification results are sent to Suzhou Jinweizhi Biotechnology Co., Ltd. for sequencing. The colonies that match the theoretical sequence are the colonies that have successfully knocked out ptsG and integrated glf.

[0076] (6) The successfully verified bacteria were inoculated in a 4 mL test tube and cultured for 12 h (50 μg / L kanamycin and a final concentration of 10 mM rhamnose were added during inoculation) to remove the pcrEG-ΔptsG plasmid. The target bacteria were further inoculated into liquid LB medium containing 5 g / L glucose and the bacterial liquid was further streaked on a plate containing 5 g / L glucose and 15 g / L sucrose to remove the pEcCpf1 plasmid. The final strain MG1655ΔptsG::glf was named (EG01).

[0077] The same operation as above was performed to complete the transformation of strains EG02, EG06, EG07, EG08, EG09 and EG10.

[0078] Example 4: Fermentation of recombinant strains to produce galactose and galactitol

[0079] The strains EG03 to EG11 constructed above were subjected to fermentation verification.

[0080] First, the above-mentioned recombinant strains EG03, EG04 and EG10 were grown on resistance agar LB plates overnight, and then inoculated into 4 mL LB medium as the initial seed solution. The culture was carried out at 37°C and 200 rpm for 8-12 hours. 400 μL of the seed solution was inoculated into 20 mL of fermentation medium and cultured at 37°C and 200 rpm until the OD 600The pH value was 0.6-0.8, and glucose was added to a final concentration of 5 g / L. Induction culture was continued at 30°C and 200 rpm for 72 hours. One mL of the fermentation broth was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was collected for HPLC analysis. The results are shown in Table 6, indicating that the galactose-producing chassis strain EG04 was successfully constructed, with a galactose yield of 2.96 g / L.

[0081] Based on the above EG04, by knocking out the ilvG site gene, the genes NmlgtB, glf and galE were integrated respectively. Under the same fermentation conditions as above, the two-copy gene NmlgtB strain produced the highest galactose of 4.88g / L.

[0082] Based on the above EG05, the rph site gene was knocked out and the integrated gene galE and the fuck site gene was knocked out and the integrated gene NmlgtB were knocked out respectively. Under the same fermentation conditions as above, the EG08 strain produced the highest galactose of 6.18 g / L.

[0083] Based on the above EG05, the xylB locus gene was knocked out and the GRE3 gene was integrated. The fermentation conditions were the same as above, and the maximum yield of galactitol was 3.98 g / L.

[0084] It should be noted that increasing the number of integrated NmlgtBs helps to increase galactose production (e.g., see E05, 4.88 g / L). In addition, increasing the number of integrated galEs also helps to increase galactose production (e.g., see E08, 6.18 g / L). The galactose production after ΔlacZh is 0, and ΔxylB::GRE3 in E11 can specifically produce galactitol. Table 6 Shake flask fermentation information of engineered strains for galactose and galactitol production

[0085]

[0086] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A recombinant Escherichia coli for producing galactose, characterized in that: It was prepared according to the following steps: using Escherichia coli MG1655 as the starting strain, the competitive pathway glucose-specific PTS enzyme IIBC component gene ptsG, glucose kinase gene glk, UDP-glucose 6-dehydrogenase gene ugd, galactokinase gene galK, gene ilvG, and gene rph were knocked out from the genome; and the UDP-glucose 4-isomerase gene galE was homologously overexpressed; and the exogenous UDP-galactopyranose mutase gene glf and β-1,4-galactosyltransferase gene NmLgtB were integrated and overexpressed.

2. A recombinant Escherichia coli for producing galactitol, characterized in that: It was prepared according to the following steps: Escherichia coli MG1655 was used as the starting strain, and the glucose-specific PTS enzyme IIBC component gene ptsG, glucose kinase gene glk, UDP-glucose 6-dehydrogenase gene ugd, galactokinase gene galK, gene ilvG, and xylulokinase xylB on the E. coli genome were knocked out from the genome; and the endogenous gene UDP-glucose-4-isomerase gene galE was overexpressed; and the exogenous genes UDP-galactopyranose mutase gene glf, β-1,4-galactosyltransferase gene NmlgtB and aldose reductase gene GRE3 were integrated and overexpressed.

3. The recombinant Escherichia coli according to claim 1, characterized in that The glf gene is integrated at the ptsG site on the E. coli genome, the NmlgtB gene is integrated at the glk site on the E. coli genome, the galE gene is integrated at the ugd site on the E. coli genome, the NmlgtB gene is integrated at the ilvG site on the E. coli genome, and the galE gene is integrated at the rph site on the E. coli genome.

4. The recombinant Escherichia coli according to claim 2, characterized in that The genetically engineered bacteria is Escherichia coli MG1655, in which galK is knocked out, the glf gene is integrated into the ptsG site on the Escherichia coli genome, the NmlgtB gene is integrated into the glk site on the Escherichia coli genome, the galE gene is integrated into the ugd site on the Escherichia coli genome, the NmlgtB gene is integrated into the ilvG site on the Escherichia coli genome, and the GRE3 gene is integrated into the xylB site on the Escherichia coli genome.

5. The recombinant Escherichia coli according to claim 1 or 2, characterized in that The nucleotide sequence of ptsG is shown in Gene ID: 945651, the nucleotide sequence of glk is shown in Gene ID: 946858, the nucleotide sequence of ugd is shown in Gene ID: 946571, the nucleotide sequence of galK is shown in Gene ID: 945358, the nucleotide sequence of ilvG is shown in Gene ID: 2847699, the nucleotide sequence of rph is shown in Gene ID: 948156, the nucleotide sequence of galE is shown in Gene ID: 945354, the nucleotide sequence of glf is shown in Gene ID: 79904430, and the nucleotide sequence of NmlgtB is shown in SEQ ID NO. 1; The nucleotide sequence of xylB is shown in Gene ID: 948133, and the nucleotide sequence of GRE3 is shown in Gene ID: 856504.

6. A method for producing galactose or galactitol, characterized in that: The recombinant Escherichia coli according to claim 1 or 3 is used to ferment and produce galactose, and the recombinant Escherichia coli according to claim 2 or 4 is used to ferment and produce galactitol.

7. The method according to claim 6, characterized in that In a culture medium containing 30 g / L glycerol, the recombinant Escherichia coli according to any one of claims 1 to 4 was used for fermentation at 37°C with a shaking speed of 200 rpm until the OD 600 After the pH value reached 0.6 to 0.8, the culture was induced at 30°C for at least 72 h; the amount of substrate glucose added was 5 g / L.

8. The method according to claim 7, characterized in that: The culture medium also contains 4.0 g / L diammonium hydrogen phosphate, 13.5 g / L potassium dihydrogen phosphate, 1.4 g / L magnesium sulfate heptahydrate, 1.7 g / L citric acid, and 10 ml / L trace metal elements; the trace metal elements include: 0.35 g / L manganese sulfate monohydrate, 10 g / L ferrous sulfate, 1.0 g / L anhydrous copper sulfate, 2.25 g / L zinc sulfate heptahydrate, 2.0 g / L calcium chloride dihydrate, 0.23 g / L sodium borate decahydrate, and 0.11 g / L ammonium molybdate.

9. Use of the recombinant Escherichia coli according to claim 1 or 3 in the preparation of galactose products.

10. Use of the recombinant Escherichia coli according to claim 2 or 4 in the preparation of galactitol products.

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