Production method of 1, 3-dihydroxyacetone

Abstract

The invention discloses a production method of 1, 3-dihydroxy acetone. The invention provides a method for constructing an engineering strain capable of producing 1, 3-dihydroxyacetone by using glucose, which comprises the following steps: A, inhibiting the expression of the following endogenous gene A on the basis of recipient bacteria, and enabling the recipient bacteria to express the coding gene of T7RNA polymerase T7RNAP and the coding gene of 1, 3-dihydroxyacetone phosphatase HdpA; in a 5L tank fermentation test for producing 1, 3-dihydroxyacetone by using glucose, the yield of the 1, 3-dihydroxyacetone can reach 30.1 g / L, and the conversion rate from the glucose to the 1, 3-dihydroxyacetone can reach 0.95 mol / mol. The method disclosed by the invention is of great significance to synthesis of 1, 3-dihydroxyacetone from glucose.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and specifically relates to a method for producing 1,3-dihydroxyacetone. Background Art

[0002] 1,3-Dihydroxyacetone (DHA) is the simplest tricarbon ketose with three active groups, enabling it to participate in various reactions. It possesses sun protection, UV protection, moisturizing, skin disease treatment, and promotes weight loss and increased endurance, making it widely used in the chemical, pharmaceutical, feed, cosmetic, and food industries. DHA synthesis methods mainly include chemical synthesis and microbial synthesis. Due to drawbacks such as low conversion efficiency, low selective oxidation efficiency, and high catalyst costs, researchers prefer microbial synthesis methods, which offer environmental optimization and higher conversion efficiency. Currently, industrial DHA production primarily utilizes *Gluconobacter* sp. for efficient conversion of glycerol substrates. However, this method suffers from problems such as high concentrations of glycerol substrate and DHA product inhibiting cell growth, the presence of glyceric acid as a byproduct, and complex fermentation processes. In recent years, with advancements in synthetic biology, constructing efficient microbial cell factories to synthesize DHA using inexpensive carbon source glucose as a substrate has become a research trend. Summary of the Invention

[0003] The purpose of this invention is to provide a method for producing 1,3-dihydroxyacetone.

[0004] In a first aspect, the present invention provides a method for constructing an engineered strain that produces 1,3-dihydroxyacetone using glucose, comprising the following step A:

[0005] A. Based on the recipient bacteria, inhibit the expression of the following endogenous gene A, and cause the recipient bacteria to express the encoding gene of T7 RNA polymerase T7RNAP and the encoding gene of phosphate dihydroxyacetone phosphatase HdpA.

[0006] The endogenous genes are as follows: the gene encoding histidine phosphocarrier protein PtsH, the gene encoding phosphoenolpyruvate-protein phosphotransferase IPtsI, the gene encoding glucose-specific enzyme II complex EIIA Crr, the gene encoding dihydroxyacetone kinase DhaRKLM, the gene encoding glycerol kinase GlpK, the gene encoding methylglyoxal synthase MgsA, the gene encoding glycerol dehydrogenase GldA, the gene encoding L-arabinose isomerase AraA, the gene encoding ribulose kinase AraB, the gene encoding triose phosphate isomerase TpiA, the gene encoding glucose-specific PTS enzyme IIBC component PtsG, the gene encoding pyruvate oxidase PoxB, and the gene encoding pyruvate formate lyase PflB.

[0007] The resulting strain was named Engineered Bacteria 1 (corresponding to TZ-222); Engineered Bacteria 1 is an engineered strain capable of producing 1,3-dihydroxyacetone using glucose. The recipient strain can use glucose as a carbon source.

[0008] The method described above also includes step B:

[0009] B. Based on step A, inhibit the expression of the encoding genes for endogenous 6-phosphofructoaldehyde 1FsaA and 6-phosphofructoaldehyde 2FsaB in the recipient bacteria.

[0010] The resulting strain was named Engineered Bacterium 2 (corresponding to TZ-223); Engineered Bacterium 2 is an engineered strain capable of producing 1,3-dihydroxyacetone from glucose.

[0011] The method described above also includes step C:

[0012] C. Based on step B, inhibit the expression of the gene encoding the endogenous glycerol transporter GlpF in the recipient bacteria.

[0013] The resulting strain was named Engineered Bacterium 3 (corresponding to TZ-231); Engineered Bacterium 3 is an engineered strain capable of producing 1,3-dihydroxyacetone from glucose.

[0014] The method described above also includes step D:

[0015] D. Based on step C, the recipient bacteria express the gene encoding NADH oxidase NOX and the expression of the gene encoding alcohol dehydrogenase AdhE in the recipient bacteria is inhibited.

[0016] The resulting strain was named Engineered Bacterium 4 (corresponding to TZ-236); Engineered Bacterium 4 is an engineered strain capable of producing 1,3-dihydroxyacetone from glucose.

[0017] The method described above also includes step E:

[0018] E. Based on step D, inhibit the expression of the encoding genes for endogenous glucose-6-phosphate dehydrogenase Zwf, phosphoglucuronide dehydratase Edd, and 2-keto-3-deoxy-6-phosphoglucuronide Eda in the recipient bacteria.

[0019] The resulting strain was named Engineered Bacterium 5 (corresponding to TZ-237); Engineered Bacterium 5 is an engineered strain capable of producing 1,3-dihydroxyacetone from glucose.

[0020] In the above text, the inhibition of the encoding gene of glucose-specific PTS enzyme IIBC component PtsG and the expression of the encoding gene of T7 RNA polymerase T7RNAP in the recipient bacteria are achieved by integrating an expression cassette containing the T7 RNA polymerase T7RNAP gene into the encoding gene of glucose-specific PTS enzyme IIBC component PtsG in the host bacteria, thereby disrupting the expression of the PtsG encoding gene ptsG.

[0021] In the above text, the inhibition of the coding genes for pyruvate oxidase PoxB (poxB) and pyruvate formate lyase PflB, and the expression of the coding gene for phosphate dihydroxypyruvate phosphatase HdpA in the recipient bacteria, are described by integrating an expression cassette containing the coding gene for phosphate dihydroxypyruvate phosphatase HdpA into the coding gene for pyruvate oxidase PoxB and the coding gene for pyruvate formate lyase PflB in the recipient bacteria, respectively, thereby disrupting the expression of the coding genes for PoxB (poxB) and PflB (pflB).

[0022] In the above text, the process of causing the recipient bacteria to express the NADH oxidase NOX gene and inhibiting the expression of the gene encoding alcohol dehydrogenase AdhE in the recipient bacteria involves integrating an expression cassette containing the NADH oxidase NOX gene into the gene encoding alcohol dehydrogenase AdhE in the recipient bacteria, thereby disrupting the expression of the AdhE gene.

[0023] The promoter driving the expression of the T7 RNA polymerase T7RNAP gene in the expression cassette (SEQ ID No. 4, positions 78-2759) is the P93 promoter;

[0024] Alternatively, the promoter driving the expression of the HdpA-encoding gene of phosphate dihydroxyacetone phosphatase in the expression cassette (positions 56-1158 of SEQ ID No. 5 or positions 56-1158 of SEQ ID No. 7) is the T7 promoter;

[0025] Alternatively, the promoter driving the expression of the NADH oxidase NOX gene in the expression cassette (positions 86-1621 of SEQ ID No. 6) is the P46 promoter.

[0026] The T7 RNA polymerase T7RNAP gene is derived from Escherichia coli BL21(DE3);

[0027] Alternatively, the gene encoding dihydroxyacetone phosphatase HdpA is derived from Corynebacterium glutamicum;

[0028] Alternatively, the NADH oxidase NOX gene may be derived from Lactobacillus rhamnosus.

[0029] The suppression of the expression of each endogenous gene is to knock out all or part of each endogenous gene.

[0030] In the method described above, the recipient bacterium is *Escherichia coli* capable of using glucose as a carbon source. In a specific embodiment of the present invention, the *Escherichia coli* is *Escherichia coli* ATCC 8739.

[0031] The T7 RNA polymerase may be a protein with the amino acid sequence shown in SEQ ID No. 1, or a protein of SEQ ID No. 1 with the same function after substitution and / or deletion and / or addition of one or more amino acid residues, or a protein with the same function as SEQ ID No. 1 having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology, or a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 1.

[0032] The DHAP phosphatase HdpA may be a protein with the amino acid sequence shown in SEQ ID No. 2, or a protein of SEQ ID No. 2 with the same function after substitution and / or deletion and / or addition of one or more amino acid residues, or a protein with the same function as SEQ ID No. 2 having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology, or a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 2.

[0033] The NADH oxidase Nox may be a protein with the amino acid sequence shown in SEQ ID No. 3, or a protein of SEQ ID No. 3 with the same function after substitution and / or deletion and / or addition of one or more amino acid residues, or a protein with the same function as SEQ ID No. 3 having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology, or a fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 3.

[0034] The substitution and / or deletion and / or addition of one or more amino acid residues refers to the substitution and / or deletion and / or addition of no more than ten amino acid residues.

[0035] In the aforementioned proteins, the tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0036] In the proteins mentioned above, homology refers to the similarity of amino acid sequences. The similarity of amino acid sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI website. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search for the similarity of a pair of amino acid sequences, the homology value (%) can then be obtained.

[0037] In the aforementioned proteins, the homology of 95% or more can be at least 96%, 97%, or 98%. The homology of 90% or more can be at least 91%, 92%, 93%, or 94%. The homology of 85% or more can be at least 86%, 87%, 88%, or 89%. The homology of 80% or more can be at least 81%, 82%, 83%, or 84%.

[0038] The amino acid sequence of the endogenous histidine phosphocarrier protein PtsH is ACA76929.1 (submission date: February 14, 2008). The amino acid sequence of the endogenous phosphoenolpyruvate-protein phosphotransferase IPtsI is ACA76928.1 (submission date: February 14, 2008). The amino acid sequence of the endogenous glucose-specific enzyme II complex EIIACrr is ACA76927.1 (submission date: February 14, 2008). The amino acid sequences of the endogenous 1,3-dihydroxyacetone kinase DhaRKLM are ACA78059.1 (submission date: February 14, 2008), ACA78060.1 (submission date: February 14, 2008), ACA78061.1 (submission date: February 14, 2008), and ACA78062.1 (submission date: February 14, 2008). The amino acid sequence of the endogenous glycerol kinase GlpK is ACA79690.1 (submission date: February 14, 2008). The amino acid sequence of the endogenous methylglyoxal synthase MgsA is ACA78263.1 (submission date: February 14, 2008). The amino acid sequence of the endogenous glycerol dehydrogenase GldA is ACA79669.1 (submission date: February 14, 2008). The amino acid sequence of the endogenous L-arabinose isomerase AraA is ACA79209.1 (submission date: February 14, 2008). The amino acid sequence of the endogenous ribulose kinase AraB is ACA79208.1 (submission date: February 14, 2008). The amino acid sequence of the endogenous triose phosphate isomerase TpiA is ACA79697.1 (submission date: February 14, 2008). The amino acid sequence of PtsG, a component of the endogenous glucose-specific PTS enzyme IIBC, is ACA78131.1 (submission date: February 14, 2008). The amino acid sequence of PflB, an endogenous pyruvate-formate lyase, is ACA78322.1 (submission date: February 14, 2008). The amino acid sequence of PoxB, an endogenous pyruvate oxidase, is ACA78353.1 (submission date: February 14, 2008). The amino acid sequence of AdhE, an endogenous alcohol dehydrogenase, is ACA78022.1 (submission date: February 14, 2008). All of the above sequences are corresponding sequences in the NCBI database.

[0039] The nucleotide sequence of the T7 promoter is shown in positions 98-165 of SEQ ID No. 4.

[0040] The gene encoding the T7 RNA polymerase T7RNAP may be a DNA molecule with a nucleotide sequence as shown in positions 166-2682 of SEQ ID No. 4, or a DNA molecule that hybridizes under stringent conditions to the DNA molecule shown in positions 166-2682 of SEQ ID No. 4 and encodes the protein shown in SEQ ID No. 1, or a DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the DNA sequence defined in positions 166-2682 of SEQ ID No. 4 and encodes the protein shown in SEQ ID No. 1.

[0041] The gene encoding the DHAP phosphatase (i.e., the hdpA gene) may be a DNA molecule with a nucleotide sequence as shown in positions 165-992 of SEQ ID No. 5, or a DNA molecule that hybridizes under stringent conditions to the DNA molecule shown in positions 165-992 of SEQ ID No. 5 and encodes the protein shown in SEQ ID No. 2, or a DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the DNA sequence defined in positions 165-992 of SEQ ID No. 5 and encodes the protein shown in SEQ ID No. 2.

[0042] The gene encoding the NADH oxidase Nox (i.e., the nox gene) may be a DNA molecule with a nucleotide sequence as shown in SEQ ID No. 6, 174-1535, or a DNA molecule that hybridizes under stringent conditions with the DNA molecule shown in SEQ ID No. 6, 174-1535 and encodes the protein shown in SEQ ID No. 3, or a DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology with the DNA sequence defined in SEQ ID No. 6, and encodes the protein shown in SEQ ID No. 3.

[0043] The stringent conditions for the above-mentioned encoding gene can be as follows: hybridization at 50°C in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 2×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 1×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.5×SSC and 0.1% SDS; or hybridization at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4, and 1mM EDTA, followed by rinsing at 50°C in 0.1×SSC and 0.1% SDS. Alternatively, the membrane can be rinsed in SDS; or hybridized at 50°C in a mixed solution of 7% SDS, 0.5M Na3PO4 and 1mM EDTA, and then rinsed at 65°C in 0.1×SSC and 0.1% SDS; or hybridized at 65°C in a solution of 6×SSC and 0.5% SDS, and then washed once each with 2×SSC and 0.1% SDS and 1×SSC and 0.1% SDS.

[0044] In the aforementioned coding genes, homology refers to the similarity of nucleotide sequences. The similarity of nucleotide sequences can be determined using homology search sites on the internet, such as the BLAST page on the NCBI website. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search for the similarity of a pair of nucleotide sequences, the homology value (%) can be obtained.

[0045] In the aforementioned coding genes, the homology of 95% or more can be at least 96%, 97%, or 98%. The homology of 90% or more can be at least 91%, 92%, 93%, or 94%. The homology of 85% or more can be at least 86%, 87%, 88%, or 89%. The homology of 80% or more can be at least 81%, 82%, 83%, or 84%.

[0046] In a specific embodiment of the present invention, knocking out a target gene in the genome is achieved by a method comprising the following steps: (a1) using pXZ-CS plasmid as a template, PCR amplification is performed using long primers Target-CS-up and Target-CS-down containing upstream and downstream homologous arm sequences of the target gene to be knocked out, to obtain a homologous recombination cat-sacB fragment I (a PCR product of approximately 2.7 kb), and this fragment is integrated into the complete coding region of the target gene in the *E. coli* genome (from the start codon ATG to the stop codon TAA or TAG); (a2) using the *E. coli* genome as a template, upstream and downstream homologous arm fragments are amplified by PCR using primers Target-YZ-up and Target-r1, and primers Target-YZ-down and Target-f1, respectively. These two homologous arm fragments are then subjected to overlapping extension PCR amplification to form a second-step homologous recombination fragment containing only upstream and downstream homologous arm sequences of the target gene, without the target gene sequence. This second homologous recombination replaces the cat-sacB fragment from the first-step recombination. The primer sequences are shown in Table 2.

[0047] In a specific embodiment of the present invention, the expression cassette of the T7 RNA polymerase encoding gene is integrated into the genome at the location of the gene encoding PtsG (i.e., the ptsG gene) of the glucose-specific PTS enzyme IIBC component; the gene encoding HdpA (i.e., the hdpA gene) of the DHAP phosphatase is integrated into the genome at two locations: the gene encoding PflB (i.e., the pflB gene) of the endogenous pyruvate formate lyase and the gene encoding PoxB (i.e., the poxB gene); and the gene encoding Nox (i.e., the nox gene) of the NADH oxidase is integrated into the genome. The method of completing these three steps, which involves assembling the gene encoding the endogenous alcohol dehydrogenase AdhE (i.e., the adhE gene) in the genome, differs from the previously described method of "knocking out a target gene in the genome" only in step (a2) from the construction of an expression cassette for knocking out the target gene: using E. coli BL21(DE3) genomic DNA as a template, the T7-RNAP gene fragment is amplified using primers T7RNAP-CF and T7RNAP-CR, and then assembled into a cassette containing the constitutive promoter P using circular polymerase extension cloning (CPEC). 93 The recombinant plasmid pSC106-T7RNAP was obtained from the plasmid vector pSC106. The plasmid containing P was amplified using primers ptsG-pSC-up and ptsG-pSC-down. 93The expression cassette fragment of the T7-RNAP promoter was obtained; using Corynebacterium glutamicum ATCC13032 genomic DNA as a template, the HdpA gene fragment was amplified using primers HdpA-CF and HdpA-CR. The HdpA gene fragment was then assembled into the plasmid vector pET30a containing the T7 promoter using CPEC, resulting in the recombinant plasmid pET30-CgHdpA. The HdpA expression cassette fragment containing the T7 promoter was amplified using primers pflB-pET-up and pflB-pET-down, and primers poxB-pET-up and poxB-pET-down, respectively. The Nox gene fragment was amplified using Lactobacillus rhamnosus genomic DNA as a template using primers Nox-CF and Nox-CR. The Nox gene fragment was then assembled into the plasmid vector pET30a containing the P7 promoter using CPEC. 46 The recombinant plasmid pSC103-Nox was obtained from the plasmid vector pSC103 containing the promoter. The plasmid carrying the P promoter was then amplified using primers adhE-pSC-up and adhE-pSC-down. 46 The expression cassette fragment of the Nox promoter. Primer sequences are shown in Table 2.

[0048] In a second aspect, the present invention provides recombinant bacteria prepared by the method described in the first aspect.

[0049] Thirdly, the present invention provides the application of the recombinant bacteria described in the third aspect in the production of 1,3-dihydroxyacetone;

[0050] Alternatively, the present invention provides the application of the recombinant bacteria described in the third aspect in increasing the yield of 1,3-dihydroxyacetone.

[0051] In the above-described application, the production of 1,3-dihydroxyacetone or the increase of 1,3-dihydroxyacetone yield refers to the production of 1,3-dihydroxyacetone using glucose.

[0052] Fourthly, the present invention provides a method for producing 1,3-dihydroxyacetone, comprising the following steps: fermenting the recombinant bacteria described in the second aspect, collecting the fermentation product, and obtaining 1,3-dihydroxyacetone.

[0053] In the method described above, the fermentation is carried out in a fermentation medium containing glucose.

[0054] Furthermore, the fermentation medium CM9 used in the fermentation culture has the following formulation: per 1L, it contains 50g of glucose, 5g of corn steep liquor powder, 1g of NH4Cl, 0.87g of (NH4)2SO4, 0.5g of MgSO4·7H2O, 0.47g of KCl, 0.14g of KH2PO4, and trace elements ZnCl2 1.47μM, H3BO3 0.81μM, CoCl2·6H2O 0.84μM, CuCl2·2H2O 0.59μM, FeCl3·6H2O 5.92μM, Na2MoO4·2H2O 0.83μM, with the balance being water.

[0055] In the method described above, the fermentation conditions can be: 37°C, 250 rpm for 72 h.

[0056] Experiments of this invention demonstrate that the engineered *E. coli* strain TZ-237 constructed in this invention achieves a 12.99 g / L yield of 1,3-dihydroxyacetone and a glucose-to-1,3-dihydroxyacetone conversion rate of 0.95 mol / mol in shake-flask fermentation experiments using glucose to produce 1,3-dihydroxyacetone. This invention is of great significance for the production of 1,3-dihydroxyacetone from glucose. Attached Figure Description

[0057] Figure 1 This is the biosynthetic route for 1,3-dihydroxyacetone in this invention.

[0058] Abbreviations: PEP, phosphoenolpyruvate; ATP, adenine triphosphate; ADP, adenine diphosphate; G6P, glucose-6-phosphate; F6P, fructose-6-phosphate; FBP, fructose-1,6-bisphosphate; DHAP, dihydroxyacetone phosphate; G3P, glyceraldehyde-3-phosphate; DHA, 1,3-dihydroxyacetone; MG, methylglyoxal; PTS, phosphotransferase system; Glk, glucokinase; Zwf, glucose-6-phosphate dehydrogenase; Edd, phosphoglucuronide dehydratase; Eda, 2-keto-4-hydroxyglutarate aldolase; Pgi, glucose-6-phosphate isomerase; Pfk, fructose-6-phosphate... Glucokinase; Fba, fructose-1,6-bisphosphate aldolase; FasA / FsaB, fructose-6-phosphate aldolase A / B; MgsA, methylglyoxal synthase; TpiA, triose phosphate isomerase; HdpA, dihydroxyacetone phosphate phosphatase; DhaRKLM, dihydroxyacetone kinase; GlpK, glycerol kinase; GldA, glycerol dehydrogenase; Nox, NADH oxidase; GlpF, glycerol transporter; PPP pathway, pentose phosphate pathway; ED pathway, Energäuhl and Dotloff pathway, also known as the 2-keto-3-deoxy-6-phosphogluconic acid (KDPG) pathway; TCA cycle, tricarboxylic acid cycle, also known as the citric acid cycle. Detailed Implementation

[0059] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0061] The biosynthetic route for 1,3-dihydroxyacetone in this invention is as follows: Figure 1 As shown (this invention relates to four key points: overexpression of HdpA, knockout of TpiA, knockout of FasA and FsaB, and overexpression of Nox; among which overexpression of HdpA is the most important). The strains used in this invention are shown in Table 1, the primers used are shown in Table 2, and the plasmids involved are shown in Table 3.

[0062] Table 1 lists the strains constructed in this invention.

[0063]

[0064] Table 2 lists the primers used in this invention.

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072] Table 3 lists the plasmids used and constructed in this invention.

[0073]

[0074]

[0075] In the following examples, all gene IDs are from the NCBI database (all gene ID information is from the NCBI database, web link: https: / / www.ncbi.nlm.nih.gov / nuccore / CP000946.1 / ), specifically as follows: The nucleotide sequence of the gene encoding PtsG, a component of the endogenous glucose-specific PTS enzyme IIBC (i.e., the ptsG gene), is Gene ID: EcolC_2500 (submission date: February 14, 2008). The nucleotide sequence of the gene encoding PflB, an endogenous pyruvate-formate lyase (i.e., the pflB gene), is Gene ID: EcolC_2693 (submission date: February 14, 2008). The nucleotide sequence of the gene encoding PoxB, an endogenous pyruvate oxidase (i.e., the poxB gene), is Gene ID: EcolC_2725 (submission date: February 14, 2008). The nucleotide sequence of the gene encoding endogenous alcohol dehydrogenase AdhE (i.e., the adhE gene) is Gene ID: EcolC_2387 (submission date: February 14, 2008).

[0076] The nucleotide sequence of the gene encoding the endogenous histidine phosphocarrier protein PtsH (i.e., the ptsH gene) is Gene ID: EcolC_1263 (submission date: February 14, 2008). The nucleotide sequence of the gene encoding the endogenous phosphoenolpyruvate-protein phosphotransferase IPtsI (i.e., the ptsI gene) is Gene ID: EcolC_1262 (submission date: February 14, 2008). The nucleotide sequence of the gene encoding the endogenous glucose-specific enzyme II complex EIIA Crr (i.e., the crr gene) is Gene ID: EcolC_1261 (submission date: February 14, 2008). The nucleotide sequences of the encoding genes for endogenous dihydroxyacetone kinase DhaRKLM (i.e., the dhaRKLM gene) are Gene ID: EcolC_2425 (submission date: February 14, 2008), EcolC_2426 (submission date: February 14, 2008), EcolC_2427 (submission date: February 14, 2008), and EcolC_2428 (submission date: February 14, 2008). The nucleotide sequence of the encoding gene for endogenous glycerol kinase GlpK (i.e., the glpK gene) is Gene ID: EcolC_4092 (submission date: February 14, 2008). The nucleotide sequence of the encoding gene for methylglyoxal synthase MgsA (i.e., the mgsA gene) is Gene ID: EcolC_2633 (submission date: February 14, 2008). The nucleotide sequence of the gene encoding glycerol dehydrogenase GldA (i.e., the gldA gene) is Gene ID: EcolC_4070 (submission date: February 14, 2008). The nucleotide sequence of the gene encoding L-arabinose isomerase AraA (i.e., the araA gene) is Gene ID: EcolC_3595 (submission date: February 14, 2008). The nucleotide sequence of the gene encoding ribulose kinase AraB (i.e., the araB gene) is Gene ID: EcolC_3594 (submission date: February 14, 2008). The nucleotide sequence of the gene encoding endogenous triose phosphate isomerase TpiA (i.e., the tpiA gene) is Gene ID: EcolC_4099 (submission date: February 14, 2008).

[0077] The nucleotide sequence of the gene encoding endogenous fructose-6-phosphate aldolase 1 (FsaA) (i.e., the fsaA gene) is Gene ID: EcolC_2819 (submission date: February 14, 2008). The nucleotide sequence of the gene encoding endogenous fructose-6-phosphate aldolase 2 (FsaB) (i.e., the fsaB gene) is Gene ID: EcolC_4069 (submission date: February 14, 2008). The nucleotide sequence of the gene encoding endogenous glycerol transporter GlpF (i.e., the glpF gene) is Gene ID: EcolC_4091 (submission date: February 14, 2008). The nucleotide sequence of the gene encoding endogenous glucose-6-phosphate dehydrogenase Zwf (i.e., the zwf gene) is Gene ID: EcolC_1780 (submission date: February 14, 2008). The nucleotide sequence of the gene encoding endogenous phosphoglucuronide dehydratase Edd (i.e., the edd gene) is GeneID: EcolC_1781 (submission date: February 14, 2008). The nucleotide sequence of the gene encoding endogenous 2-keto-3-deoxy-6-phosphoglucuronide Eda (i.e., the eda gene) is GeneID: EcolC_1782 (submission date: February 14, 2008). All GeneIDs are from the NCBI database.

[0078] Example 1: Construction of recombinant Escherichia coli TZ-222

[0079] Starting from *Escherichia coli* ATCC 8739 (Gunsalus IC, Hand DB (1941) The use of bacteria in the chemical determination of total vitamin CJ BiolChem 141:853-858.), the T7RNAP gene (GenBank No: WP_001092355.1), derived from *Escherichia coli* BL21 (DE3), was integrated using the λRed homologous recombination plasmid tool; the phosphate hydroxypyruvate phosphatase HdpA gene (GenBank No: WP_003859582.1), derived from *Corynebacterium glutamicum* ATCC13032, was integrated; the gene encoding histidine phosphate carrier protein PtsH (i.e., ptsH gene) was knocked out, the gene encoding phosphoenolpyruvate-protein phosphotransferase IPtsI (i.e., ptsI gene) was knocked out, and the glucose-specific enzyme II complex EIIA was knocked out. The following gene sequences were used to obtain recombinant Escherichia coli TZ-222 (Table 1): * **crr gene** (the gene encoding Crr), * **dhaRKLM gene** (the gene encoding dihydroxyacetone kinase DhaRKLM), * **glpK gene** (the gene encoding glycerol kinase GlpK), * **mgsA gene** (the gene encoding methylglyoxal synthase MgsA), * **gldA gene** (the gene encoding glycerol dehydrogenase GldA), * **araA gene** (the gene encoding L-arabinose isomerase AraA), * **araB gene** (the gene encoding ribulose kinase AraB), and * **tpiA gene** (the gene encoding triose phosphate isomerase TpiA).

[0080] I. Integration of T7RNAP gene and phosphate hydroxypyruvate phosphatase HdpA gene using λRed homologous recombination plasmid tool

[0081] 1. Constructing exogenous gene recombination expression plasmids

[0082] The first step is to construct the vector backbone for expressing exogenous genes.

[0083] Using empty plasmids pSC103 and pSC106 as templates (Table 3), PCR amplification was performed using primers pSC103-CF and pSC103-CR, and pSC106-CF and pSC106-CR (Table 2), respectively, yielding a 3517 bp vector backbone fragment 1 (containing amp). R pSC101 ori, P 46 (promoter) and 3429bp vector backbone fragment 2 (containing amp) R pSC101 ori, P93 (Promoter).

[0084] Using the pET30a(+) empty plasmid as a template (Table 3), the vector backbone 3 containing kan was amplified using primers pET30-CF and pET30-CR. R pBR322 ori, P T7lac Promoter.

[0085] The amplification system consisted of: 10 μl New England Biolabs Phusion 5X buffer, 1 μl dNTPs (2.5 mM each), 20 ng DNA template, 2 μl primers (10 μM each), 0.5 μl Phusion High-Fidelity DNA polymerase (2.5 U / μl), and 33.5 μl distilled water, for a total volume of 50 μl.

[0086] The amplification conditions were: 98℃ pre-denaturation for 2 minutes (1 cycle); 98℃ denaturation for 10 seconds, 60℃ annealing for 10 seconds, 72℃ extension for 2.5 minutes (30 cycles); 72℃ extension for 5 minutes (1 cycle).

[0087] The second step is to obtain the nox gene, T7RNAP expression cassette, and hdpA gene fragment.

[0088] Using the Lactobacillus plantarum genome as a template, PCR amplification was performed using primers Nox-p103-CF and Nox-p103-CR (Table 2) to obtain a 1412 bp PCR product, which is DNA fragment I (nox gene). The amplification system and conditions are the same as those described in the first step above.

[0089] Using the Escherichia coli BL(DE3) genome as a template, PCR amplification was performed using primers T7RNAP-p106-CF and T7RNAP-p106-CR (Table 2) to obtain a PCR product of approximately 2.5 kb, which is DNA fragment II (T7RNAP expression cassette). The amplification system and conditions are the same as those described in the first step above.

[0090] Using the genome of Corynebacterium glutamicum ATCC13032 as a template, PCR amplification was performed using primers CgHdpA-pET30-CF and CgHdpA-pET30-CR (Table 2) to obtain an 868 bp PCR product, which is DNA fragment III (hdpA gene fragment). The amplification system and conditions are the same as those in the first step above.

[0091] In the third step, vector backbone fragment 1 and DNA fragment I, vector backbone fragment 2 and DNA fragment II, and vector backbone fragment 3 and DNA fragment III were assembled using a seamless cloning kit (sequentially seamlessly linked in the order of I, II, and III), and transformed into TransT1 competent cells (Beijing TransGen Biotech Co., Ltd.). The resulting clones were verified by PCR using primers pSC-YZ-F and pSC-YZ-R (to identify pSC106-T7RNAP or pSC103-NOX, with a positive result of 2969 bp) and primers pET30-YZ-F and pET30-YZ-R (to identify pET30-CgHdpA, with a positive result of 1094 bp) (Table 2). Plasmid DNA was extracted from the positive clones and sent for sequencing analysis to obtain the correct recombinant plasmids pSC106-T7RNAP, pSC103-NOX, and pET30-CgHdpA.

[0092] 2. Integration of exogenous genes

[0093] The integration of exogenous genes was achieved using a two-step homologous recombination method, the specific method of which can be found in the literature (Tan et al., 2016, Biotechnol Biofuels 9; Chen et al., 2014, Appl Microbiol Biotechnol 95:2197-2205).

[0094] 1) PtsG site integration P 93 -T7RNAP

[0095] First step: Homologous recombination:

[0096] Using pXZ-CS plasmid as a template, PCR amplification was performed using primers ptsG-CS-up / ptsG-CS-down (Table 2) to obtain the first homologous recombination fragment ptsG-CS fragment I, which was then integrated into the ptsG gene from position 151 upstream to position 1434 upstream.

[0097] Starting with *E. coli* ATCC 8739, electrotransformation competent cells were prepared. Plasmid pKD46 was transformed into *E. coli* ATCC 8739 electrotransformation competent cells, plated on ampicillin plates, and incubated overnight at 30°C. Single colonies were picked and cultured in 2 mL LB agar (containing ampicillin and 5% L(+)-arabinose) at 250 rpm for 6 hours at 30°C. Competent cells were then prepared, transformed with ptsG-CS fragment I, and cultured at 75 rpm for 4 hours at 30°C. The cells were then plated on LB agar (containing ampicillin and chloramphenicol) and incubated at 30°C for 24 hours. Ten single colonies were selected for colony PCR verification. Primers were CS-YZ-up and ptsG-YZ-down (Table 2). Cells approximately 0.8 kb in size were considered positive and named the first homologous recombinant strain.

[0098] The second step is homologous recombination: competent cells and transformation methods are the same as above.

[0099] Using pSC106-T7RNAP plasmid as a template, ptsG-pSC-up / ptsG-pSC-down was used to amplify P plasmids carrying upstream and downstream homologous arm sequences of the ptsG gene. 93 The -T7RNAP expression cassette, measuring 2759 bp, yielded the second homologous recombination fragment ptsG::P. 93 -T7 RNA fragment II, the nucleotide sequence of which is SEQ ID No. 4, with P at positions 78-165. 93 The promoter contains the T7RNAP fragment at positions 166-2682, and the terminator at positions 2683-2759.

[0100] Competent cells of the first homologous recombinant bacteria were prepared and transformed into ptsG::P 93 -T7 RNA fragment II, 75 rpm, 30°C for 4 hours of recovery culture, then inoculated into LB medium (containing 10% sucrose), 250 rpm, 37°C for 24 hours. The culture was diluted and plated on LB agar plates, incubated overnight at 37°C. Single colonies were picked and inoculated onto LB and chloramphenicol-LB agar plates respectively. Single colonies that grew normally on LB agar but not on chloramphenicol-LB agar were selected for PCR verification. PCR amplification was performed using primers ptsG-YZ-up / ptsG-YZ-down (Table 2). A colony size of approximately 3.3 kb was considered positive and named ATCC8739 / ptsG::P 93 -T7RNAP, this bacterium is a P-type RNAP 93 The -T7RNAP fragment (SEQ ID No. 4, positions 78-2682) replaces the fragment shown at positions 151-1434 of the ptsG gene in the ATCC 8739 genome.

[0101] 2) Integration of the phosphate hydroxyacetone phosphatase HdpA gene at the poxB site.

[0102] The first step, homologous recombination, is basically the same as step 1) above, with the only differences being the following:

[0103] The first homologous recombination fragment, poxB-CS fragment I, was obtained using the template pXZ-CS and primers poxB-CS-up and poxB-CS-down, with a size of 2729 bp.

[0104] The electroporated competent cells were ATCC8739 / ptsG::P 93 -T7RNAP;

[0105] The identification primers were CS-YZ-up and poxB-CS-down. A sample size of 820 bp was considered positive, and the positive strain was named the first homologous recombinant strain.

[0106] The second step is homologous recombination:

[0107] The second homologous recombination fragment poxB::P T7 Obtaining the -CgHdpA fragment II: The template was pET30-CgHdpA, the primers were poxB-pET-up / poxB-pET-down, and the size was 1158bp; the P T7 The nucleotide sequence of fragment II of -CgHdpA is shown in SEQ ID No. 5, where segments 56-164 are P. T7 The promoter is CgHdpA, with bits 165-992 being the promoter and bits 993-1158 being the terminator.

[0108] The competent cells for electroporation were the aforementioned first homologous recombinant bacteria;

[0109] The identification primers were poxB-YZ-up and poxB-YZ-down. A sample size of 1849 bp was considered positive, and the positive strain was named ATCC 8739 / ptsG::P 93 -T7RNAP / poxB::P T7 -hdpA, this bacterium is P 93 The -T7RNAP fragment (positions 78-2682 of SEQ ID No. 4) replaces the fragment shown at positions 151-1434 of the ptsG gene in the ATCC 8739 genome, and the poxB::P T7 The CgHdpA fragment (SEQ ID No. 5, positions 56-1158) replaces the fragment shown at positions 396-1083 of the poxB gene in the ATCC 8739 genome.

[0110] 3) Integration of the phosphate hydroxypyruvate phosphatase hdpA gene at the pflB site.

[0111] The first step, homologous recombination, is basically the same as step 1) above, with the only differences being the following:

[0112] The first homologous recombination fragment pflB-CS fragment I was obtained: the template was pXZ-CS, the primers were pflB-CS-up and pflB-CS-down, and the size was 2729bp;

[0113] The electroporated competent cells were ATCC 8739 / ptsG::P 93 -T7RNAP / poxB::P T7 -hdpA;

[0114] The identification primers were CS-YZ-up and pflB-CS-down. A sample size of 671 bp was considered positive, and the positive strain was named the first homologous recombinant strain.

[0115] The second step is homologous recombination:

[0116] The second homologous recombination fragment pflB::P T7 Obtaining the -hdpA fragment II: The template was pET30-CgHdpA, and the primers were pflB-pET-up / pflB-pET-down, with a size of 1157bp; the pflB::P T7 The nucleotide sequence of fragment II of -hdpA is shown in SEQ ID No. 7, where segments 56-164 are P. T7 The promoter is CgHdpA, with bits 165-992 being the promoter and bits 993-1158 being the terminator.

[0117] The competent cells for electroporation were the aforementioned first homologous recombinant bacteria;

[0118] The identification primers were pflB-YZ-up and pflB-YZ-down. A sample size of 1433 bp was considered positive, and the positive strain was named ATCC 8739 / ptsG::P 93 -T7RNAP / poxB::P T7 -hdpA / pflB::P T7 -hdpA, this bacterium is P 93 The -T7RNAP fragment (positions 78-2682 of SEQ ID No. 4) replaces the fragment shown at positions 151-1434 of the ptsG gene in the ATCC 8739 genome, and the poxB::P T7The -hdpA fragment (positions 56-1158 of SEQ ID No. 5) replaces the fragment shown at positions 396-1083 of the poxB gene in the ATCC 8739 genome, and pflB::P T7 The -hdpA fragment (SEQ ID No. 7, positions 56-1158) replaces the fragment shown in positions 1-2283 of the pflB gene in the ATCC8739 genome.

[0119] II. Gene Knockout

[0120] 1. Knockout of the ptsHI-crr gene

[0121] Gene knockout of the recombinant bacterium ATCC 8739 / ptsG::P, which integrates the exogenous genes T7RNAP and HdpA expression cassettes from step 2 of the previous step, and the gene knockout of step 3 of the previous step. 93 -T7RNAP / poxB::P T7 -hdpA / pflB::P T7 Based on -hdpA, the specific steps of gene knockout are exemplified by knocking out the ptsHI-crr tri-gene. Specifically, this involves knocking out the gene encoding the endogenous histidine phosphotransferase PtsH (i.e., the ptsH gene), the gene encoding the phosphoenolpyruvate-protein phosphotransferase IPtsI (i.e., the ptsI gene), and the gene encoding the glucose-specific enzyme II complex EIIA Crr (i.e., the crr gene). The details are as follows:

[0122] 1) First homologous recombination

[0123] Obtaining the first homologous recombination fragment ptsHIcrr-CS: Using pXZ-CS plasmid as a template, PCR amplification was performed using primers ptsHIcrr-CS-up / ptsHIcrr-CS-down (Table 2) to obtain the first homologous recombination fragment ptsHIcrr-CS.

[0124] From Escherichia coli ATCC 8739 / ptsG::P 93 -T7RNAP / poxB::P T7 -hdpA / pflB::P T7 Starting with -hdpA, electrotransformation competent cells were prepared, and plasmid pKD46 was transformed into ATCC 8739 / ptsG::P 93 -T7RNAP / poxB::P T7 -hdpA / pflB::P T7-hdpA electroporation of competent cells was performed, and the cells were plated on ampicillin plates and incubated overnight at 30°C. Single colonies were picked and cultured in 2 mL LB agar (containing ampicillin and 5% L(+)-arabinose) at 250 rpm for 6 hours at 30°C. Competent cells were then prepared and transformed with the first homologous recombination fragment ptsHIcrr-CS, and cultured at 75 rpm for 4 hours at 30°C. The cells were then plated on LB agar (containing ampicillin and chloramphenicol) and incubated at 30°C for 24 hours. Ten single colonies were selected for colony PCR verification using primers CS-YZ-up and ptsHIcrr-YZ-down (Table 2). A colony size of 755 bp was considered positive. These colonies were named the first homologous recombination strain.

[0125] 2) Second homologous recombination

[0126] Using the Escherichia coli ATCC 8739 genome as a template, a 300bp upstream homologous arm fragment was amplified using primers ptsHIcrr-YZ-up and ptsHIcrr-r1, and a 321bp downstream homologous arm fragment was amplified using primers ptsHIcrr-YZ-down and ptsHIcrr-f1. The upstream and downstream homologous arm fragments were then superimposed and amplified using overlap extension PCR to obtain a second homologous recombination fragment △ptsHIcrr that does not contain the ptsHI-crr trigene. This fragment was then used for the second homologous recombination step.

[0127] The competent cells and transformation methods for the second homologous recombination were the same as above. Competent cells of the bacteria from the first homologous recombination were prepared, transformed with the ΔptsHIcrr fragment, and cultured at 75 rpm for 4 hours at 30°C. The revived bacterial culture was then inoculated into LB medium (containing 10% sucrose) and cultured at 250 rpm for 24 hours at 37°C. The bacterial culture was diluted and plated on LB plates and cultured overnight at 37°C. Single colonies were picked and inoculated onto LB plates and chloramphenicol-LB plates, respectively. Single colonies that grew normally on LB plates but not on chloramphenicol-LB plates were selected for PCR verification. PCR amplification was performed using primers ptsHIcrr-YZ-up / ptsHIcrr-YZ-down (Table 2). A colony with a size of 559 bp was considered positive and named ATCC 8739 / ptsG::P 93 -T7RNAP / poxB::P T7 -hdpA / pflB::P T7 -hdpA / △ptsHI-crr.

[0128] 2. Knockout of the dhaRKLM gene

[0129] 1) First homologous recombination: Basically the same as 1 above, except for the following:

[0130] Obtaining the dhaRKLM-CS fragment: Using pXZ-CS plasmid as a template, PCR amplification was performed using primers dhaRKLM-CS-up / dhaRKLM-CS-down (Table 2) to obtain the first homologous recombination fragment dhaRKLM-CS.

[0131] The electroporated competent cells were ATCC 8739 / ptsG::P 93 -T7RNAP / poxB::P T7 -hdpA / pflB::P T7 -hdpA / △ptsHI-crr;

[0132] The identification primers were CS-YZ-up and dhaRKLM-YZ-down. A sample size of 796 bp was positive, and the first homologous recombinant bacteria were obtained.

[0133] 2) Second homologous recombination: Basically the same as 1 above, except for the following:

[0134] Obtaining the second homologous recombination fragment △dhaRKLM: Using the Escherichia coli ATCC 8739 genome as a template, a 357bp upstream homologous arm fragment was amplified using primers dhaRKLM-YZ-up and dhaRKLM-r1, and a 375bp downstream homologous arm fragment was amplified using primers dhaRKLM-YZ-down and dhaRKLM-f1. The second homologous recombination fragment △dhaRKLM, which does not contain the dhaRKLM gene, was obtained by overlapping extension PCR.

[0135] The competent cells for electroporation were the aforementioned first homologous recombinant bacteria;

[0136] The identification primers were dhaRKLM-YZ-up / dhaRKLM-YZ-down, and a size of 712 bp was positive, yielding ATCC8739 / ptsG::P. 93 -T7RNAP / poxB::P T7 -hdpA / pflB::P T7 -hdpA / ΔptsHI-crr / ΔdhaRKLM.

[0137] 3. Knock out the glpK gene

[0138] 1) First homologous recombination: Basically the same as 1 above, except for the following:

[0139] Obtaining the glpK-CS fragment: Using pXZ-CS plasmid as a template, PCR amplification was performed using primers glpK-CS-up / glpK-CS-down (Table 2) to obtain the first homologous recombination fragment glpK-CS.

[0140] The electroporated competent cells were ATCC 8739 / ptsG::P 93 -T7RNAP / poxB::P T7 -hdpA / pflB::P T7 -hdpA / △ptsHI-crr / △dhaRKLM;

[0141] The identification primers were CS-YZ-up and glpK-YZ-down. A sample size of 772bp was positive, and the first homologous recombinant bacteria were obtained.

[0142] 2) Second homologous recombination: Basically the same as 1 above, except for the following:

[0143] Obtaining the second homologous recombination fragment △glpK: Using the Escherichia coli ATCC 8739 genome as a template, a 330bp upstream homologous arm fragment was amplified using primers glpK-YZ-up and glpK-r1, and a 351bp downstream homologous arm fragment was amplified using primers glpK-YZ-down and glpK-f1. The second homologous recombination fragment △glpK, which does not contain the glpK gene, was obtained by overlapping extension PCR.

[0144] The competent cells for electroporation were the aforementioned first homologous recombinant bacteria;

[0145] The identification primers were glpK-YZ-up / glpK-YZ-down, with a size of 661 bp indicating a positive result, yielding ATCC 8739 / ptsG::P 93 -T7RNAP / poxB::P T7 -hdpA / pflB::P T7 -hdpA / ΔptsHI-crr / ΔdhaRKLM / ΔglpK.

[0146] 4. Knockout of the mgsA gene

[0147] 1) First homologous recombination: Basically the same as 1 above, except for the following:

[0148] Obtaining the mgsA-CS fragment: Using pXZ-CS plasmid as a template, PCR amplification was performed using primers mgsA-CS-up / mgsA-CS-down (Table 2) to obtain the first homologous recombination fragment mgsA-CS.

[0149] The electroporated competent cells were ATCC 8739 / ptsG::P 93 -T7RNAP / poxB::P T7 -hdpA / pflB::P T7 -hdpA / △ptsHI-crr / △dhaRKLM / △glpK;

[0150] The identification primers were CS-YZ-up and mgsA-YZ-down. A sample size of 741 bp was positive, and the first homologous recombinant bacteria were obtained.

[0151] 2) Second homologous recombination: Basically the same as 1 above, except for the following:

[0152] Obtaining the second homologous recombination fragment △mgsA: Using the E. coli ATCC 8739 genome as a template, a 300bp upstream homologous arm fragment was amplified using primers mgsA-YZ-up and mgsA-r1, and a 320bp downstream homologous arm fragment was amplified using primers mgsA-YZ-down and mgsA-f1. The second homologous recombination fragment △mgsA, which does not contain the mgsA gene, was obtained by overlapping extension PCR.

[0153] The competent cells for electroporation were the aforementioned first homologous recombinant bacteria;

[0154] The identification primers were mgsA-YZ-up / mgsA-YZ-down, with a size of 600 bp indicating a positive result, yielding ATCC 8739 / ptsG::P 93 -T7RNAP / poxB::P T7 -hdpA / pflB::P T7 -hdpA / ΔptsHI-crr / ΔdhaRKLM / ΔglpK / ΔmgsA.

[0155] 5. Knock out the gldA gene

[0156] 1) First homologous recombination: Basically the same as 1 above, except for the following:

[0157] Obtaining the gldA-CS fragment: Using pXZ-CS plasmid as a template, PCR amplification was performed using primers gldA-CS-up / gldA-CS-down (Table 2) to obtain the first homologous recombination fragment gldA-CS.

[0158] The electroporated competent cells were ATCC 8739 / ptsG::P 93 -T7RNAP / poxB::P T7 -hdpA / pflB::P T7-hdpA / △ptsHI-crr / △dhaRKLM / △glpK / △mgsA;

[0159] The identification primers were CS-YZ-up and gldA-YZ-down. A sample size of 791 bp was positive, and the first homologous recombinant bacteria were obtained.

[0160] 2) Second homologous recombination: Basically the same as 1 above, except for the following:

[0161] Obtaining the second homologous recombination fragment △gldA: Using the Escherichia coli ATCC 8739 genome as a template, a 305bp upstream homologous arm fragment was amplified using primers gldA-YZ-up and gldA-r1, and a 375bp downstream homologous arm fragment was amplified using primers gldA-YZ-down and gldA-f1. The second homologous recombination fragment △gldA, which does not contain the gldA gene, was obtained by overlapping extension PCR.

[0162] The competent cells for electroporation were the aforementioned first homologous recombinant bacteria;

[0163] The identification primers were gldA-YZ-up / gldA-YZ-down, and a size of 655bp was considered positive, yielding ATCC 8739 / ptsG::P. 93 -T7RNAP / poxB::P T7 -hdpA / pflB::P T7 -hdpA / ΔptsHI-crr / ΔdhaRKLM / ΔglpK / ΔmgsA / ΔgldA.

[0164] 6. Knock out the araBA gene

[0165] 1) First homologous recombination: Basically the same as 1 above, except for the following:

[0166] Obtaining the araBA-CS fragment: Using pXZ-CS plasmid as a template, PCR amplification was performed using primers araB-CS-up / araA-CS-down (Table 2) to obtain the first homologous recombination fragment araBA-CS.

[0167] The electroporated competent cells were ATCC 8739 / ptsG::P 93 -T7RNAP / poxB::P T7 -hdpA / pflB::P T7 -hdpA / △ptsHI-crr / △dhaRKLM / △glpK / △mgsA / △gldA;

[0168] The identification primers were CS-YZ-up and araA-YZ-down. A sample size of 818 bp was positive, and the first homologous recombinant bacteria were obtained.

[0169] 2) Second homologous recombination: Basically the same as 1 above, except for the following:

[0170] Obtaining the second homologous recombination fragment △araBA: Using the Escherichia coli ATCC 8739 genome as a template, a 300bp upstream homologous arm fragment was amplified using primers araB-YZ-up and araB-r1, and a 397bp downstream homologous arm fragment was amplified using primers araA-YZ-down and araA-f1. The second homologous recombination fragment △araBA, which does not contain the araBA gene, was obtained by overlapping extension PCR.

[0171] The competent cells for electroporation were the aforementioned first homologous recombinant bacteria;

[0172] The identification primers were araB-YZ-up / araA-YZ-down, and a sample size of 677bp was positive, yielding ATCC 8739 / ptsG::P. 93 -T7RNAP / poxB::P T7 -hdpA / pflB::P T7 -hdpA / △ptsHI-crr / △dhaRKLM / △glpK / △mgsA / △gldA / △araBA.

[0173] 7. Knock out the tpiA gene

[0174] 1) First homologous recombination: Basically the same as 1 above, except for the following:

[0175] Obtaining the tpiA-CS fragment: Using pXZ-CS plasmid as a template, PCR amplification was performed using primers tpiA-CS-up / tpiA-CS-down (Table 2) to obtain the first homologous recombination fragment tpiA-CS.

[0176] The electroporated competent cells were ATCC 8739 / ptsG::P 93 -T7RNAP / poxB::P T7 -hdpA / pflB::P T7 -hdpA / △ptsHI-crr / △dhaRKLM / △glpK / △mgsA / △gldA / △araBA;

[0177] The identification primers were CS-YZ-up and tpiA-YZ-down. A sample size of 741bp was positive, and the first homologous recombinant bacteria were obtained.

[0178] 2) Second homologous recombination: Basically the same as 1 above, except for the following:

[0179] Obtaining the second homologous recombination fragment △tpiA: Using the Escherichia coli ATCC 8739 genome as a template, a 303bp upstream homologous arm fragment was amplified using primers tpiA-YZ-up and tpiA-r1, and a 320bp downstream homologous arm fragment was amplified using primers tpiA-YZ-down and tpiA-f1. The second homologous recombination fragment △tpiA, which does not contain the tpiA gene, was obtained by overlapping extension PCR.

[0180] The competent cells for electroporation were the aforementioned first homologous recombinant bacteria;

[0181] The identification primers were tpiA-YZ-up / tpiA-YZ-down, and a size of 603 bp was positive, yielding ATCC 8739 / ptsG::P 93 -T7RNAP / poxB::P T7 -hdpA / pflB::P T7 -hdpA / △ptsHI-crr / △dhaRKLM / △glpK / △mgsA / △gldA / △araBA / △tpiA, named recombinant Escherichia coli TZ-222.

[0182] Recombinant Escherichia coli TZ-222 is derived from ATCC 8739 Escherichia coli. The full-length ptsHI-crr gene (positions 1-2622, composed of three genes: ptsH, ptsI, and crr, with sequence numbers EcolC_1261, EcolC_1262, EcolC_1263), the full-length dhaRKLM gene (positions 1-5290, composed of four genes: dhaR, dhaK, dhaL, and dhaM, with sequence numbers EcolC_2425, EcolC_2426, EcolC_2427, EcolC_2428), the glpK gene (Gene ID: EcolC_4092) (positions 1-1509), and the mgsA gene (Gene ID: EcolC_2425, EcolC_2426, EcolC_2427, EcolC_2428) are derived from these genes. The knockout of gene ID: EcolC_2633 (positions 1-459), gldA gene (positions 46-1104), araBA gene (positions 274-3159; araBA gene is composed of araB and araA genes, the knockout portion is from position 274 of araB gene to the end of araA gene; the two genes are Gene ID: EcolC_3594, EcolC_3595) and tpiA gene (positions 1-758), achieves the purpose of inactivating the function of these genes, and also removes P 93 The -T7RNAP fragment (positions 78-2682 of SEQ ID No. 4) replaces the fragment shown at positions 151-1434 of the ptsG gene in the ATCC 8739 genome, and the poxB::P T7 The -hdpA fragment (positions 56-1158 of SEQ ID No. 5) replaces the fragment shown at positions 396-1083 of the poxB gene in the ATCC 8739 genome, and pflB::P T7 The -hdpA fragment (SEQ ID No. 7, positions 56-1158) replaces the fragment shown in positions 1-2283 of the pflB gene in the ATCC 8739 genome.

[0183] Example 2: Fermentation of recombinant Escherichia coli TZ-222

[0184] The fermentation of recombinant Escherichia coli TZ-222 prepared in Example 1 to produce 1,3-dihydroxyacetone was evaluated.

[0185] CM9 fermentation medium: Each 1L contains 50g glucose, 5g corn steep liquor powder, 1g NH4Cl, 0.87g (NH4)2SO4, 0.5g MgSO4·7H2O, 0.47g KCl, 0.14g KH2PO4, and trace elements ZnCl2 1.47μM, H3BO3 0.81μM, CoCl2·6H2O 0.84μM, CuCl2·2H2O 0.59μM, FeCl3·6H2O 5.92μM, Na2MoO4·2H2O 0.83μM, with the remainder being water.

[0186] Shake-flask fermentation: Monoclonal recombinant Escherichia coli TZ-222 from LB agar plates was transferred to 10 ml of LB medium and cultured at 37°C and 250 rpm for 18 h to obtain shake-flask fermentation seed culture. The seed culture was then transferred to 50 ml of CM9 medium at an inoculum rate of 1% (v / v) and cultured at 37°C and 250 rpm for 72 h. The culture was then centrifuged at 10000 g for 2 min, and the supernatant was collected.

[0187] The yield of 1,3-dihydroxyacetone in the supernatant was analyzed by HPLC, and the conversion rate was calculated. *Escherichia coli* ATCC 8739 was used as a control.

[0188] HPLC detection conditions: using Yuexu The fermentation products were analyzed by HPLC using a Sugar-Ca column. The column temperature was controlled at 70℃, the mobile phase was 0.5 g / L EDTA-Ca aqueous solution, the flow rate was 0.4 ml / min, the injection volume for each sample was 20 μl, and the detection time was 30 minutes.

[0189] 1,3-Dihydroxyacetone standard was purchased from Shanghai Mairui Biochemical Technology Co., Ltd., product catalog number B66080-500G. The elution time of 1,3-dihydroxyacetone was 9.5 min. The HPLC quantitative detection standard curve equation for 1,3-dihydroxyacetone was: y = 346656x + 12665, R0 2 =0.9999. After diluting the supernatant by a certain factor, perform HPLC quantitative detection. The peak area obtained is the y-value. Substitute it into the 1,3-dihydroxyacetone standard curve equation to obtain the x-value. Multiply the x-value by the dilution factor to obtain the yield.

[0190] The molar conversion rate is calculated as follows: Conversion rate (mol / mol) = (Dihydroxyacetone yield / 90) / ((Total glucose - Residual sugar) / 180) * 100%

[0191] The results are as follows: the yield of 1,3-dihydroxyacetone in the control strain Escherichia coli ATCC 8739 was 0, while the yield of 1,3-dihydroxyacetone in TZ-222 was 9.23 g / L (supernatant), with a conversion rate of 0.9 mol / mol.

[0192] Example 3: Construction and fermentation of recombinant Escherichia coli TZ-223

[0193] I. Construction of Recombinant Escherichia coli TZ-223

[0194] Starting from the recombinant Escherichia coli TZ-222 prepared in Example 1, the gene encoding 6-phosphofructoaldehyde 1 FsaA (i.e., fsaA gene) (Gene ID: EcolC_2819) and the gene encoding 6-phosphofructoaldehyde 2 FsaB (i.e., fsaB gene) (Gene ID: EcolC_4069) were knocked out sequentially to obtain recombinant Escherichia coli TZ-223.

[0195] The gene knockout procedure is the same as step 1 of Example 1, as follows:

[0196] 1. fsaA gene knockout

[0197] 1) First homologous recombination: Basically the same as 1 above, except for the following:

[0198] Obtaining the fsaA-CS fragment: Using pXZ-CS plasmid as a template, PCR amplification was performed using primers fsaA-CS-up / fsaA-CS-down (Table 2) to obtain the first homologous recombination fragment fsaA-CS.

[0199] The electroporated competent cells were TZ-222 cells prepared in Example 1;

[0200] The identification primers were CS-YZ-up and fsaA-YZ-down. A sample size of 741bp was positive, and the first homologous recombinant bacteria were obtained.

[0201] 2) Second homologous recombination: Basically the same as 1 above, except for the following:

[0202] Obtaining the second homologous recombination fragment △fsaA: Using the Escherichia coli ATCC 8739 genome as a template, a 300bp upstream homologous arm fragment was amplified using primers fsaA-YZ-up and fsaA-r1, and a 320bp downstream homologous arm fragment was amplified using primers fsaA-YZ-down and fsaA-f1. The second homologous recombination fragment △fsaA, which does not contain the fsaA gene, was obtained by overlapping extension PCR.

[0203] The competent cells for electroporation were the aforementioned first homologous recombinant bacteria;

[0204] The identification primers were fsaA-YZ-up / fsaA-YZ-down, and a size of 600bp was considered positive, yielding TZ-222 / △fsaA.

[0205] 2. fsaB gene knockout

[0206] 1) First homologous recombination: Basically the same as 1 above, except for the following:

[0207] Obtaining the fsaA-CS fragment: Using pXZ-CS plasmid as a template, PCR amplification was performed using primers fsaB-CS-up / fsaB-CS-down (Table 2) to obtain the first homologous recombination fragment fsaB-CS.

[0208] The electroporated competent cells were the TZ-222 / △fsaA cells prepared above;

[0209] The identification primers were CS-YZ-up and fsaB-YZ-down. A sample size of 741bp was positive, and the first homologous recombinant bacteria were obtained.

[0210] 2) Second homologous recombination: Basically the same as 1 above, except for the following:

[0211] Obtaining the second homologous recombination fragment △fsaB: Using the Escherichia coli ATCC 8739 genome as a template, a 351bp upstream homologous arm fragment was amplified using primers fsaB-YZ-up and fsaB-r1, and a 320bp downstream homologous arm fragment was amplified using primers fsaB-YZ-down and fsaB-f1. The second homologous recombination fragment △fsaB, which does not contain the fsaB gene, was obtained by overlapping extension PCR.

[0212] The competent cells for electroporation were the aforementioned first homologous recombinant bacteria;

[0213] The identification primers were fsaB-YZ-up / fsaB-YZ-down, with a size of 651bp indicating a positive result, yielding TZ-222 / △fsaA△fsaB, which was named TZ-223.

[0214] Recombinant Escherichia coli TZ-223 is a recombinant bacterium obtained by knocking out positions 1-663 of the fsaA gene and positions 1-663 of the fsaB gene in Escherichia coli TZ-222.

[0215] II. Fermentation with Recombinant Escherichia coli TZ-223

[0216] The shake-flask fermentation was performed and then tested according to the method in Example 2.

[0217] The results are as follows: the yield of 1,3-dihydroxyacetone from TZ-223 was 9.07 g / L, and the conversion rate of glucose to 1,3-dihydroxyacetone was 0.95 mol / mol.

[0218] Example 4: Construction and fermentation of recombinant Escherichia coli TZ-231

[0219] I. Construction of Recombinant Escherichia coli TZ-231

[0220] Starting from the Escherichia coli TZ-223 constructed in Example 3, the gene encoding the glycerol transporter GlpF (i.e., the glpF gene) (Gene ID: EcolC_4091) was knocked out to obtain recombinant Escherichia coli TZ-231.

[0221] The glpF gene knockout procedure is the same as step 1 of Example 1, as follows:

[0222] 1) First homologous recombination: Basically the same as 1 above, except for the following:

[0223] Obtaining the glpF-CS fragment: Using pXZ-CS plasmid as a template, PCR amplification was performed using primers glpF-CS-up / glpF-CS-down (Table 2) to obtain the first homologous recombination fragment glpF-CS.

[0224] The electroporated competent cells were TZ-223 cells prepared in Example 3;

[0225] The identification primers were CS-YZ-up and glpF-YZ-down. A sample size of 841 bp was positive, and the first homologous recombinant bacteria were obtained.

[0226] 2) Second homologous recombination: Basically the same as 1 above, except for the following:

[0227] Obtaining the second homologous recombination fragment △glpF: Using the Escherichia coli ATCC 8739 genome as a template, a 297bp upstream homologous arm fragment was amplified using primers glpF-YZ-up and glpF-r1, and a 420bp downstream homologous arm fragment was amplified using primers glpF-YZ-down and glpF-f1. The second homologous recombination fragment △glpF, which does not contain the glpF gene, was obtained by overlapping extension PCR.

[0228] The competent cells for electroporation were the aforementioned first homologous recombinant bacteria;

[0229] The identification primers were glpF-YZ-up / glpF-YZ-down, and a size of 697bp was positive, yielding TZ-223 / △glpF, which was named TZ-231.

[0230] Recombinant Escherichia coli TZ-231 is a recombinant bacterium obtained by knocking out positions 1-813 of the glpF gene in Escherichia coli TZ-223.

[0231] II. Fermentation with Recombinant Escherichia coli TZ-231

[0232] The shake-flask fermentation was performed and then tested according to the method in Example 2.

[0233] Fermentation results showed that TZ-231 produced 9.57 g / L of 1,3-dihydroxyacetone, and the conversion rate of glucose to 1,3-dihydroxyacetone was 0.93 mol / mol.

[0234] Example 5: Construction and fermentation of recombinant Escherichia coli TZ-236

[0235] I. Construction of Recombinant Escherichia coli TZ-236

[0236] Starting from the Escherichia coli TZ-231 constructed in Example 4, the NADH oxidase NOX expression cassette was integrated into the adhE site to obtain recombinant Escherichia coli TZ-236.

[0237] The integration of the NADH oxidase NOX expression cassette into the adhE site is the same as step 2 of Example 1;

[0238] The first step, homologous recombination, is essentially the same as step 2(1) of Example 1, except for the following:

[0239] The first homologous recombination fragment, adhE-CS fragment I, was obtained: the template was pXZ-CS, the primers were adhE-CS-up and adhE-CS-down, and the size was 2729bp.

[0240] The competent cells for electroporation were TZ-231;

[0241] The identification primers were CS-YZ-up and adhE-CS-down. A sample size of 594 bp was considered positive, and the positive strain was named the first homologous recombinant strain.

[0242] The second step, homologous recombination, is basically the same as step 1) above, with the only differences being the following:

[0243] Second homologous recombination fragment P 46 Obtaining the -nox fragment II: The template was pSC103-NOX, the primers were adhE-pSC-up / adhE-pSC-down, and the size was 1621bp; homologous recombination fragment P 46 The nucleotide sequence of the -nox fragment II is SEQ ID No. 6, and nucleotides 86-173 of this sequence are P. 46 The promoter is NOX, with bits 174-1535 being the NOX terminator and bits 1536-1621 being the terminator.

[0244] The competent cells for electroporation were first-time homologous recombinant bacteria;

[0245] The identification primers were adhE-YZ-up and adhE-YZ-down. A sample size of 1750 bp was considered positive, and the positive strain was named TZ-231 / adhE::P 46 -nox refers to recombinant Escherichia coli TZ-236.

[0246] Recombinant Escherichia coli TZ-236 is used to make P 46 The recombinant bacteria were obtained by replacing the 102-2676 fragment of the adhE gene of E. coli TZ-231 with -nox (86-1621 of SEQ ID No. 6).

[0247] II. Fermentation of Recombinant Escherichia coli TZ-236

[0248] The shake-flask fermentation was performed and then tested according to the method in Example 2.

[0249] Fermentation results showed that TZ-236 produced 12.64 g / L of 1,3-dihydroxyacetone, and the conversion rate of glucose to 1,3-dihydroxyacetone was 0.95 mol / mol.

[0250] Example 6: Construction and fermentation of recombinant Escherichia coli TZ-237

[0251] I. Construction of Recombinant Escherichia coli TZ-237

[0252] Starting from the *E. coli* TZ-236 constructed in Example 5, the gene encoding glucose-6-phosphate dehydrogenase Zwf (i.e., the zwf gene) (Gene ID: EcolC_1780), the gene encoding phosphogluconic acid dehydratase Edd (i.e., the edd gene) (Gene ID: EcolC_1781), and the gene encoding 2-keto-3-deoxy-6-phosphate glucuronide Eda (i.e., the eda gene) (Gene ID: EcolC_1782) were simultaneously knocked out to obtain recombinant *E. coli* TZ-237.

[0253] The zwf-edd-eda gene was knocked out, and the knockout procedure was the same as step 1 of Example 1, as follows:

[0254] 1) First homologous recombination: Basically the same as 1 above, except for the following:

[0255] Obtaining the zwf-edd-eda-CS fragment: Using pXZ-CS plasmid as a template, PCR amplification was performed using primers zwf-CS-up / eda-CS-down (Table 2) to obtain the first homologous recombination fragment zwf-edd-eda-CS.

[0256] The competent cells for electroporation were TZ-236;

[0257] The identification primers were CS-YZ-up and eda-YZ-down. A sample size of 894bp was positive, and the first homologous recombinant bacteria were obtained.

[0258] 2) Second homologous recombination: Basically the same as 1 above, except for the following:

[0259] Obtaining the second homologous recombination fragment △zwf-edd-eda: Using the Escherichia coli ATCC 8739 genome as a template, a 305bp upstream homologous arm fragment was amplified using primers zwf-YZ-up and zwf-r1, and a 473bp downstream homologous arm fragment was amplified using primers eda-YZ-down and edaF-f1. The second homologous recombination fragment △zwf-edd-eda, which does not contain the zwf-edd-eda gene, was obtained by overlapping extension PCR.

[0260] The competent cells for electroporation were the aforementioned first homologous recombinant bacteria;

[0261] The identification primer was zwf-YZ-up / eda-YZ-down, and a size of 758bp was positive, yielding TZ-236 / △zwf-edd-eda, which is TZ-237.

[0262] Recombinant Escherichia coli TZ-237 is a recombinant bacterium obtained by knocking out the zwf-edd-eda genes in Escherichia coli TZ-236, specifically by knocking out positions 1-1476 of the zwf gene, positions 1-1812 of the edd gene, and positions 1-642 of the eda gene.

[0263] II. Fermentation of Recombinant Escherichia coli TZ-237

[0264] The shake-flask fermentation was performed and then tested according to the method in Example 2.

[0265] Fermentation results showed that the yield of 1,3-dihydroxyacetone from TZ-237 was 12.99 g / L, and the conversion rate of glucose to 1,3-dihydroxyacetone was 0.95 mol / mol.

[0266] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A method for constructing an engineered strain for producing 1,3-dihydroxyacetone using glucose, comprising the following steps A: A. Inhibiting the expression of the following endogenous gene A on the basis of the recipient bacteria, and causing the recipient bacteria to express the coding gene of T7 RNA polymerase T7RNAP and the coding gene of dihydroxyacetone phosphate phosphatase HdpA; The endogenous genes are as follows: the gene encoding the histidine phosphate carrier protein PtsH, the gene encoding the phosphoenolpyruvate-protein phosphotransferase IPtsI, the gene encoding the glucose-specific enzyme II complex EIIA Crr, the gene encoding the dihydroxyacetone kinase DhaRKLM, the gene encoding the glycerol kinase GlpK, the gene encoding the methylglyoxal synthase MgsA, the gene encoding the glycerol dehydrogenase GldA, the gene encoding the L-arabinose isomerase AraA, the gene encoding the ribulose kinase AraB, the gene encoding the triosephosphate isomerase TpiA, the gene encoding the glucose-specific PTS enzyme IIBC component PtsG, the gene encoding the pyruvate oxidase PoxB and the gene encoding the pyruvate formate lyase PflB.

2. The method according to claim 1, characterized in that: The method further comprises step B: B. Based on step A, inhibiting the expression of the endogenous gene encoding 6-phosphofructoyl aldolase 1FsaA and the gene encoding 6-phosphofructoyl aldolase 2FsaB in the recipient bacteria.

3. The method according to claim 2, characterized in that: The method further comprises step C: C. Based on step B, inhibiting the expression of the gene encoding the endogenous glycerol transporter GlpF in the recipient bacteria.

4. The method according to claim 3, characterized in that: The method further comprises step D: D. Based on step C, the recipient bacteria is made to express the NADH oxidase NOX gene and the expression of the gene encoding the alcohol dehydrogenase AdhE in the recipient bacteria is inhibited.

5. The method according to claim 4, characterized in that: The method further comprises step E: E. Based on step D, inhibiting the expression of the endogenous genes encoding 6-phosphogluconate dehydrogenase Zwf, phosphogluconate dehydratase Edd and 2-keto-3-deoxy-6-phosphogluconate aldehyde Eda in the recipient bacteria.

6. The method according to any one of claims 1 to 5, characterized in that: The recipient bacteria is Escherichia coli that can use glucose as a carbon source.

7. A recombinant bacterium prepared by the method described in any one of claims 1 to 6.

8. Use of the recombinant bacteria according to claim 7 in the production of 1,3-dihydroxyacetone; Or the use of the recombinant bacteria according to claim 7 in increasing the production of 1,3-dihydroxyacetone.

9. A method for producing 1,3-dihydroxyacetone, comprising the following steps: fermenting the recombinant bacteria according to claim 7, collecting the fermentation product, and obtaining 1,3-dihydroxyacetone.

10. The method according to claim 9, characterized in that: The fermentation is carried out in a fermentation medium containing glucose.

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