Construction and application of engineering strain for synthesizing 1, 3-propylene glycol by using carbon dioxide and mono-carbon raw materials

Abstract

The invention discloses construction and application of an engineering strain for synthesizing 1, 3-propylene glycol by using carbon dioxide and mono-carbon raw materials, and belongs to the field of genetic engineering recombinant microorganisms. The invention aims to solve the technical problem of how to synthesize 1, 3-propylene glycol by taking carbon dioxide, methanol, formaldehyde and the like as raw materials. In order to solve the technical problem, the invention provides a recombinant bacterium which is used for synthesizing 1, 3-propylene glycol by taking methanol as a raw material. The invention also provides an application of the recombinant bacterium in preparation of 1, 3-propylene glycol. The invention also provides a method for preparing 1, 3-propylene glycol, and the method comprises the step of preparing 1, 3-propylene glycol by taking the recombinant strain as a fermentation strain and methanol as a raw material. The invention provides a recombinant bacterium capable of efficiently synthesizing 1, 3-propylene glycol by using methanol, formaldehyde and other raw materials, establishes a route for synthesizing 1, 3-propylene glycol by using carbon dioxide, methanol, formaldehyde and other raw materials, and has great economic value and social value.

Description

Technical Field

[0001] The present invention belongs to the field of genetically engineered recombinant microorganisms, and specifically relates to the construction and application of an engineered strain for synthesizing 1,3-propylene glycol using carbon dioxide and a one-carbon raw material. Background Art

[0002] 1,3-Propanediol is an important chemical raw material, widely used in the cosmetics, lubricant, food, and pharmaceutical industries. More importantly, 1,3-Propanediol can be used as a monomer to synthesize the polymer polytrimethylene terephthalate (PTT). PTT is a new polyester polymer material with excellent performance. Compared to materials such as nylon, polyester, and acrylic, PTT boasts strong toughness, good dyeability, antistatic properties, and UV resistance. PPT also has improved biodegradability and recyclability, and has broad application prospects in textiles, carpets, leather, engineering thermoplastics, and other fields.

[0003] Currently, most bulk platform chemicals are produced using fossil resources as raw materials. The use of biomanufacturing production methods using biomass as raw materials will help alleviate pressure from both the environment and resources. Diol compounds containing two hydroxyl groups at the end can be widely used in polymers, energy fuels, cosmetics, and pharmaceuticals. Among them, 1,3-propylene glycol is mainly used to synthesize polytrimethylene terephthalate (PTT) fibers with excellent performance. 1,3-propylene glycol (1,3-PDO) is expensive, and the market urgently needs a low-cost production process for 1,3-PDO. Currently, the main methods for producing 1,3-propylene glycol are chemical synthesis and biological methods. The main chemical synthesis processes are acrolein hydration hydrogenation and ethylene oxide carbonyl synthesis.

[0004] Currently, there are two main biofermentation routes for producing 1,3-propylene glycol (PD) using glucose and glycerol. Natural strains of Klebsiella pneumoniae, for example, can synthesize 1,3-PD using glycerol as a raw material. Their maximum theoretical conversion rate is 0.60 g / g. However, glycerol is relatively expensive. Engineered strains of Escherichia coli can synthesize 1,3-PD using relatively inexpensive glucose as a raw material, with a theoretical conversion rate of 0.50 g / g. However, these 1,3-PD synthesis routes have limited potential due to limitations in raw material costs and conversion rates, leading to the urgent need for new raw material routes and synthetic pathways.

[0005] Methanol and formaldehyde are both inexpensive, readily available organic one-carbon feedstocks. They have higher energy densities than feedstocks like glucose and can provide more reducing power for biosynthesis, thereby increasing the conversion rate of biomanufacturing. Methanol is primarily produced synthetically. Currently, industrial production primarily utilizes pressurized catalytic hydrogenation of carbon monoxide, where the feedstock can come from processed products such as coal, petroleum, and natural gas. Alternatively, methanol production through carbon dioxide hydrogenation coupled with water electrolysis is a potential and highly efficient methanol synthesis pathway. Methanol feedstock costs less than glucose and is only one-fifth the cost of glycerol. As a cheap, readily available, and environmentally friendly renewable carbon resource, carbon dioxide's utilization not only reduces CO2 emissions but also provides a green production technology route, significantly contributing to green and sustainable development. However, a key technical limitation limiting industrial application is the lack of efficient synthetic routes and practically applicable microbial strains. Existing technologies are unable to achieve high-conversion microbial synthesis of 1,3-propylene glycol using one-carbon compounds (carbon dioxide, methanol, and formaldehyde) as the sole carbon feedstock. Summary of the Invention

[0006] The technical problem to be solved by the present invention is: how to realize the synthesis of 1,3-propylene glycol using carbon dioxide, methanol, formaldehyde and the like as raw materials.

[0007] In order to solve the above technical problems, the present invention provides a recombinant bacterium, which synthesizes 1,3-propylene glycol using methanol as a raw material.

[0008] Furthermore, the following metabolic pathways (reactions) exist in the recombinant bacteria:

[0009] (a) converting methanol into formaldehyde;

[0010] (b) synthesizing D-arabino-hex-3-ulose 6-phosphate from formaldehyde and D-ribulose 5-phosphate;

[0011] (c) converting D-arabino-hex-3-ketose 6-phosphate into fructose 6-phosphate;

[0012] (d) converting fructose 6-phosphate into fructose 1,6-bisphosphate;

[0013] (e) converting fructose 1,6-diphosphate into dihydroxyacetone phosphate;

[0014] (f) converting dihydroxyacetone phosphate into glycerol triphosphate;

[0015] (g) converting glycerol-3-phosphate into glycerol;

[0016] (h) converting glycerol into 3-hydroxypropionaldehyde;

[0017] (i) Conversion of 3-hydroxypropanal to 1,3-propanediol.

[0018] Furthermore, in the recombinant bacteria:

[0019] The (a) is catalyzed by methanol dehydrogenase,

[0020] The (b) is catalyzed by 3-hexulose-6-phosphate synthase;

[0021] Said (c) is catalyzed by 6-phospho-3-hexose isomerase (Phi);

[0022] Said (d) is catalyzed by 6-phosphofructokinase;

[0023] The (e) is catalyzed by fructose bisphosphate aldolase;

[0024] The (f) is catalyzed by dihydroxyacetone phosphate reductase;

[0025] The (g) is catalyzed by 3-phosphoglycerate phosphatase;

[0026] The (h) is catalyzed by glycerol dehydratase;

[0027] The step (i) is catalyzed by alcohol dehydrogenase or 1,3-propanediol oxidoreductase.

[0028] Furthermore, the recombinant bacteria include the following characteristics:

[0029] (1) Containing a methanol dehydrogenase gene or / and a substance that regulates the expression of the methanol dehydrogenase gene or / and a substance that regulates the activity or content of methanol dehydrogenase;

[0030] (2) containing a 3-hexulose-6-phosphate synthase gene or / and a substance that regulates the expression of the 3-hexulose-6-phosphate synthase gene or / and a substance that regulates the activity or content of 3-hexulose-6-phosphate synthase;

[0031] (3) containing a 6-phospho-3-hexuloisomerase gene or / and a substance that regulates the expression of the 6-phospho-3-hexuloisomerase gene or / and a substance that regulates the activity or content of 6-phospho-3-hexol isomerase (Phi);

[0032] (4) containing the 6-phosphofructokinase gene or / and substances that regulate the expression of the 6-phosphofructokinase gene or / and substances that regulate the activity or content of 6-phosphofructokinase;

[0033] (5) containing a fructose-bisphosphate aldolase gene or / and a substance that regulates the expression of the fructose-bisphosphate aldolase gene or / and a substance that regulates the activity or content of fructose-bisphosphate aldolase;

[0034] (6) containing a dihydroxyacetone phosphate dehydrogenase gene or / and a substance that regulates the expression of the dihydroxyacetone phosphate reductase gene or / and a substance that regulates the activity or content of dihydroxyacetone phosphate reductase;

[0035] (7) containing the glycerol 3-phosphate phosphatase gene or / and substances that regulate the expression of the glycerol 3-phosphate phosphatase gene or / and substances that regulate the activity or content of glycerol 3-phosphate phosphatase;

[0036] (8) containing a glycerol dehydratase gene or / and a substance that regulates the expression of the glycerol dehydratase gene or / and a substance that regulates the activity or content of the glycerol dehydratase;

[0037] (9) containing a glycerol dehydratase activating factor gene or / and a substance that regulates the expression of the glycerol dehydratase activating factor gene or / and a substance that regulates the activity or content of the glycerol dehydratase activating factor;

[0038] (10) Containing an alcohol dehydrogenase gene or a 1,3-propanediol oxidoreductase gene or / and a substance that regulates the expression of the alcohol dehydrogenase or 1,3-propanediol oxidoreductase gene or / and a substance that regulates the activity or content of the alcohol dehydrogenase or 1,3-propanediol oxidoreductase.

[0039] Furthermore, the recombinant bacteria is a recombinant Bacillus, which is prepared by performing the following operations on the Bacillus as a recipient bacteria:

[0040] A1) introducing a dihydroxyacetone phosphate reductase gene and / or a substance that regulates the expression of the dihydroxyacetone phosphate reductase gene and / or a substance that regulates the activity or content of the dihydroxyacetone phosphate reductase into the recipient bacterium;

[0041] A2) introducing a glycerol 3-phosphate phosphatase gene and / or a substance that regulates the expression of the glycerol 3-phosphate phosphatase gene and / or a substance that regulates the activity or content of glycerol 3-phosphate phosphatase;

[0042] A3) introducing a glycerol dehydratase gene and / or a substance that regulates the expression of the glycerol dehydratase gene and / or a substance that regulates the activity or content of the glycerol dehydratase;

[0043] A4) introducing a glycerol dehydratase activating factor gene and / or a substance that regulates the expression of the glycerol dehydratase activating factor gene and / or a substance that regulates the activity or content of the glycerol dehydratase activating factor;

[0044] A5) Introduction of an alcohol dehydrogenase gene and / or a substance that regulates alcohol dehydrogenase gene expression and / or a substance that regulates alcohol dehydrogenase activity or content.

[0045] Furthermore, the Bacillus as the recipient bacterium contains a methanol dehydrogenase gene, a 3-hexulose-6-phosphate synthase gene, a 6-phosphate-3-hexose isomerase gene, a 6-phosphofructokinase gene, and a fructose bisphosphate aldolase gene.

[0046] The coding sequence of the methanol dehydrogenase gene (Mdh) is a nucleotide sequence of SEQ ID No. 43. The methanol dehydrogenase having an amino acid sequence of SEQ ID No. 44 can be expressed.

[0047] The coding sequence of the 3-hexulose-6-phosphate synthase gene (Hps) is a DNA molecule with a nucleotide sequence of SEQ ID No. 15. The 3-hexulose-6-phosphate synthase with an amino acid sequence of SEQ ID No. 33 can be expressed.

[0048] The nucleotide sequence of the 6-phosphate-3-hexose isomerase (Phi) gene is SEQ ID No. 18. The 6-phosphate-3-hexose isomerase having the amino acid sequence of SEQ ID No. 34 can be expressed.

[0049] The nucleotide sequence of the 6-phosphofructokinase gene (Pfk) is SEQ ID No. 39. The 6-phosphofructokinase having the amino acid sequence of SEQ ID No. 40 can be expressed.

[0050] The nucleotide sequence of the fructose bisphosphate aldolase gene (Fba) is SEQ ID No. 41. The fructose bisphosphate aldolase having an amino acid sequence of SEQ ID No. 42 can be expressed.

[0051] In one embodiment of the present invention, the recipient bacteria may specifically be Bacillus YL23 strain.

[0052] Furthermore, in the recombinant bacteria, A1) the dihydroxyacetonephosphate dehydrogenase is derived from Saccharomyces cerevisiae; A2) the glycerol 3-phosphate phosphatase is derived from Saccharomyces cerevisiae; A3) the glycerol dehydratase is derived from Klebsiella pneumoniae; A4) the glycerol dehydratase activating factor is derived from Klebsiella pneumoniae; and A5) the alcohol dehydrogenase is derived from Escherichia coli.

[0053] Furthermore, in the recombinant bacteria, the dihydroxyacetone dehydrogenase in A1) is any one of the following:

[0054] A1-1), a protein having an amino acid sequence of SEQ ID No. 24;

[0055] A1-2) or a protein related to dihydroxyacetone dehydrogenase obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in A1-1) having an identity of 80% or more to the protein shown in A1-1);

[0056] A1-3), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in A1-1) or A1-2);

[0057] A2) The 3-phosphoglycerate phosphatase is any one of the following:

[0058] A2-1), a protein having an amino acid sequence of SEQ ID No. 25;

[0059] A protein related to 3-phosphoglycerate phosphatase obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A2-2) and A2-1) having an identity of 80% or more to the protein shown in A2-1);

[0060] A2-3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in 2-1) or A2-2);

[0061] A3) The glycerol dehydratase includes glycerol dehydratase DhaB1, glycerol dehydratase DhaB2 and glycerol dehydratase DhaB3, wherein the glycerol dehydratase DhaB1 is any one of the following:

[0062] A3-1), a protein having an amino acid sequence of SEQ ID No. 26;

[0063] A protein related to glycerol dehydratase obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A3-2) and A3-1) having an identity of 80% or more to the protein shown in A3-1);

[0064] A3-3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in A3-1) or A3-2);

[0065] The glycerol dehydratase DhaB2 is any one of the following:

[0066] A3-4), a protein having an amino acid sequence of SEQ ID No. 27;

[0067] A protein related to glycerol dehydratase obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A3-5) and A3-4) and having an identity of more than 80% with the protein shown in A3-4);

[0068] A3-6) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in A3-4) or A3-5);

[0069] The glycerol dehydratase DhaB3 is any one of the following:

[0070] A3-7), a protein with an amino acid sequence of SEQ ID No. 28;

[0071] A protein related to glycerol dehydratase obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A3-8) and A3-7) having an identity of 80% or more to the protein shown in A3-7);

[0072] A3-9) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in A3-7) or A3-8);

[0073] A4) The dehydratase activating factor includes a dehydratase activating factor GdrA and a dehydratase activating factor GdrB, wherein the dehydratase activating factor GdrA is any one of the following:

[0074] A4-1), a protein with an amino acid sequence of SEQ ID No. 29;

[0075] A protein related to a dehydratase activating factor obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A4-2) and A4-1) having an identity of 80% or more to the protein shown in A4-1);

[0076] A4-3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in A4-1) or A4-2);

[0077] The dehydratase activating factor GdrB is any one of the following:

[0078] A4-4), a protein having an amino acid sequence of SEQ ID No. 30;

[0079] A4-5) or a protein related to a dehydratase activating factor obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in A4-4) having an identity of more than 80% with the protein shown in A4-4);

[0080] A4-6), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in A4-4) or A4-6);

[0081] A5) The alcohol dehydrogenase is any one of the following:

[0082] A5-1), a protein having an amino acid sequence of SEQ ID No. 31;

[0083] A5-2) or a protein related to alcohol dehydrogenase obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in A5-1) and having an identity of 80% or more to the protein shown in A5-1);

[0084] A5-3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in A5-1) or A5-2).

[0085] Furthermore, in the recombinant bacteria, A1) the coding sequence of the dihydroxyacetone dehydrogenase gene is a DNA molecule with a nucleotide sequence of SEQ ID No. 4; A2) the coding sequence of the 3-phosphoglycerol phosphatase gene is a DNA molecule with a nucleotide sequence of SEQ ID No. 5; A3) the coding sequence of the glycerol dehydratase gene is a DNA molecule with a nucleotide sequence of SEQ ID No. 6; A4) the coding sequence of the dehydratase activator gene is a DNA molecule with a nucleotide sequence of SEQ ID No. 7; A5) the coding sequence of the alcohol dehydrogenase gene is a DNA molecule with a nucleotide sequence of SEQ ID No. 8;

[0086] Furthermore, in the recombinant bacteria, the substance described in any one of A1) to A5) is one of the following:

[0087] g1) a promoter or terminator, wherein the promoter or terminator is derived from Bacillus methanolicus;

[0088] g2) a promoter or a terminator, wherein the promoter is a DNA molecule having a nucleotide sequence of positions 1 to 1023 of SEQ ID No. 3, and the terminator is a DNA molecule having a nucleotide sequence of positions 2173 to 2344 of SEQ ID No. 3.

[0089] Furthermore, the recombinant bacteria also includes the following characteristics:

[0090] (11) does not contain the endotoxin lpxM gene;

[0091] (12) does not contain the hydroxymethyl glutathione dehydrogenase (frmA) gene;

[0092] (13) It does not contain the triose-phosphate isomerase tpiA gene.

[0093] The recombinant bacteria further include the features described in (12) and (13) or the recombinant bacteria further include the feature described in (14):

[0094] (14) It does not contain the outer membrane protein ompT gene.

[0095] Furthermore, the recombinant bacteria is recombinant Escherichia coli, and the recombinant Escherichia coli is prepared by performing the following operations on Escherichia coli as a recipient bacteria:

[0096] B1) introducing a methanol dehydrogenase gene and / or introducing a substance that regulates the expression of the methanol dehydrogenase gene and / or introducing a substance that regulates the activity or content of methanol dehydrogenase;

[0097] B2) introducing a 3-hexulose-6-phosphate synthase gene (hps) and / or introducing a substance that regulates the expression of 3-hexulose-6-phosphate synthase and / or introducing a substance that regulates the activity or content of 3-hexulose-6-phosphate synthase;

[0098] B3) knocking out the hydroxymethyl-glutathione dehydrogenase gene (frmA) of the recipient bacteria;

[0099] B4) introducing and / or introducing a substance that regulates the expression of the 6-phosphate-3-hexose isomerase gene (phi) and / or introducing a substance that regulates the activity or content of 6-phosphate-3-hexose isomerase;

[0100] B5) knocking out the triose phosphate isomerase gene (tpiA) of the recipient bacteria;

[0101] B6) introducing a dihydroxyacetone phosphate reductase gene and / or introducing a substance that regulates the expression of the dihydroxyacetone phosphate reductase gene and / or introducing a substance that regulates the activity or content of the dihydroxyacetone phosphate reductase;

[0102] B7) introducing a 3-phosphoglycerate phosphatase gene and / or introducing a substance that regulates the expression of the 3-phosphoglycerate phosphatase gene and / or introducing a substance that regulates the activity or content of 3-phosphoglycerate phosphatase;

[0103] B8) introducing a glycerol dehydratase gene and / or introducing a substance that regulates the expression of glycerol dehydratase and / or introducing a substance that regulates the activity or content of glycerol dehydratase;

[0104] B9) Introduction of an alcohol dehydrogenase gene and / or a substance that regulates alcohol dehydrogenase gene expression and / or a substance that regulates alcohol dehydrogenase activity or content.

[0105] B10) introducing a glycerol dehydratase activating factor gene and / or introducing a substance that regulates the expression of the glycerol dehydratase activating factor gene and / or introducing a substance that regulates the activity or content of the glycerol dehydratase activating factor.

[0106] Furthermore, the recipient bacteria contains an endotoxin gene (lpxM), a hydroxymethyl-glutathione dehydrogenase gene (frmA), a triosephosphate isomerase gene (tpiA), a 6-phospho-fructose kinase gene (Pfk), and a fructose bisphosphate aldolase gene (Fba).

[0107] The reference gene sequence of the endotoxin gene (lpxM) is NC_000913.3 (1,939,222-1,940,193, complement) (09-

[0108] The amino acid sequence of the expressed endotoxin is NP_416369.1 (09-MAR-2022).

[0109] The reference gene sequence of the hydroxymethyl-glutathione dehydrogenase gene (frmA) is NC_000913.3 (378, 462-379, 571, complement) (09-MAR-2022). The amino acid sequence of the expressed hydroxymethyl-glutathione dehydrogenase is NP_414890.1 (09-MAR-2022).

[0110] The reference gene sequence of the triosephosphate isomerase gene (tpiA) is NC_000913.3 (4,110,740-4,111,507, complement) (09-MAR-2022). The amino acid sequence of the triosephosphate isomerase expressed is NP_418354.1 (09-MAR-2022).

[0111] The 6-phosphofructokinase gene includes PfkA and PfkB. The reference gene sequence of PfkA is NC_000913.3 (4,107,552-4,108,514) (09-MAR-2022). The amino acid sequence of the 6-phosphofructokinase PfkA is NP_418351.1 (09-MAR-2022). The reference gene sequence of PfkB is NC_000913.3 (1,806,370-1,807,299) (09-MAR-2022). The amino acid sequence of the 6-phosphofructokinase PfkB is NP_416237.1 (09-MAR-2022).

[0112] The fructose bisphosphate aldolase gene includes FbaA and FbaB. The reference gene sequence of FbaA is NC_000913.3 (3,070,165-3,071,244, complement) (09-MAR-2022). The reference sequence number of the amino acid sequence expressing fructose bisphosphate aldolase FbaA is NP_417400.1 (09-MAR-2022). The reference gene sequence of FbaB is NC_000913.3 (2,177,512-2,178,564) (09-MAR-2022). The amino acid sequence expressing fructose bisphosphate aldolase FbaB is NP_416600.1 (09-MAR-2022).

[0113] The reference gene sequence of the outer membrane protein gene (ompT) is NC_000913.3 (584, 680-585, 633, complement) (09-MAR-2022). The amino acid sequence of the expressed outer membrane protein is NP_415097.1 (09-MAR-2022).

[0114] In one embodiment of the present invention, the receptor may be Escherichia coli BW25113 strain.

[0115] Furthermore, in the above recombinant bacteria, the methanol dehydrogenase gene is introduced into the endotoxin lpxM gene site of the recipient bacteria, and the glycerol dehydratase activator gene is introduced into the outer membrane protein gene (ompT) site of the recipient bacteria.

[0116] In one embodiment of the present invention, the methanol dehydrogenase gene is introduced into the endotoxin lpxM gene site of the recipient bacteria through a targeting fragment. The targeting fragment is, from upstream to downstream, the upstream homology arm sequence of the knockout gene lpxM (SEQ ID No. 13), the P119 promoter (SEQ ID No. 9), the methanol dehydrogenase Bstmdh gene from Bacillus stearothermophilus (SEQ ID No. 12), the TrrnB terminator (SEQ ID No. 10), the kanamycin resistance gene with FRT flanks (FRT-kan-FRT) (SEQ ID No. 11) and the downstream homology arm sequence of the knockout gene lpxM (SEQ ID No. 14), and the target gene knocked out by the targeting fragment is the endotoxin lpxM gene.

[0117] In one embodiment of the present invention, the glycerol dehydratase activator gene is introduced into the outer membrane protein gene (ompT) site of the recipient bacteria via a targeting fragment. The targeting fragment comprises, from upstream to downstream, an upstream homology arm sequence of the knockout gene ompT (SEQ ID No. 21), a P119 promoter, a Klebsiella pneumoniae glycerol dehydratase activator GdrAB gene cluster, a TrrnB terminator, a kanamycin resistance gene with FRT flanks (FRT-kan-FRT), and a downstream homology arm sequence of the knockout gene ompT (SEQ ID No. 22). The target gene to be knocked out by the targeting fragment is the triose phosphate isomerase tpiA gene.

[0118] Furthermore, in the recombinant bacteria, B1) the methanol dehydrogenase is derived from Geobacillus stearothermophilus; B2) the 3-hexulose-6-phosphate synthase hps is derived from Bacillus; B4) the 6-phosphate-3-hexose isomerase is derived from Bacillus; B6) the dihydroxyacetone phosphate reductase is derived from Saccharomyces cerevisiae; B7) the 3-phosphoglycerol phosphatase is derived from Saccharomyces cerevisiae; B8) the glycerol dehydratase is derived from Klebsiella pneumoniae; B9) the alcohol dehydrogenase is derived from Escherichia coli; B10) the glycerol dehydratase activator is derived from Klebsiella pneumoniae.

[0119] Furthermore, in the recombinant bacteria,

[0120] B1) The methanol dehydrogenase is any one of the following:

[0121] B1-1), a protein having an amino acid sequence of SEQ ID No. 32;

[0122] B1-2) A protein related to methanol dehydrogenase obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in B1-1) having an identity of 80% or more to the protein shown in B1-1);

[0123] B1-3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in B1-1) or B1-2);

[0124] B2) The 3-hexulose-6-phosphate synthase is any one of the following:

[0125] B2-1), a protein having an amino acid sequence of SEQ ID No. 33;

[0126] B2-2) A protein related to 3-hexulose-6-phosphate synthase obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in B2-1) having an identity of 80% or more to the protein shown in B2-1);

[0127] B2-3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in B2-1) or B2-2);

[0128] B4) The 6-phosphate-3-hexose isomerase is any one of the following:

[0129] B4-1), a protein having an amino acid sequence of SEQ ID No. 34;

[0130] B4-2) A protein related to 6-phospho-3-hexose isomerase, obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in B4-1) and having an identity of 80% or more to the protein shown in B4-1);

[0131] B4-3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in B4-1) or B4-2);

[0132] B6) the dihydroxyacetone dehydrogenase is any one of the following:

[0133] B6-1), a protein having an amino acid sequence of SEQ ID No. 24;

[0134] B6-2) A protein related to alcohol dehydrogenase obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in B6-1) having an identity of 80% or more to the protein shown in B6-1);

[0135] B6-3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in B6-1) or B6-2);

[0136] B7) The 3-phosphoglycerate phosphatase is any one of the following:

[0137] B7-1), a protein with the amino acid sequence of SEQ ID No. 25;

[0138] B7-2) A protein related to alcohol dehydrogenase obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in B7-1) having an identity of 80% or more to the protein shown in B7-1);

[0139] B7-3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in B7-1) or B7-2);

[0140] B8) The glycerol dehydratase includes glycerol dehydratase DhaB1, glycerol dehydratase DhaB2 and glycerol dehydratase DhaB3, wherein the glycerol dehydratase DhaB1 is any one of the following:

[0141] B8-1), a protein having an amino acid sequence of SEQ ID No. 26;

[0142] B8-2) A protein related to glycerol dehydratase obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in B8-1) having an identity of 80% or more to the protein shown in B8-1);

[0143] B8-3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in B8-1) or B82);

[0144] The glycerol dehydratase DhaB2 is any one of the following:

[0145] B8-4), a protein having an amino acid sequence of SEQ ID No. 27;

[0146] B8-5) and B8-4) amino acid sequences obtained by substitution and / or deletion and / or addition of amino acid residues, and proteins having 80% or more identity with the protein shown in B8-4) and related to glycerol dehydratase;

[0147] B8-6) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in B8-4) or B8-5);

[0148] The glycerol dehydratase DhaB3 is any one of the following:

[0149] B8-7), a protein with the amino acid sequence of SEQ ID No. 28;

[0150] B8-8) and B8-7) wherein the amino acid sequences are substituted and / or deleted and / or added to obtain a protein having 80% or greater identity with the protein shown in B8-7) and related to glycerol dehydratase;

[0151] B8-9), a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in B8-7) or B8-8);

[0152] B9) The alcohol dehydrogenase is any one of the following:

[0153] B9-1), a protein with the amino acid sequence of SEQ ID No. 31;

[0154] B9-2) A protein related to alcohol dehydrogenase obtained by substitution and / or deletion and / or addition of amino acid residues in the amino acid sequence shown in B9-1) having an identity of 80% or more to the protein shown in B9-1);

[0155] B9-3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in B9-1) or B9-2);

[0156] B10) The dehydratase activating factor includes a dehydratase activating factor GdrA and a dehydratase activating factor GdrB, wherein the dehydratase activating factor GdrA is any one of the following:

[0157] B10-1), a protein with the amino acid sequence of SEQ ID No. 29;

[0158] B10-2) A protein related to a dehydratase activating factor obtained by substitution and / or deletion and / or addition of amino acid residues of the amino acid sequence shown in B10-1) having an identity of 80% or more to the protein shown in B10-1);

[0159] B10-3) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in B10-1) or B10-2);

[0160] The dehydratase activating factor GdrB is any one of the following:

[0161] B10-4), a protein having an amino acid sequence of SEQ ID No. 30;

[0162] B10-5) and B10-4) amino acid sequences obtained by substitution and / or deletion and / or addition of amino acid residues, and proteins having more than 80% identity with the protein shown in B10-4) and related to dehydratase activating factors;

[0163] B10-6) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in B10-4) or B10-5).

[0164] Furthermore, in the recombinant bacteria, B1) the coding sequence of the methanol dehydrogenase gene is a DNA molecule with a nucleotide sequence of SEQ ID No. 12; B2) the coding sequence of the 3-hexulose-6-phosphate synthase gene is a DNA molecule with a nucleotide sequence of SEQ ID No. 15; B4) the coding sequence of the 6-phosphate-3-hexose isomerase gene is a DNA molecule with a nucleotide sequence of SEQ ID No. 18; B6) the coding sequence of the dihydroxyacetone dehydrogenase gene is a DNA molecule with a nucleotide sequence of SEQ ID No. 4; B7) the coding sequence of the 3-phosphoglycerophosphatase gene is a DNA molecule with a nucleotide sequence of SEQ ID No. 5; B8) the coding sequence of the glycerol dehydratase gene is a DNA molecule with a nucleotide sequence of SEQ ID No. 6; B9) the coding sequence of the alcohol dehydrogenase gene is a DNA molecule with a nucleotide sequence of SEQ ID No. 8; and B10) the coding sequence of the glycerol dehydratase activator gene is a DNA molecule with a nucleotide sequence of SEQ ID No. 7.

[0165] Furthermore, in the recombinant bacteria, the substance described in any one of B1), B2), B4) and B6)-B10) is any one of the following:

[0166] g3) a promoter or terminator, wherein the promoter described in any one of B1), B2), B4) and B10) is the P119 promoter, and the promoter described in B6), B7), B8) and B9) is the PBAD promoter; and the terminator is the TrrnB terminator;

[0167] g4) a promoter or terminator, wherein the promoter described in any one of B1), B2), B4) and B10) is the P119 promoter whose nucleotide sequence is SEQ ID No. 9, the promoter described in B6), B7), B8) and B9) is the PBAD promoter whose nucleotide sequence is positions 1238-1266 of SEQ ID No. 1, and the terminator is the TrrnB terminator whose nucleotide sequence is SEQ ID No. 10.

[0168] Furthermore, in the recombinant bacteria, the 3-hexulose-6-phosphate synthase gene (hps) is introduced at the site of the hydroxymethyl-glutathione dehydrogenase gene (frmA) of the recipient bacteria. The 6-phosphate-3-hexose isomerase gene (phi) is introduced at the site of the triose phosphate isomerase gene (tpiA) of the recipient bacteria.

[0169] Furthermore, in the recombinant bacteria, the knockout in any one of B3) and B5) is achieved by introducing a targeting fragment, and the targeting fragment can knock out the target gene.

[0170] Furthermore, in the above-mentioned recombinant bacteria, the glycerol dehydratase gene may be a gene cluster of three genes, glycerol dehydratase DhaB1, DhaB2, and DhaB3, named the DhaB123 gene cluster, and the nucleotide sequence of the DhaB123 gene cluster is SEQ ID No. 6. Among them, positions 1-1668 of SEQ ID No. 6 are the glycerol dehydratase dhaB1 gene, which encodes the glycerol dehydratase dhaB1 with an amino acid sequence of SEQ ID No. 26. Positions 1681-2265 of SEQ ID No. 6 are the glycerol dehydratase dhaB2 gene, which encodes the glycerol dehydratase dhaB2 with an amino acid sequence of SEQ ID No. 27. Positions 2268-2693 of SEQ ID No. 6 are the glycerol dehydratase dhaB3 gene, which encodes the glycerol dehydratase dhaB3 with an amino acid sequence of SEQ ID No. 28.

[0171] In the present invention, the glycerol dehydratase activator gene may be a gene cluster consisting of two glycerol dehydratase activator genes, GdrA and GdrB, designated as the GdrAB gene cluster. The nucleotide sequence of the GdrAB gene cluster is SEQ ID No. 7. Positions 1-1824 of SEQ ID No. 7 represent the glycerol dehydratase activator GdrA gene, encoding the glycerol dehydratase activator GdrA with an amino acid sequence of SEQ ID No. 29. Positions 1845-2198 of SEQ ID No. 7 represent the glycerol dehydratase activator GdrB gene, encoding the glycerol dehydratase activator GdrB with an amino acid sequence of SEQ ID No. 30.

[0172] In one embodiment of the present invention, the targeting fragment used in B3) is, from upstream to downstream, the upstream homology arm sequence of the knockout gene frmA (SEQ ID No. 16), the P119 promoter, the Bacillus-derived 3-hexulose-6-phosphate synthase hps gene (SEQ ID No. 15), the TrrnB terminator, the kanamycin resistance gene with FRT flanks (FRT-kan-FRT), and the downstream homology arm sequence of the knockout gene frmA (SEQ ID No. 17). The target gene knocked out by the targeting fragment is the hydroxymethyl-glutathione dehydrogenase frmA gene.

[0173] In one embodiment of the present invention, the targeting fragment used in B5) is, from upstream to downstream, the upstream homology arm sequence of the knockout gene tpiA (SEQ ID No. 19), the P119 promoter, the Bacillus-derived 6-phosphate-3-hexose isomerase phi gene (SEQ ID No. 18), the TrrnB terminator, the kanamycin resistance gene with FRT flanks (FRT-kan-FRT), and the downstream homology arm sequence of the knockout gene tpiA (SEQ ID No. 20). The target gene knocked out by the targeting fragment is the triose phosphate isomerase tpiA gene.

[0174] Furthermore, the recombinant bacteria also includes the following characteristics:

[0175] (15) containing a promoter that enhances the expression of a transketolase gene or / and enhances the content and / or activity of transketolase;

[0176] (16) A promoter that enhances the expression of a ribulose-phosphate 3-epimerase gene or / and enhances the content and / or activity of ribulose-phosphate 3-epimerase.

[0177] The substance described in (15) and (16) may be an enhanced promoter P119 promoter. The nucleotide sequence of the P119 promoter is SEQ ID No. 9.

[0178] Furthermore, the recombinant bacteria are obtained by replacing the promoters of transketolase and ribulose phosphate epimerase in Escherichia coli as a recipient bacterium with P119 promoter.

[0179] The transketolase genes include tktA and tktB. The reference gene sequence of tktA is NC_000913.3 (3,079,644-3,081,635, complement) (09-MAR-2022). The amino acid sequence expressing the transketolase tktA is YP_026188.1 (09-MAR-2022). The reference gene sequence of tktB is NC_000913.3 (2,579,636-2,581,639) (09-MAR-2022). The amino acid sequence expressing the transketolase tktB is NP_416960.1 (09-MAR-2022).

[0180] The reference gene sequence of the ribulose phosphate epimerase gene (rpe) is NC_000913.3 (3,514,382-3,515,059, complement) (09-MAR-2022). The amino acid sequence expressing the ribulose phosphate epimerase is NP_417845.1 (09-MAR-2022).

[0181] In a second aspect, the present invention provides an application, which is the application of the above-mentioned recombinant bacteria in the preparation of 1,3-propanediol.

[0182] In a third aspect, the present invention provides a method for preparing 1,3-propylene glycol, which comprises using the above-mentioned recombinant bacteria as a fermentation strain and methanol as a raw material to prepare 1,3-propylene glycol.

[0183] In a fourth aspect, the present invention provides a method for constructing the above-mentioned recombinant bacteria, comprising constructing a metabolic pathway in a recipient bacterium to obtain a recombinant bacterium that synthesizes 1,3-propanediol using methanol as a raw material,

[0184] The metabolic pathways are (a)-(i):

[0185] (a) converting methanol into formaldehyde;

[0186] (b) synthesizing D-arabino-hex-3-ulose 6-phosphate from formaldehyde and D-ribulose 5-phosphate;

[0187] (c) converting D-arabino-hex-3-ketose 6-phosphate into fructose 6-phosphate;

[0188] (d) converting fructose 6-phosphate into fructose 1,6-bisphosphate;

[0189] (e) converting fructose 1,6-diphosphate into dihydroxyacetone phosphate;

[0190] (f) converting dihydroxyacetone phosphate into glycerol triphosphate;

[0191] (g) converting glycerol-3-phosphate into glycerol;

[0192] (h) converting glycerol into 3-hydroxypropionaldehyde;

[0193] (i) Conversion of 3-hydroxypropanal to 1,3-propanediol.

[0194] The microbial strain of the present invention can be a Bacillus sp. strain. The genus Bacillus contains a variety of strains that can naturally grow using methanol as the only carbon source, such as Bacillus methanolicus. Bacillus YL23 (Bacillus sp. YL23, deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms, with a deposit number of CGMCC NO: 29579) is a strain that can grow rapidly using methanol as a carbon source. Its advantage is that it has a high efficiency in assimilating methanol and can be genetically modified efficiently using conventional operating elements under normal conditions. However, Bacillus YL23 does not have a metabolic pathway for synthesizing 1,3-propanediol. By Figure 2 Genetic modification can provide a production route for synthesizing 1,3-propylene glycol using methanol as raw material.

[0195] The microbial strain of the present invention can also be Escherichia coli. Escherichia coli has a clear genetic background, a short generation time, is easy to culture, and has low-cost culture medium raw materials. Natural Escherichia coli does not have a metabolic pathway for utilizing methanol and formaldehyde, nor a metabolic pathway for synthesizing 1,3-propanediol. By Figure 3 and Figure 4 Genetic modification can provide a production route for synthesizing 1,3-propylene glycol using methanol as raw material.

[0196] The methanol raw material of the present invention can be derived from carbon dioxide and gases containing carbon dioxide. The methanol can be produced by electrolyzing water to produce hydrogen, using hydrogen and carbon dioxide as raw materials for chemical reaction.

[0197] The beneficial technical effects of the present invention are as follows:

[0198] The present invention provides a recombinant bacterium that can efficiently synthesize 1,3-propylene glycol using raw materials such as methanol and formaldehyde, and establishes a route for synthesizing 1,3-propylene glycol using carbon dioxide, methanol, formaldehyde, etc. as raw materials, which has great economic and social value.

[0199] Preservation Instructions

[0200] Bacteria name: Bacillus

[0201] Latin name: Bacillus sp.

[0202] Strain ID: YL23

[0203] Depository: General Microbiology Center of China Culture Collection Administration

[0204] Abbreviation of depository institution: CGMCC

[0205] Address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0206] Deposit date: January 12, 2024

[0207] CGMCC registration number: CGMCC NO:29579 BRIEF DESCRIPTION OF THE DRAWINGS

[0208] Figure 1 A synthetic pathway for the synthesis of 1,3-propanediol from methanol using the RuMP cycle.

[0209] Figure 2 This is the synthetic pathway for synthesizing 1,3-propanediol using methanol as raw material in Bacillus and Escherichia coli (the dark target is the heterologous 1,3-propanediol synthesis pathway).

[0210] Figure 3 This is a synthetic pathway for 1,3-propanediol synthesis in Escherichia coli using methanol as a raw material (targets marked with five stars are heterologous 1,3-propanediol synthesis pathways, targets marked with triangles are heterologous methanol assimilation pathways, and targets marked with crosses are knockout targets). In the examples, the DAR1 gene is introduced to exert DAR activity; the GPP1 gene exerts G3PP activity; the DhaB123 and GdrAB genes exert GDH activity; and the yqhD gene exerts ADH activity.

[0211] Figure 4 This is a pathway related to the replenishment of ribulose 5-phosphate from fructose 6-phosphate in Escherichia coli, in which transketolase and ribulose phosphate epimerase need to be enhanced.

[0212] Figure 5 This is the pHP-M05 plasmid map. DETAILED DESCRIPTION

[0213] The present invention constructs the following metabolic pathway in the recipient bacteria: the organism can use the ribulose phosphate cycle (RuMP cycle) to assimilate methanol and formaldehyde. The RuMP cycle has a high activity in prokaryotic microorganisms. Therefore, carbon dioxide, methanol, and formaldehyde can be used as raw materials to synthesize fructose 6-phosphate through the ribulose phosphate cycle (RuMP cycle); fructose 6-phosphate is synthesized into dihydroxyacetone phosphate through partial reactions in the glycolysis pathway; dihydroxyacetone phosphate is synthesized into glycerol through dihydroxyacetone phosphate reductase (dihydroxyacetone phosphate dehydrogenase) and 3-phosphoglycerol phosphatase (glycerol 3-phosphate phosphatase); glycerol is then synthesized into 1,3-propanediol (1,3-propanediol oxidoreductase) through glycerol dehydratase (glycerol dehydratase), specific 1,3-propanediol oxidoreductase (1,3-propanediol oxidoreductase) or non-specific alcohol dehydrogenase (alcohol dehydrogenase) Figure 1 ).

[0214] The microbial strain of the present invention may be a Bacillus sp. strain. The genus Bacillus contains a variety of strains that can naturally utilize methanol, such as Bacillus methanolicus. Bacillus YL23 (Bacillus sp. YL23, deposited in the General Microbiology Center of the China Culture Collection Administration, with a deposit number of CGMCC NO: 29579, is a strain that can grow rapidly with methanol as a carbon source. Its advantage is that it has a high efficiency in assimilating methanol and can be genetically modified efficiently under normal conditions using conventional operating elements. However, natural Bacillus YL23 does not have a metabolic pathway for synthesizing 1,3-propanediol. By Figure 2 Genetic modification can obtain a metabolic pathway for synthesizing 1,3-propanediol using methanol as raw material. Figure 2 This is the synthetic pathway for synthesizing 1,3-propanediol using methanol as raw material in Bacillus (the dark target is the heterologous 1,3-propanediol synthetic pathway).

[0215] The microbial strain of the present invention can also be Escherichia coli. Escherichia coli has a clear genetic background, a short generation time, is easy to culture, and has low-cost culture medium raw materials. Natural Escherichia coli does not have a metabolic pathway for utilizing methanol and formaldehyde, nor a metabolic pathway for synthesizing 1,3-propanediol. By Figure 3 and Figure 4 Genetic modification can obtain a metabolic pathway for synthesizing 1,3-propanediol using methanol as raw material. Figure 3This is a synthetic pathway for 1,3-propanediol synthesis in Escherichia coli using methanol as a raw material (targets marked with five stars are heterologous 1,3-propanediol synthesis pathways, targets marked with triangles are heterologous methanol assimilation pathways, and targets marked with crosses are knockout targets). In the examples, the DAR1 gene is introduced to exert DAR activity; the GPP1 gene exerts G3PP activity; the DhaB123 and GdrAB genes exert GDH activity; and the yqhD gene exerts ADH activity. Figure 4 This is a pathway related to the replenishment of ribulose 5-phosphate from fructose 6-phosphate in Escherichia coli, in which the transketolase core and ribulose phosphate epimerase need to be strengthened.

[0216] The present invention provides a method for transforming the microbial strain and the transformed microbial strain.

[0217] The present invention also relates to the use of the modified microbial strain in the preparation of 1,3-propylene glycol.

[0218] The present invention also provides a method for synthesizing 1,3-propylene glycol using the modified microbial strain and methanol and formaldehyde as raw materials.

[0219] The methanol raw material of the present invention can be derived from carbon dioxide and gases containing carbon dioxide. The methanol can be produced by electrolyzing water to produce hydrogen, using hydrogen and carbon dioxide as raw materials for chemical reaction.

[0220] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0221] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0222] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0223] Among them, the Bacillus sp. YL23 strain is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration Committee with the deposit number CGMCC NO: 29579. Escherichia coli BW25113 was kindly donated by Tao Yong's group at the Institute of Microbiology, Chinese Academy of Sciences, and is disclosed in the document "Xiaoyu Piao, Lei Wang, Baixue Lin, Hao Chen, Weifeng Liu, Yong Tao. Metabolic engineering of Escherichia coli for production of L-aspartate and its derivative β-alanine with high stoichiometric yield. Metabolic Engineering. 2019 54: 244-254." The public can obtain the above-mentioned biological materials from the applicant. The obtained biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0224] Plasmids pBM19 (Genebank sequence number NC_005328.1, 02-MAY-2023), pHP13 (Genebank sequence number DQ297764.1, 07-APR-2006), pYB1s (SEQ ID No. 1), and pLB1a (SEQ ID No. 2) can be constructed by whole gene synthesis.

[0225] The pUC57 plasmid is a product of Shanghai Dibai Biotechnology Co., Ltd., with the product number S201464A.

[0226] The culture medium used for culturing Bacillus YL23 is nutrient medium and growth medium.

[0227] The basic nutrient medium formula is: 1% tryptone, 0.5% yeast powder, 0.5% NaCl, pH 7.0.

[0228] The basic growth medium formula is: 1L contains: 8.5g Na2HPO4·2H2O, 3g KH2PO4, 2.4g NH4Cl, 0.5g NaCl, 0.246g MgSO4·7H2O, 1.7mg ZnCl2, 0.43mg CuCl2·2H2O, 0.6mg CoCl2·6H2O, 0.6mg Na2MoO4·2H2O, 1.11mg CaCl2, 4.3mg FeCl2, 0.5g yeast powder, 0.5mg vitamin B1, 0.05mg riboflavin, pH 7.0.

[0229] The culture medium used for Escherichia coli was LB medium.

[0230] LB medium contained the following ingredients: 1% tryptone, 0.5% yeast extract, 1% NaCl, and the remainder was water, pH 7.0.

[0231] Plasmids constructed during genetic manipulation of Bacillus are constructed in Escherichia coli DH5α. E. coli is typically grown at 37°C in liquid or solid Luria-Bertani (LB) medium supplemented with appropriate antibiotics. The pUC18 plasmid (Genebank sequence number L08752.1, 27-APR-1993) is used as a helper plasmid during genetic manipulation and can be constructed using whole-genome synthesis.

[0232] E. coli plasmid transformation was performed using the CaCl2 transformation method, and E. coli chromosome editing was performed using the electroporation method. The relevant methods can be performed according to the literature (Molecular Cloning: A Laboratory Manual (Fourth Edition). ISBN 978-1-936113-42-2).

[0233] Plasmid transformation of Bacillus YL23 was performed by electroporation.

[0234] Table 1: Primers used in the examples

[0235]

[0236]

[0237] Example 1. Construction of recombinant Bacillus engineered strain BM-G01

[0238] 1.1. Obtaining DNA fragment PU-M:

[0239] Using plasmid pBM19 as a template and primers MDH-01F and MDH-01R, high-fidelity TransStart Fast Pfu DNA polymerase (Beijing Quanshijin Biotechnology Co., Ltd., catalog number AP221) was used to amplify the mdh gene fragment (including the promoter and terminator) of Bacillus methanolicus, MDH-T (SEQ ID No. 3). The target fragment was recovered by agarose gel electrophoresis. Sequence ID No. 3 contains the mdh promoter at positions 1-1023, the mdh gene at positions 1024-2172 (encoding the methanol dehydrogenase of SEQ ID No. 23), and the mdh terminator at positions 2173-2344.

[0240] Using primers PU-MDH-01F and PU-MDH-01R, plasmid pUC18 was used as a template to PCR amplify the plasmid backbone PU fragment, and the target fragment was recovered by agarose gel electrophoresis. The MDH-T fragment and the PU fragment were ligated using the Gibson assembly method (Gibson DG, Young L, et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. methods. 2009; 6(5): 343-345). Escherichia coli DH5α competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd., product catalog CD201) were transformed using the CaCl2 method. They were evenly spread on LB plates containing ampicillin and cultured at 37°C overnight. Clones were selected and identified using primers PU-F1 / MDH-01R to amplify the target fragment and sequenced. The target fragment of the positive clone was approximately 2400 bp. Positive clones were selected for plasmid extraction, and the resulting positive plasmid was named pUC-M01. The plasmid backbone PU-M fragment was amplified by PCR using primers PU-MDH-02F and PU-MDH-02R and plasmid pUC-M01 as template, and the target fragment was recovered by agarose gel electrophoresis.

[0241] 1.2 Construction of plasmid pUC-M-DAR:

[0242] The Saccharomyces cerevisiae dihydroxyacetone phosphate reductase DAR1 gene (SEQ ID No. 4, synthesized by Beijing Qingke Biotechnology Co., Ltd.) was artificially synthesized and used to replace the fragment between the EcoRI and PstI restriction sites of the backbone vector pUC57 to obtain the pUC57-DAR1 plasmid. Using primers MDAR-01F and MDAR-01R and the pUC57-DAR1 plasmid as a template, the MDAR gene fragment was amplified by PCR using high-fidelity TransStart FastPfu DNA polymerase (Beijing Quanshijin Biotechnology Co., Ltd., product catalog number AP221). The target fragment was recovered by agarose gel electrophoresis. The MDAR fragment was ligated with the PU-M fragment using the Gibson assembly method. Escherichia coli DH5α competent cells were transformed using the CaCl2 method, following the procedure described in Example 1, "Obtaining the DNA Fragment PU-M." Clones were selected and identified using primers MDH-01F / MDAR-01R for amplification of the target fragment. Positive clones had a target fragment of approximately 2200 bp. Positive clones were selected and plasmids were extracted. The obtained positive plasmid was named pUC-M-DAR. pUC-M-DAR was mainly used for the subcloning operation of constructing pHP-M01.

[0243] 1.3 Construction of plasmid pHP-M01:

[0244] Using primers HP-01F and HP-R, and plasmid pHP13 as a template, PCR amplify the plasmid backbone HP13 fragment, and recover the target fragment by agarose gel electrophoresis. Using primers MDH-AF and MDH-AR, and plasmid pUC-M-DAR as a template, PCR amplify the MDH-DAR fragment containing the Pmdh-DAR1-Tmdh expression cassette, and recover the target fragment by agarose gel electrophoresis. The MDH-DAR fragment and the HP13 fragment were ligated using the Gibson assembly method. Escherichia coli DH5α competent cells were transformed using the CaCl2 method, following the procedure described in Example 1, "Obtaining DNA Fragment PU-M." Clones were selected, and clones capable of amplifying the target fragment were identified using primers MDAR-01F / HP13-R and sequenced. The target fragment of the positive clones was approximately 1500 bp. Positive clones were selected for plasmid extraction, and the resulting positive plasmid was designated pHP-M01. pHP-M01 contains a DAR1 expression cassette (from upstream to downstream: mdh promoter, Saccharomyces cerevisiae dihydroxyacetone phosphate reductase DAR1 gene and mdh terminator), and can express Saccharomyces cerevisiae dihydroxyacetone phosphate reductase with an amino acid sequence of SEQ ID No. 24.

[0245] 1.4. Construction of plasmid pUC-M-GPP:

[0246] The Saccharomyces cerevisiae 3-phosphoglycerophosphatase (GPP1) gene (SEQ ID No. 5, synthesized by Beijing Qingke Biotechnology Co., Ltd.) was artificially synthesized and used to replace the fragment between the EcoRI and PstI restriction sites of the backbone vector pUC57, generating the pUC57-GPP1 plasmid. The MGPP gene fragment was amplified by PCR using the high-fidelity TransStart FastPfu DNA polymerase (Beijing Quanshijin Biotechnology Co., Ltd., catalog number AP221) using primers MGPP-01F and MGPP-01R. The target fragment was recovered by agarose gel electrophoresis. The MGPP fragment was ligated with the PU-M fragment using the Gibson assembly method. Escherichia coli DH5α competent cells were transformed using the CaCl2 method, following the procedure described in Example 1, "Obtaining the PU-M DNA Fragment." Clones were selected and identified using primers MDH-01F / MGPP-01R to amplify the target fragment. Positive clones had a target fragment of approximately 1800 bp. Positive clones were selected and plasmids were extracted. The obtained positive plasmid was named pUC-M-GPP. pUC-M-DAR was mainly used for the subcloning operation of constructing pHP-M02.

[0247] 1.5. Construction of plasmid pHP-M02:

[0248] Using primers HP-02F and HP-R, and plasmid pHP-M01 as a template, the plasmid backbone HP-M01 fragment was PCR amplified. The target fragment was recovered by agarose gel electrophoresis. Using primers MDH-BF and MDH-BR, and plasmid pUC-M-GPP as a template, the MDH-GPP fragment containing the Pmdh-GPP1-Tmdh expression cassette was PCR amplified. The target fragment was recovered by agarose gel electrophoresis. The MDH-GPP fragment was ligated with the HP-M01 fragment using the Gibson assembly method. Competent E. coli DH5α cells were transformed using the CaCl2 method, following the procedure described in Example 1, "Obtaining DNA Fragment PU-M." Clones were selected and identified using primers MGPP-01F / HP13-R. Positive clones were sequenced, and the target fragment was approximately 1100 bp. Positive clones were selected for plasmid extraction, and the resulting plasmid was designated pHP-M02. pHP-M02 contains a DAR1 expression cassette and a GPP1 expression cassette (from upstream to downstream: the mdh promoter, the Saccharomyces cerevisiae 3-phosphoglycerol phosphatase GPP1 gene, and the mdh terminator), and can express the Saccharomyces cerevisiae dihydroxyacetone phosphate reductase with an amino acid sequence of SEQ ID No. 24 and the Saccharomyces cerevisiae 3-phosphoglycerol phosphatase with an amino acid sequence of SEQ ID No. 25.

[0249] 1.6. Construction of engineered strain BM-G01:

[0250] Bacillus YL23 was used as a recipient bacterium. Bacillus YL23 contained a methanol dehydrogenase gene, a 3-hexulose-6-phosphate synthase gene, a 6-phosphate-3-hexose isomerase gene, a 6-phospho-fructose kinase gene, and a fructose bisphosphate aldolase gene.

[0251] The coding sequence of the methanol dehydrogenase gene is the nucleotide sequence of SEQ ID No. 43. The methanol dehydrogenase having the amino acid sequence of SEQ ID No. 44 can be expressed.

[0252] The nucleotide sequence of the 3-hexulose-6-phosphate synthase gene is SEQ ID No. 15. The 3-hexulose-6-phosphate synthase having the amino acid sequence of SEQ ID No. 33 can be expressed.

[0253] The nucleotide sequence of the 6-phosphate-3-hexose isomerase (Phi) gene is SEQ ID No. 18. The 6-phosphate-3-hexose isomerase (Phi) having the amino acid sequence of SEQ ID No. 34 can be expressed.

[0254] The nucleotide sequence of the 6-phosphofructokinase gene is SEQ ID No. 39. The 6-phosphofructokinase having the amino acid sequence of SEQ ID No. 40 can be expressed.

[0255] The nucleotide sequence of the fructose bisphosphate aldolase gene is SEQ ID No. 41. The fructose bisphosphate aldolase having the amino acid sequence of SEQ ID No. 42 can be expressed.

[0256] Competent Bacillus subtilis YL23 was prepared and electroporated with the plasmid pHP-M02. The resulting strain was named BM-G01 and stored at -80°C. The electroporation method for Bacillus subtilis plasmids is as follows:

[0257] a. Prepare competent cells: Take Bacillus YL23 strain from the freeze-dried tube and inoculate it into 100 mL of basic nutrient medium and grow it at 37°C for 16 hours to obtain a culture. Transfer 2 mL of culture into 100 mL of nutrient medium (i.e., basic nutrient medium supplemented with 10% sucrose at a final concentration) and continue growing at 37°C until the OD 600 = 0.25. Centrifuge at 3,000 g for 5 min to collect the cells, wash twice in 3.5 ml of EP buffer (1 mM HEPES, 25% polyethylene glycol 8000, pH 7.0), and resuspend in 0.2 ml of EP buffer. After treatment with liquid nitrogen, competent cells are obtained. Competent cells are stored at 80°C.

[0258] b. Electroporation: Competent cells (100 μL) were mixed with approximately 1 μg of plasmid DNA and incubated on ice for 30 min. The mixture was transferred to a pre-chilled electroporation cuvette (0.2 cm; Bio-Rad Laboratories) and electroporated using a Bio-Rad Gene-Pulser (200 Ω; 25 μF; 2.5 kV).

[0259] c. Recovery culture: After electroporation, culture the cells in 5 mL of nutrient medium at 37°C for 16 h. Transfer 4 mL of the cell culture to 100 mL of nutrient medium supplemented with chloramphenicol and continue culturing at 37°C for 6 h. Spread the culture onto a solid basal nutrient medium containing chloramphenicol and incubate at 37°C overnight to obtain a recovered bacterial plate.

[0260] d. Strain preservation: Single colonies were picked from the plates and transferred to shake flasks containing 100 mL of growth medium with antibiotics and 1% glucose. The culture was incubated overnight at 37°C, and then 15% glycerol was added to preserve the strains. The strains were then stored at -80°C to obtain Bacillus BM-G01.

[0261] Compared to Bacillus YL23, BM-G01 contains DAR1 expression cassette and GPP1 expression cassette, and can express Saccharomyces cerevisiae dihydroxyacetone phosphate reductase with amino acid sequence of SEQ ID No. 24 and Saccharomyces cerevisiae 3-phosphate glycerol phosphatase with amino acid sequence of SEQ ID No. 25.

[0262] Example 2: Construction of recombinant Bacillus engineered strain BM-G02

[0263] 2.1. Construction of plasmid pUC-M-DHB:

[0264] The gene cluster DhaB123 (SEQ ID No. 6, synthesized by Beijing Qingke Biotechnology Co., Ltd.) encoding the three genes of Klebsiella pneumoniae glycerol dehydratase DhaB1, DhaB2, and DhaB3 was artificially synthesized, and the fragment between the EcoRI and PstI restriction sites of the backbone vector pUC57 was replaced with the DhaB123 gene to obtain the pUC57-DhaB123 plasmid. Among them, SEQ ID No. 6 positions 1-1668 are the glycerol dehydratase dhaB1 gene, which encodes the glycerol dehydratase dhaB1 of SEQ ID No. 26. SEQ ID No. 6 positions 1681-2265 are the glycerol dehydratase dhaB2 gene, which encodes the glycerol dehydratase dhaB2 of SEQ ID No. 27. SEQ ID No. 6 positions 2268-2693 are the glycerol dehydratase dhaB3 gene, which encodes the glycerol dehydratase dhaB3 of SEQ ID No. 28.

[0265] Using MDHB-01F and MDHB-01R as primers, the high-fidelity TransStart FastPfu DNA polymerase (Beijing Quanshijin Biotechnology Co., Ltd., product catalog number AP221) was used to amplify the MDHB gene fragment by PCR. The target fragment was recovered by agarose gel electrophoresis. The MDHB fragment was ligated with the PU-M fragment using the Gibson assembly method. E. coli DH5α competent cells were transformed using the CaCl2 method, as described in Example 1, "Obtaining the DNA fragment PU-M." Clones were selected and identified using primers MDH-01F / MDHB-01R. Clones that amplified the target fragment were sequenced. The target fragment of the positive clones was 3700 bp. Plasmids were extracted from the selected positive clones, and the resulting positive plasmid was named pUC-M-DHB. pUC-M-DHB contains the DhaB123 expression cassette (from upstream to downstream: the mdh promoter, the gene cluster DhaB123 of the three genes of Klebsiella pneumoniae glycerol dehydratase DhaB1, DhaB2, and DhaB3, and the mdh terminator), and can express the Klebsiella pneumoniae glycerol dehydratase DhaB1, DhaB2, and DhaB3 whose amino acid sequences are SEQ ID No. 26, 27, and 28, respectively.

[0266] 2.2 Construction of plasmid pHP-M03:

[0267] Using primers HP-03F and HP-R, and plasmid pHP-M02 as a template, the plasmid backbone HP-M02 fragment was PCR amplified. The target fragment was recovered by agarose gel electrophoresis. Using primers MDH-CF and MDH-CR, and plasmid pUC-M-DHB as a template, the MDH-DHB fragment containing the Pmdh-DhaB123-Tmdh expression cassette was PCR amplified. The target fragment was recovered by agarose gel electrophoresis. The MDH-DHB fragment was ligated with the HP-M02 fragment using the Gibson assembly method. Competent E. coli DH5α cells were transformed using the CaCl2 method, following the procedure described in Example 1, "Obtaining DNA Fragment PU-M." Clones were selected and identified using primers MDHB-01F / HP13-R. Positive clones were sequenced, and the target fragment was 3000 bp. Positive clones were selected for plasmid extraction, resulting in the positive plasmid designated pHP-M03. pHP-M03 contains the DAR1 expression cassette, the GPP1 expression cassette, and the DhaB123 expression cassette, and can express the Saccharomyces cerevisiae dihydroxyacetone phosphate reductase with the amino acid sequence of SEQ ID No. 24, the Saccharomyces cerevisiae 3-phosphate glycerol phosphatase with the amino acid sequence of SEQ ID No. 25, and the Klebsiella pneumoniae glycerol dehydratase DhaB1, DhaB2, and DhaB3 with the amino acid sequences of SEQ ID Nos. 26, 27, and 28, respectively.

[0268] 2.3 Construction of plasmid pUC-M-GDR:

[0269] The gene cluster GdrAB (SEQ ID No. 7, synthesized by Beijing Qingke Biotechnology Co., Ltd.), encoding the two genes for the glycerol dehydratase activator GdrA and GdrB from Klebsiella pneumoniae, was artificially synthesized. The GdrAB gene was used to replace the fragment between the EcoRI and PstI restriction sites of the backbone vector pUC57 to obtain the pUC57-GdrAB plasmid. Sequence ID No. 7 positions 1-1824 represent the glycerol dehydratase activator GdrA gene, encoding the glycerol dehydratase activator GdrA with the amino acid sequence of SEQ ID No. 29. Sequence ID No. 7 positions 1845-2198 represent the glycerol dehydratase activator GdrB gene, encoding the glycerol dehydratase activator GdrB with the amino acid sequence of SEQ ID No. 30.

[0270] Using MGDR-01F and MGDR-01R as primers, the high-fidelity TransStart FastPfu DNA polymerase (Beijing Quanshijin Biotechnology Co., Ltd., catalog number AP221) was used to amplify the MGDR gene fragment by PCR. The target fragment was recovered by agarose gel electrophoresis. The MGDR fragment was ligated with the PU-M fragment using the Gibson assembly method. Escherichia coli DH5α competent cells were transformed using the CaCl2 method, following the procedure described in Example 1, "Obtaining the DNA fragment PU-M." Clones were selected and identified using primers MDH-01F / MGDR-01R. Clones that amplified the target fragment were sequenced. The target fragment of the positive clones was 3200 bp. Plasmids were extracted from the selected positive clones, and the resulting plasmid was named pUC-M-GDR. pUC-M-DHB contains the GdrAB expression cassette (from upstream to downstream: the mdh promoter, the gene cluster GdrAB of the two genes of the Klebsiella pneumoniae glycerol dehydratase activator GdrA and GdrB, and the mdh terminator), and can express the amino acid sequences of SEQ ID No. 29 and 30, respectively, of the Klebsiella pneumoniae glycerol dehydratase activator GdrA and GdrB.

[0271] 2.4 Construction of plasmid pHP-M04:

[0272] Using primers HP-04F and HP-R, and plasmid pHP-M03 as a template, PCR amplified the plasmid backbone HP-M03 fragment. The target fragment was recovered by agarose gel electrophoresis. Using primers MDH-DF and MDH-DR, and plasmid pUC-M-GDR as a template, PCR amplified the MDH-GDR fragment containing the Pmdh-GdrAB-Tmdh expression cassette. The target fragment was recovered by agarose gel electrophoresis. The MDH-GDR fragment was ligated with the HP-M03 fragment using the Gibson assembly method. E. coli DH5α competent cells were transformed using the CaCl2 method, following the procedure described in Example 1, "Obtaining DNA Fragment PU-M." Clones were selected and identified using primers MGDR-01F / HP13-R. Positive clones were sequenced, and the target fragment was 2500 bp. Positive clones were selected for plasmid extraction, and the resulting plasmid was designated pHP-M04. pHP-M04 contains a DAR1 expression cassette, a GPP1 expression cassette, a DhaB123 expression cassette, and a GdrAB expression cassette, and can express the Saccharomyces cerevisiae dihydroxyacetone phosphate reductase with an amino acid sequence of SEQ ID No. 24, the Saccharomyces cerevisiae 3-phosphate glycerol phosphatase with an amino acid sequence of SEQ ID No. 25, the Klebsiella pneumoniae glycerol dehydratase DhaB1, DhaB2, and DhaB3 with amino acid sequences of SEQ ID Nos. 26, 27, and 28, respectively, and the Klebsiella pneumoniae glycerol dehydratase activators GdrA and GdrB with amino acid sequences of SEQ ID Nos. 29 and 30, respectively.

[0273] 2.5. Construction of plasmid pUC-M-YQHD:

[0274] The Escherichia coli alcohol dehydrogenase YqhD gene (SEQ ID No. 8, synthesized by Beijing Qingke Biotechnology Co., Ltd.) was artificially synthesized and used to replace the fragment between the EcoRI and PstI restriction sites of the backbone vector pUC57 to obtain the pUC57-YQHD plasmid. Using MYQD-01F and MYQD-01R as primers, the high-fidelity TransStart FastPfu DNA polymerase (Beijing Quanshijin Biotechnology Co., Ltd., product catalog number AP221) was used to PCR amplify the gene fragment MYQD. The target fragment was recovered by agarose gel electrophoresis. The MYQD fragment was ligated with the PU-M fragment using the Gibson assembly method. Escherichia coli DH5α competent cells were transformed using the CaCl2 method. The transformation was carried out according to the "Obtaining DNA Fragment PU-M" section of Example 1. Clones were selected and clones that were able to amplify the target fragment were identified using primers MDH-01F / MYQD-01R and sequenced. The target fragment of the positive clones was 2100bp. Positive clones were selected and plasmids were extracted. The resulting plasmid was named pUC-M-YQHD. pUC-M-YQHD contains the YqhD expression cassette (from upstream to downstream: the mdh promoter, the E. coli alcohol dehydrogenase YqhD gene, and the mdh terminator), which can express the E. coli alcohol dehydrogenase with the amino acid sequence of SEQ ID No. 31.

[0275] 2.6 Construction of plasmid pHP-M05:

[0276] Using primers HP-05F and HP-R, and plasmid pHP-M04 as a template, PCR amplify the plasmid backbone HP-M04 fragment, and recover the target fragment by agarose gel electrophoresis. Using primers MDH-EF and MDH-ER, and plasmid pUC-M-YQHD as a template, PCR amplify the MDH-YQHD fragment containing the Pmdh-YqhD-Tmdh expression cassette, and recover the target fragment by agarose gel electrophoresis. The MDH-YQD fragment and the HP-M04 fragment were ligated using the Gibson assembly method. Escherichia coli DH5α competent cells were transformed using the CaCl2 method. Transformation was performed according to the "Obtaining DNA Fragment PU-M" section of Example 1. Clones were selected, and clones capable of amplifying the target fragment were identified using primers MYQD-01F / HP13-R and sequenced. The target fragment of the positive clones was 1400 bp. Positive clones were selected for plasmid extraction, and the resulting positive plasmid was named pHP-M05. From upstream to downstream, pHP-M05 contains the Saccharomyces cerevisiae dihydroxyacetone phosphate reductase DAR1 gene expression cassette (DAR1 expression cassette), the Saccharomyces cerevisiae 3-phosphate glycerol phosphatase GPP1 gene expression cassette (GPP1 expression cassette), the Klebsiella pneumoniae glycerol dehydratase gene cluster DhaB123 expression cassette (DhaB123 expression cassette), the Klebsiella pneumoniae glycerol dehydratase activator gene cluster GdrA and GdrB gene cluster GdrAB expression cassette (GdrAB expression cassette), and the Escherichia coli alcohol dehydrogenase YqhD gene expression cassette (YqhD expression cassette) ( Figure 5 ).

[0277] pHP-M05 can express the Saccharomyces cerevisiae dihydroxyacetone phosphate reductase with the amino acid sequence of SEQ ID No. 24, the Saccharomyces cerevisiae 3-phosphate glycerol phosphatase with the amino acid sequence of SEQ ID No. 25, the Klebsiella pneumoniae glycerol dehydratase DhaB1, DhaB2, DhaB3 with the amino acid sequences of SEQ ID Nos. 26, 27, and 28, respectively, the Klebsiella pneumoniae glycerol dehydratase activators GdrA and GdrB with the amino acid sequences of SEQ ID Nos. 29 and 30, respectively, and the Escherichia coli alcohol dehydrogenase with the amino acid sequence of SEQ ID No. 31.

[0278] 2.7 Construction of engineered strain BM-G02:

[0279] Following the method in 1.6, electroporation of plasmid pHP-M05 was performed using Bacillus subtilis YL23 as the recipient strain. The resulting strain was named BM-G02 and stored at -80°C.

[0280] Compared with Bacillus YL23, BM-G02 also contains the Saccharomyces cerevisiae dihydroxyacetone phosphate reductase DAR1 gene expression cassette (DAR1 expression cassette), the Saccharomyces cerevisiae 3-phosphate glycerol phosphatase GPP1 gene expression cassette (GPP1 expression cassette), the Klebsiella pneumoniae glycerol dehydratase gene cluster DhaB123 expression cassette (DhaB123 expression cassette), the Klebsiella pneumoniae glycerol dehydratase activator gene cluster GdrA and GdrB gene expression cassette (GdrAB expression cassette), and the Escherichia coli alcohol dehydrogenase YqhD gene expression cassette (YqhD expression cassette). The invention can express the yeast cerevisiae dihydroxyacetone phosphate reductase with the amino acid sequence of SEQ ID No. 24, the yeast cerevisiae 3-phosphate glycerol phosphatase with the amino acid sequence of SEQ ID No. 25, the Klebsiella pneumoniae glycerol dehydratase DhaB1, DhaB2, DhaB3 with the amino acid sequences of SEQ ID Nos. 26, 27, and 28 respectively, the Klebsiella pneumoniae glycerol dehydratase activating factors GdrA and GdrB with the amino acid sequences of SEQ ID Nos. 29 and 30 respectively, and the Escherichia coli alcohol dehydrogenase with the amino acid sequence of SEQ ID No. 31.

[0281] Example 3: Preparation of 1,3-propanediol using methanol as raw material using recombinant Bacillus engineered strain BM-G02

[0282] (1) Bacterial culture and enzyme induction

[0283] BM-G02 was inoculated at 1% inoculum into a shake flask containing 50 mL of growth medium containing 1% methanol, cultured at 50°C for 8 h, methanol was added to a final concentration of 2%, and cultured for another 16 h. The cells were then collected.

[0284] (2) Whole-cell catalysis of 1,3-propanediol

[0285] The collected cells were resuspended in a shake flask containing 20 mL of growth medium, methanol was added to a final concentration of 3%, and cultured at 50°C for 24 h. The supernatant was collected by centrifugation and filtered.

[0286] (3) Methanol and 1,3-propylene glycol content were determined using a gas chromatograph with a Shimadzu wax column (SHIMADZU InertCap WAX). The mobile phase was high-purity nitrogen at a flow rate of 500 mL / min, and the detector was a hydrogen flame ionization detector. Methanol (Jizhi Chemical, product number M14855) and 1,3-propylene glycol (Jizhi Chemical, product number P30040) were used as standards. The methanol and 1,3-propylene glycol contents were qualitatively analyzed based on the retention time of the standards and quantitatively analyzed using a standard curve method. The conversion rate was calculated using the ratio of 1,3-propylene glycol production to methanol consumption.

[0287] The bacterial culture and enzyme induction of BM-G01 and the whole-cell catalysis of 1,3-propanediol were carried out according to steps (1) and (2), with the only difference being that the strain BM-G02 was replaced by BM-G01.

[0288] The bacterial culture and enzyme induction of Bacillus YL23 and the whole-cell catalysis of 1,3-propanediol were carried out according to steps (1) and (2), with the only difference being that the strain BM-G02 was replaced by Bacillus YL23.

[0289] The results showed that strain BM-G02 produced 15.5 g / L of 1,3-propylene glycol, with a conversion rate of 53%. In contrast, strains BM-G01 and Bacillus YL23 produced no significant product. These results indicate that strain BM-G02, derived from the modified Bacillus YL23 strain, is capable of efficiently synthesizing 1,3-propylene glycol using methanol as the sole carbon source with high conversion rates.

[0290] Example 4: Construction of recombinant Escherichia coli engineered strain EC-G06

[0291] 4.1. Replacement of the endotoxin lpxM gene with the methanol dehydrogenase Bstmdh gene from Geobacillus stearothermophilus

[0292] The reference sequence number of the endotoxin lpxM gene is NC_000913.3 (1,939,222-1,940,193, complement) (09-MAR-2022), and the reference sequence number of the amino acid sequence encoded by it is NP_416369.1 (09-MAR-2022). The coding sequence of the methanol dehydrogenase gene from Geobacillus stearothermophilus, Bstmdh, is SEQ ID No. 12. The amino acid sequence of the methanol dehydrogenase from Geobacillus stearothermophilus encoded by it is SEQ ID No. 32.

[0293] The construction adopts the "λ-Red homologous recombination method", which includes four molecular biological operations: "(a) preparation of host bacteria, (b) preparation of targeting fragments, (c) homologous recombination, and (d) resistance elimination."

[0294] (a) Host Bacterial Preparation: The pKD46 plasmid (Clontech) was chemically transformed into the recipient Escherichia coli BW25113 to generate recombinant E. coli pKD46 / BW containing the plasmid pKD46. This recombinant bacterium was inoculated in LB medium containing 0.02% arabinose at a final concentration of 30°C to induce expression of the λ phage Red recombinant protein, conferring homologous recombination capability. The bacterium was then rendered electrocompetent, resulting in recombinant E. coli pKD46 / BW expressing the recombinase.

[0295] (b) Preparation of the targeting fragment lpxMTup-P119-Bstmdh-kan-lpxMdown: The lpxMTup-P119-Bmdh-kan-lpxMdown fragment consists of the upstream homology arm sequence of the knockout gene lpxM (SEQ ID No. 13), the P119 promoter (SEQ ID No. 9), the methanol dehydrogenase Bstmdh gene from Bacillus stearothermophilus (SEQ ID No. 12), the TrrnB terminator (SEQ ID No. 10), the kanamycin resistance gene with FRT flanks (FRT-kan-FRT, SEQ ID No. 11, wherein positions 15-48 of SEQ ID No. 11 are FRT sites, positions 409-1203 are kan resistance genes, and positions 1394-1427 are FRT sites), and the downstream homology arm sequence of the knockout gene lpxM (SEQ ID No. 14). This targeting fragment can be obtained by whole-genome synthesis and constructed on the plasmid pUC57, designated pUC-L-MDH. pUC-L-MDH is a plasmid created by replacing the fragment between the EcoRI and PstI restriction sites of the backbone vector pUC57 with the targeting fragment lpxMTup-P119-Bstmdh-kan-lpxMdown. PCR amplification using primers LPX-F / LPX-R and plasmid pUC-L-MDH as a template yielded the targeting fragment lpxMTup-P119-Bstmdh-kan-lpxMdown, which was then recovered by agarose gel electrophoresis.

[0296] (c) Homologous Recombination: The targeting fragment lpxMTup-P119-Bstmdh-kan-lpxMdown was directly electroporated into recombinant E. coli pKD46 / BW cells expressing the recombinase. Positive clones were screened on LB plates containing kanamycin (50 μg / mL). PCR verification was performed using the internal forward primer Bstmdh-IF for the insertion gene Bstmdh and the downstream verification primer lpxM-dR for the knockout gene lpxM. The target fragment of the positive clone was 2700 bp. The resulting positive single clone is designated EC-G01.

[0297] (d) Resistance Elimination: The obtained EC-G01 strain was streaked onto LB plates without antibiotics and cultured in a 42°C incubator to cause the loss of the temperature-sensitive plasmid pKD46. Single clones were then spotted onto LB plates and onto LB plates supplemented with ampicillin. Single clones that grew on LB but not on ampicillin-resistant plates were selected, demonstrating the loss of the pKD46 plasmid. The selected single clones were made competent for transfection and transformed with the plasmid pCP20 (purchased from Clontech) expressing the Flp recombinase using the calcium chloride method. After overnight culture at 30°C on LB plates containing ampicillin, clones were selected to obtain recombinant Escherichia coli pCP20 / EC-G01 containing the plasmid pCP20. After culturing overnight at 30°C in LB medium containing ampicillin resistance, the cells were streaked onto non-resistance LB plates and cultured overnight at 42°C. Single clones were spotted on LB and LB plates supplemented with ampicillin and kanamycin resistance, respectively. Monoclones that grew on LB but not on either ampicillin or kanamycin resistance plates were selected, demonstrating that they had eliminated the kanamycin resistance selection marker and the temperature-sensitive plasmid pCP20. This non-resistance monoclonal clone was named EC-G02.

[0298] Compared with Escherichia coli BW25113, the endotoxin lpxM gene in EC-G02 is replaced with the methanol dehydrogenase Bstmdh gene from Bacillus stearothermophilus, which can express methanol dehydrogenase from Bacillus stearothermophilus but not endotoxin lpxM.

[0299] 4.2. Replacement of the hydroxymethyl-glutathione dehydrogenase frmA gene with the 3-hexulose-6-phosphate synthase hps gene from Bacillus

[0300] The reference sequence number of the hydroxymethyl-glutathione dehydrogenase frmA gene is NC_000913.3 (378,462-379,571, complement) (09-MAR-2022), and the reference sequence number of the amino acid encoded by it is NP_414890.1 (09-MAR-2022).

[0301] The coding sequence of the hps gene of 3-hexulose-6-phosphate synthase derived from Bacillus is SEQ ID No. 15. The amino acid sequence of the 3-hexulose-6-phosphate synthase derived from Bacillus encoded by the hps gene is SEQ ID No. 33.

[0302] The construction employed the λ-Red homologous recombination method, including the following steps: (a) Preparation of the host strain: Chemically transforming the pKD46 plasmid (purchased from Clontech) into strain EC-G02 to generate recombinant Escherichia coli pKD46 / EC-G02 containing the plasmid pKD46, and then generating electrocompetent cells. (b) Preparation of the targeting fragment frmATup-P119-Bhps-kan-frmAdown: The targeting fragment frmATup-P119-Bhps-kan-frmAdown consists, from upstream to downstream, of the upstream homology arm sequence of the knockout gene frmA (SEQ ID No. 16), the P119 promoter, the Bacillus-derived 3-hexulose-6-phosphate synthase hps gene (SEQ ID No. 15), the TrrnB terminator, the FRT-flanked kanamycin resistance gene (FRT-kan-FRT), and the downstream homology arm sequence of the knockout gene frmA (SEQ ID No. 17). This fragment can be obtained by total gene synthesis and constructed on the plasmid pUC57, and the plasmid is named pUC-F-FRM. pUC-F-FRM is a plasmid obtained by replacing the fragment between the EcoRI and PstI restriction sites of the backbone vector pUC57 with the targeting fragment frmATup-P119-Bhps-kan-frmAdown. PCR amplification is performed using primers FRM-F / FRM-R and plasmid pUC-F-FRM as a template to obtain the targeting fragment. (c) Homologous recombination: The targeting fragment is electroporated into the recombinant Escherichia coli pKD46 / EC-G02. PCR verification is performed using the internal gene forward primer Bhps-IF and the downstream verification primer frmA-dR of the knockout gene frmA. The target fragment of the positive clone is 2300bp. The resulting positive single clone is represented by EC-G03. (d) Resistance elimination: pKD46 in the obtained EC-G03 was eliminated and the plasmid pCP20 was transferred to eliminate the kanamycin resistance gene. The obtained resistance-free monoclonal clone was named EC-G04.

[0303] Compared with EC-G02, the hydroxymethyl-glutathione dehydrogenase frmA gene in EC-G04 was replaced with the hps gene for 3-hexulose-6-phosphate synthase from Bacillus, which can express 3-hexulose-6-phosphate synthase from Bacillus but not hydroxymethyl-glutathione dehydrogenase.

[0304] 4.3. Replacement of the triosephosphate isomerase tpiA gene with the 6-phosphate-3-hexose isomerase phi gene from Bacillus

[0305] The reference sequence number of the triosephosphate isomerase tpiA gene is NC_000913.3 (4,110,740-4,111,507, complement) (09-MAR-2022), and the reference sequence number of the amino acid encoded by it is NP_418354.1 (09-MAR-2022).

[0306] The coding sequence of the Bacillus-derived 6-phosphate-3-hexose isomerase phi gene is SEQ ID No. 18. The amino acid sequence of the Bacillus-derived 6-phosphate-3-hexose isomerase encoded by the phi gene is SEQ ID No. 34.

[0307] The construction employed the λ-Red homologous recombination method, including: (a) host bacterial preparation: chemically transforming the pKD46 plasmid (purchased from Clontech) into strain EC-G04 to generate recombinant E. coli pKD46 / EC-G04 containing the plasmid pKD46, and then generating electrocompetent cells. (b) preparation of the targeting fragment tpiATup-P119-Bphi-kan-tpiAdown: the targeting fragment tpiATup-P119-Bphi-kan-tpiAdown consists, from upstream to downstream, of the upstream homology arm sequence of the knockout gene tpiA (SEQ ID No. 19), the P119 promoter, the Bacillus-derived 6-phosphate-3-hexose isomerase phi gene (SEQ ID No. 18), the TrrnB terminator, the FRT-flanked kanamycin resistance gene (FRT-kan-FRT), and the downstream homology arm sequence of the knockout gene tpiA (SEQ ID No. 20). This fragment can be obtained by means of total gene synthesis and constructed on the plasmid pUC57, and the plasmid is named pUC-T-PHI. pUC-T-PHI is a plasmid obtained by replacing the fragment between the EcoRI and PstI restriction sites of the backbone vector pUC57 with the targeting fragment tpiATup-P119-Bphi-kan-tpiAdown. PCR amplification is performed using primers TPI-F / TPI-R and plasmid pUC-T-PHI as a template to obtain the targeting fragment. (c) Homologous recombination: The targeting fragment is electroporated into the recombinant Escherichia coli pKD46 / EC-G04. PCR verification is performed using the gene-internal forward primer Bphi-IF and the downstream verification primer tpiA-dR of the knockout gene tpiA. The target fragment of the positive clone is 2200bp. The obtained positive single clone is represented by EC-G05. (d) Resistance elimination: pKD46 in the obtained EC-G05 was eliminated and the plasmid pCP20 was transferred to eliminate the kanamycin resistance gene. The obtained resistance-free monoclonal clone was named EC-G06.

[0308] Compared with EC-G04, the triosephosphate isomerase tpiA gene in EC-G06 was replaced with the 6-phosphate-3-hexose isomerase phi gene from Bacillus, which can express the 6-phosphate-3-hexose isomerase from Bacillus but not the triosephosphate isomerase.

[0309] Example 5: Construction of recombinant Escherichia coli engineered strain EC-G10

[0310] 5.1. Replacing the promoter of the transketolase gene tktA with the P119 promoter to enhance its activity

[0311] The reference sequence number of the transketolase gene tktA is NC_000913.3 (3,079,644-3,081,635, complement) (09-MAR-2022), and the reference sequence number of the amino acid encoded by it is YP_026188.1 (09-MAR-2022).

[0312] The nucleotide sequence of the P119 promoter is SEQ ID No.9.

[0313] The construction utilizes the λ-Red homologous recombination method, including: (a) host bacterial preparation: chemically transforming the pKD46 plasmid (purchased from Clontech) into strain EC-G06 to generate recombinant E. coli pKD46 / EC-G06 containing the plasmid pKD46, and then generating electrocompetent cells. (b) preparation of the targeting fragment tktATup-kan-P119-tktAdown: The targeting fragment tktATup-kan-P119-tktAdown consists, from upstream to downstream, of the upstream homology arm sequence of the knockout gene tktA promoter (SEQ ID No. 35), the FRT-flanked kanamycin resistance gene (FRT-kan-FRT), the P119 promoter, and the downstream homology arm sequence of the knockout gene tktA promoter (SEQ ID No. 36). This fragment can be obtained by total gene synthesis and constructed on the plasmid pUC57, which is designated pUC-T-TKT119. pUC-T-TKT119 is a plasmid obtained by replacing the fragment between the EcoRI and PstI restriction sites of the backbone vector pUC57 with the targeting fragment tktATup-kan-P119-tktAdown. PCR amplification was performed using primers TKT-F / TKT-R and plasmid pUC-T-TKT119 as a template to obtain the targeting fragment. (c) Homologous recombination: The targeting fragment was electroporated into the recombinant Escherichia coli pKD46 / EC-G06. PCR verification was performed using the P119 promoter forward primer 119-F and the downstream verification primer tktA-dR of the gene tktA. The target fragment of the positive clone was 500bp. The obtained positive monoclonal clone was represented by EC-G07. (d) Resistance elimination: pKD46 in the obtained EC-G07 was eliminated and transferred into the plasmid pCP20 to eliminate the kanamycin resistance gene. The obtained non-resistant monoclonal clone was named EC-G08.

[0314] Compared with EC-G06, the promoter of the transketolase gene in EC-G08 was replaced with the P119 promoter, and the expression of the transketolase gene tktA was enhanced.

[0315] 5.2. Replace the promoter of the ribulose-phosphate 3-epimerase gene rpe with the P119 promoter to enhance its activity

[0316] The reference sequence number of the ribulose-phosphate 3-epimerase gene rpe is NC_000913.3 (3,514,382-3,515,059, complement) (09-MAR-2022), and the reference sequence number of the amino acid encoded by it is NP_417845.1 (09-MAR-2022).

[0317] The nucleotide sequence of the P119 promoter is SEQ ID No.9.

[0318] The construction utilizes the "λ-Red homologous recombination method," including: (a) host bacterial preparation: chemically transforming the pKD46 plasmid (purchased from Clontech) into strain EC-G08 to generate recombinant E. coli pKD46 / EC-G08 containing the plasmid pKD46, and then generating electrocompetent cells. (b) preparation of the targeting fragment rpeup-kan-P119-rpedown: The targeting fragment rpeup-kan-P119-rpedown consists, from upstream to downstream, of the upstream homology arm sequence of the knockout gene rpe promoter (SEQ ID No. 37), the FRT-flanked kanamycin resistance gene (FRT-kan-FRT), the P119 promoter, and the downstream homology arm sequence of the knockout gene rpe promoter (SEQ ID No. 38). This fragment can be obtained by total gene synthesis and constructed on plasmid pUC57, designated pUC-T-RPE119. pUC-T-RPE119 is a plasmid obtained by replacing the fragment between the EcoRI and PstI restriction sites of the backbone vector pUC57 with the targeting fragment rpeup-kan-P119-rpedown. PCR amplification was performed using primers RPE-F / RPE-R and plasmid pUC-T-RPE119 as a template to obtain the targeting fragment. (c) Homologous recombination: The targeting fragment was electroporated into the recombinant Escherichia coli pKD46 / EC-G08. PCR verification was performed using the P119 promoter forward primer 119-F and the downstream verification primer rpe-dR of the gene rpe. The target fragment of the positive clone was 500bp. The resulting positive single clone was represented by EC-G09. (d) Resistance elimination: pKD46 in the obtained EC-G11 was eliminated and transferred into the plasmid pCP20 to eliminate the kanamycin resistance gene. The resulting non-resistant single clone was named EC-G10.

[0319] Compared with EC-G08, the promoter of the ribulose-phosphate 3-epimerase gene in EC-G10 is replaced with the P119 promoter, thereby enhancing the expression of the ribulose-phosphate 3-epimerase gene.

[0320] Example 6: Construction of recombinant Escherichia coli engineered strain EC-G12

[0321] 6.1. Replacement of the outer membrane protein ompT gene with the gene cluster GdrAB containing the two genes GdrA and GdrB, which are activators of the Klebsiella pneumoniae glycerol dehydratase

[0322] The reference sequence number of the outer membrane protein ompT gene is NC_000913.3 (584, 680-585, 633, complement) (09-MAR-2022), and the reference sequence number of the amino acid encoded by it is NP_415097.1 (09-MAR-2022).

[0323] The coding sequence of the gene cluster GdrAB of the two genes of glycerol dehydratase activator GdrA and GdrB is SEQ ID No. 7, and the encoded amino acid sequence of the glycerol dehydratase activator GdrA is SEQ ID No. 29 and the amino acid sequence of the glycerol dehydratase activator GdrB is SEQ ID No. 29.

[0324] The construction utilizes the λ-Red homologous recombination method, including: (a) host bacterial preparation: chemically transforming the pKD46 plasmid (purchased from Clontech) into strain EC-G10 to generate recombinant Escherichia coli pKD46 / EC-G10 containing the plasmid pKD46, and preparing electrocompetent cells. (b) preparation of the targeting fragment ompTup-P119-PgdrAB-kan-ompTdown: the targeting fragment ompTup-P119-PgdrAB-kan-ompTdown comprises, from upstream to downstream, the upstream homology arm sequence of the knockout gene ompT (SEQ ID No. 21), the P119 promoter, the Klebsiella pneumoniae glycerol dehydratase activator GdrAB gene cluster, the TrrnB terminator, the FRT-flanked kanamycin resistance gene (FRT-kan-FRT), and the downstream homology arm sequence of the knockout gene ompT (SEQ ID No. 22). This fragment can be obtained by total gene synthesis and constructed on the plasmid pUC57, which is named pUC-O-GDR. pUC-O-GDR is a plasmid obtained by replacing the fragment between the EcoRI and PstI restriction sites of the backbone vector pUC57 with the targeting fragment ompTup-P119-PgdrAB-kan-ompTdown. PCR amplification is performed using primers OMP-F / OMP-R and plasmid pUC-O-GDR as a template to obtain the targeting fragment. (c) Homologous recombination: The targeting fragment is electroporated into the recombinant Escherichia coli pKD46 / EC-G10. PCR verification is performed using the gene-internal forward primer Pgdr-IF and the downstream verification primer ompT-dR for the knockout gene ompT. The target fragment of the positive clone is 2900bp. The resulting positive single clone is represented by EC-G11. (d) Resistance elimination: pKD46 in the obtained EC-G07 was eliminated and the plasmid pCP20 was transferred to eliminate the kanamycin resistance gene. The obtained resistance-free monoclonal clone was named EC-G12.

[0325] Compared with EC-G10, the outer membrane protein ompT gene in EC-G12 is replaced by the gene cluster GdrAB of the two genes of Klebsiella pneumoniae glycerol dehydratase activator GdrA and GdrB. It can express the Klebsiella pneumoniae glycerol dehydratase activator GdrA and GdrB, but not the outer membrane protein ompT.

[0326] 6.2 Overexpression of GdrA and GdrB in wild-type BW25113 and EC-G06 strains

[0327] The procedure was identical to that of 6.1, except that E. coli pKD46 / EC-G10 was replaced with pKD46 / EC-BW and pKD46 / EC-G06. The resulting strains were EC-GA1, which integrates the resistance-conferring GdrAB genes based on BW25113, and EC-GA2, which eliminates resistance. The strains EC-GB1, which integrates the resistance-conferring GdrAB genes based on EC-G06, and EC-GB2, which eliminates resistance, were also obtained.

[0328] Example 7: Construction of recombinant Escherichia coli engineered strain EC-G15

[0329] 7.1 Construction of plasmid pYB1s-DY

[0330] Using primers RNB-F and BAD-R, plasmid pYB1s (SEQ ID No. 1) was used as a template to PCR amplify the plasmid backbone PY-E fragment, and the target fragment was recovered by agarose gel electrophoresis. In SEQ ID No. 1, positions 86-964 represent the araC gene, positions 1238-1266 represent the PBAD promoter sequence, positions 1295-1299 represent the RBS sequence, positions 1489-1646 represent the TrrnB terminator sequence, positions 1655-2445 represent the p15A replication initiation site sequence, and positions 2556-3344 represent the streptomycin resistance gene coding sequence.

[0331] Using the pUC57-DhaB123 plasmid mentioned in "Example 2: Construction of Recombinant Bacillus Engineered Strain BM-G02" as a template and primers EDHB-01F and EDHB-01R, PCR was performed to amplify the EDHB gene cluster encoding the three genes for the Klebsiella pneumoniae glycerol dehydratase enzymes DhaB1, DhaB2, and DhaB3. The target fragment was recovered by agarose gel electrophoresis. Using the pUC57-YQHD plasmid mentioned in "Example 2: Construction of Recombinant Bacillus Engineered Strain BM-G02" as a template and primers EYQH-01F and EYQH-01R, PCR was performed to amplify the EYQH fragment encoding the Escherichia coli alcohol dehydrogenase YqhD gene. The target fragment was recovered by agarose gel electrophoresis. The EDHB and EYQH fragments were ligated with the PY-E fragment using the Gibson assembly method. E. coli DH5α competent cells were transformed with the CaCl2 method. The transformation was carried out according to the section "Obtaining DNA Fragment PU-M" in Example 1. Clones were selected and clones were identified and sequenced using primers BAD-01F / EYQH-01R that could amplify the target fragment. The target fragment of the positive clone was 3900bp. Positive clones were selected for plasmid extraction and the positive plasmid was named pYB1s-DY. pYB1s-DY contains an expression cassette (DhaB123-YqhD expression cassette) for the gene cluster DhaB123 expressing the three genes of Klebsiella pneumoniae glycerol dehydratase DhaB1, DhaB2, and DhaB3, and the E. coli alcohol dehydrogenase gene YqhD. The amino acid sequences of the Klebsiella pneumoniae glycerol dehydratase DhaB1, DhaB2, and DhaB3, respectively, are expressed, and the E. coli alcohol dehydrogenase YqhD is expressed.

[0332] 7.2 Construction of plasmid pLB1a-DG

[0333] Using primers RNB-F and BAD-R, plasmid pLB1a (SEQ ID No. 2) was used as a template to PCR amplify the plasmid backbone PL-E fragment, and the target fragment was recovered by agarose gel electrophoresis. Sequence ID No. 2 contains the araC gene coding sequence at positions 86-964, the PBAD promoter sequence at positions 1238-1266, the RBS sequence at positions 1295-1300, the TrrnB terminator sequence at positions 1501-1658, the R6K replication origin oriR6k sequence at positions 1674-2060, the R6K replication origin pir gene coding sequence at positions 2150-3067, and the ampicillin resistance gene coding sequence at positions 3333-4193.

[0334] The pUC57-DAR1 plasmid mentioned in "Example 2: Construction of Recombinant Bacillus Engineered Strain BM-G02" was used as a template. PCR amplification of the EDAR fragment encoding the Saccharomyces cerevisiae dihydroxyacetone dehydrogenase DAR1 gene was performed using primers EDAR-01F and EDAR-01R. The target fragment was recovered by agarose gel electrophoresis. The pUC57-GPP1 plasmid mentioned in "Example 2: Construction of Recombinant Bacillus Engineered Strain BM-G02" was used as a template. PCR amplification of the EGPP fragment encoding the Saccharomyces cerevisiae 3-phosphoglycerophosphatase GPP1 gene was performed using primers EGPP-01F and EGPP-01R. The target fragment was recovered by agarose gel electrophoresis. The EDAR and EGGP fragments were ligated with the PL-E fragment using the Gibson assembly method. E. coli DH5α competent cells were transformed using the CaCl2 method, following the procedure described in Example 1, "Obtaining DNA Fragment PU-M." Clones were selected and identified using primers BAD-01F / EGGP-01R to amplify the target fragment. Positive clones were identified and sequenced, yielding a 2800-bp target fragment. Plasmids were extracted from selected positive clones, resulting in the plasmid pLB1a-DG. pLB1a-DG contains an expression cassette (DAR1-GPP1 expression cassette) for the Saccharomyces cerevisiae dihydroxyacetone dehydrogenase and Saccharomyces cerevisiae 3-phosphoglycerophosphatase genes. The amino acid sequences of the Saccharomyces cerevisiae dihydroxyacetone dehydrogenase DAR1 and Saccharomyces cerevisiae 3-phosphoglycerophosphatase GPP1 are SEQ ID Nos. 24 and 25, respectively.

[0335] Plasmids pYB1s-DY and pLB1a-DG were co-transformed into strain EC-GA2. Transformation was performed according to the "Obtaining DNA fragment PU-M" section of Example 1. A clone was selected on a kanamycin / streptomycin plate and named EC-G13.

[0336] The pYB1s-DY and pLB1a-DG plasmids were co-transformed into strain EC-GB2. Transformation was performed according to the "Obtaining DNA fragment PU-M" section of Example 1. A clone was selected on a kanamycin / streptomycin plate and named EC-G14.

[0337] Plasmids pYB1s-DY and pLB1a-DG were co-transformed into strain EC-G12. Transformation was performed according to the "Obtaining DNA Fragment PU-M" section of Example 1. Clones were selected on plates resistant to both kanamycin and ampicillin and designated EC-G15. Compared to EC-G12, EC-G15 also contains the gene cluster DhaB123, which contains the three genes for the Klebsiella pneumoniae glycerol dehydratase enzymes DhaB1, DhaB2, and DhaB3, and a gene for Escherichia coli alcohol dehydrogenase. These genes express the amino acid sequences of the Klebsiella pneumoniae glycerol dehydratase enzymes DhaB1, DhaB2, and DhaB3, respectively, with enhanced expression of the alcohol dehydrogenase.

[0338] Example 8: Preparation of 1,3-propanediol using recombinant Escherichia coli engineered strain EC-G15

[0339] 8.1. Autoinduction Culture

[0340] Streak strain EC-G15 onto LB plates containing 1.5% agar (mass percentage concentration) and 50 μg / mL kanamycin and 50 μg / mL ampicillin and incubate at 37°C for 16 hours. Select a single colony from the plate and inoculate it into liquid LB medium with the same resistance, incubating overnight at 37°C with shaking at 220 rpm. Inoculate a 1% inoculum volume of the overnight culture into autoinduction medium ZYM and incubate at 20°C with shaking at 220 rpm for 16 hours to obtain the induced bacterial suspension.

[0341] The formula for the autoinduction medium ZYM is: 100 mL A + 2 mL B + 2 mL C + 200 μL D + 100 μL E. (The following percentages are all mass percentages);

[0342] A.ZY: 1% tryptone, 0.5% yeast powder;

[0343] B. 50×M: 1.25M Na2HPO4, 1.25M KH2PO4, 2.5M NH4Cl and 0.25M Na2SO4;

[0344] C.50×5052: 25% glycerol, 2.5% glucose, 10% L-arabinose;

[0345] D.500×MgSO4:1M MgSO4

[0346] E.1000× trace elements: 50mM FeCl3, 20mM CaCl2, 10mM MnCl2, 10mM ZnSO4, 2mM each of CoCl2, NiCl2, Na2Mo4, Na2SeO3 and H3BO3.

[0347] 8.2 Whole-cell catalytic production of 1,3-propanediol

[0348] The induced bacterial solution was centrifuged at 8000 g for 10 min at 4°C, and the supernatant was discarded. The bacterial solution was washed twice with 0.85% saline, and the supernatant was discarded. The bacterial solution was resuspended in 10 mL of transformation solution to a final OD of 600nm The value was 15, and the reaction was carried out at 37°C and 200 rpm for 20 hours to obtain the transformed solution.

[0349] Conversion solution: 50 mM KH2PO4 / K2HPO4 buffer pH 7.0, 10 mM glucose, 0.03 g / L vitamin B12, initial methanol concentration is 1% (mass concentration).

[0350] The converted solution was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was removed and filtered through a 0.22 μm filter membrane. The methanol and 1,3-propylene glycol contents were determined using a gas chromatograph with a Shimadzu wax column (SHIMADZU InertCap WAX). The mobile phase was high-purity nitrogen at a flow rate of 500 mL / min, and the detector was a hydrogen flame ionization detector. Methanol (Jizhi Chemical, product number M14855) and 1,3-propylene glycol (Jizhi Chemical, product number P30040) were used as standards. The methanol and 1,3-propylene glycol contents were qualitatively analyzed based on the retention time of the standards and quantitatively analyzed using a standard curve method. The conversion rate was calculated as the ratio of 1,3-propylene glycol production to methanol consumption.

[0351] 8.3 Whole-cell catalysis of control bacteria

[0352] Refer to steps 8.1 and 8.2 to autoinduce BW25113, EC-G13, and EC-G14 and perform whole-cell catalysis to produce 1,3-propanediol.

[0353] Results showed that strain EC-G15 produced 4.6 g / L of 1,3-propylene glycol, while no significant 1,3-propylene glycol was detected in strains BW25113 and EC-G13. EC-G14 produced 0.85 g / L of 1,3-propylene glycol. The conversion rate was calculated by calculating the 1,3-propylene glycol yield relative to methanol digestion. The conversion rates for BW25113, EC-G13, EC-G14, and EC-G15 were 0%, 0%, 38.4%, and 46.2%, respectively. These results indicate that only recombinant E. coli strains incorporating all relevant target genes are capable of synthesizing 1,3-propylene glycol with high conversion rates using methanol as the sole carbon source.

[0354] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. A recombinant bacterium, characterized in that: The recombinant bacteria synthesizes 1,3-propylene glycol using methanol as a raw material.

2. The recombinant bacterium according to claim 1, characterized in that: The following metabolic pathways exist in the recombinant bacteria: (a) converting methanol into formaldehyde; (b) synthesizing D-arabino-hex-3-ulose 6-phosphate from formaldehyde and D-ribulose 5-phosphate; (c) converting D-arabino-hex-3-ketose 6-phosphate into fructose 6-phosphate; (d) converting fructose 6-phosphate into fructose 1,6-bisphosphate; (e) converting fructose 1,6-diphosphate into dihydroxyacetone phosphate; (f) converting dihydroxyacetone phosphate into glycerol triphosphate; (g) converting glycerol-3-phosphate into glycerol; (h) converting glycerol into 3-hydroxypropionaldehyde; (i) Conversion of 3-hydroxypropanal to 1,3-propanediol.

3. The recombinant bacterium according to claim 2, characterized in that: The (a) is catalyzed by methanol dehydrogenase, The (b) is catalyzed by 3-hexulose-6-phosphate synthase; The (c) is catalyzed by 6-phosphate-3-hexose isomerase; Said (d) is catalyzed by 6-phosphofructokinase; The (e) is catalyzed by fructose bisphosphate aldolase; The (f) is catalyzed by dihydroxyacetone phosphate reductase; The (g) is catalyzed by 3-phosphoglycerate phosphatase; The (h) is catalyzed by glycerol dehydratase; The step (i) is catalyzed by alcohol dehydrogenase or 1,3-propanediol oxidoreductase.

4. The recombinant bacterium according to any one of claims 1 to 3, characterized in that: The recombinant bacteria include the following characteristics: (1) containing a methanol dehydrogenase gene or / and a substance that regulates the expression of the methanol dehydrogenase gene or / and a substance that regulates the activity or content of methanol dehydrogenase; (2) containing a 3-hexulose-6-phosphate synthase gene or / and a substance that regulates the expression of the 3-hexulose-6-phosphate synthase gene or / and a substance that regulates the activity or content of 3-hexulose-6-phosphate synthase; (3) containing a 6-phosphate-3-hexose isomerase gene or / and a substance that regulates the expression of the 6-phosphate-3-hexose isomerase gene or / and a substance that regulates the activity or content of 6-phosphate-3-hexose isomerase; (4) containing a 6-phosphofructokinase gene or / and a substance that regulates the expression of the 6-phosphofructokinase gene or / and a substance that regulates the activity or content of 6-phosphofructokinase; (5) containing a fructose bisphosphate aldolase gene or / and a substance that regulates the expression of the fructose bisphosphate aldolase gene or / and a substance that regulates the activity or content of fructose bisphosphate aldolase; (6) containing a dihydroxyacetone phosphate reductase gene or / and a substance that regulates the expression of the dihydroxyacetone phosphate reductase gene or / and a substance that regulates the activity or content of dihydroxyacetone phosphate reductase; (7) containing a 3-phosphoglycerate phosphatase gene or / and a substance that regulates the expression of the 3-phosphoglycerate phosphatase gene or / and a substance that regulates the activity or content of 3-phosphoglycerate phosphatase; (8) containing a glycerol dehydratase gene or / and a substance that regulates the expression of the glycerol dehydratase gene or / and a substance that regulates the activity or content of the glycerol dehydratase; (9) containing a glycerol dehydratase activator gene or / and a substance that regulates the expression of the glycerol dehydratase activator gene or / and a substance that regulates the activity or content of the glycerol dehydratase activator; (10) Containing an alcohol dehydrogenase gene or a 1,3-propanediol oxidoreductase gene and / or a substance that regulates the expression of the alcohol dehydrogenase or 1,3-propanediol oxidoreductase gene and / or a substance that regulates the activity or content of the alcohol dehydrogenase or 1,3-propanediol oxidoreductase.

5. The recombinant bacterium according to any one of claims 1 to 4, characterized in that: The recombinant bacteria is a recombinant Bacillus, which is prepared by performing the following operations on the Bacillus as a recipient bacteria: A1) introducing a dihydroxyacetone phosphate reductase gene and / or a substance that regulates the expression of the dihydroxyacetone phosphate reductase gene and / or a substance that regulates the activity or content of the dihydroxyacetone phosphate reductase into the recipient bacterium; A2) introducing a 3-phosphoglycerate phosphatase gene and / or introducing a substance that regulates the expression of the 3-phosphoglycerate phosphatase gene and / or introducing a substance that regulates the activity or content of 3-phosphoglycerate phosphatase; A3) introducing a glycerol dehydratase gene and / or introducing a substance that regulates the expression of the glycerol dehydratase gene and / or introducing a substance that regulates the activity or content of the glycerol dehydratase; A4) introducing a dehydratase activator gene and / or introducing a substance that regulates the expression of the dehydratase activator gene and / or introducing a substance that regulates the activity or content of the dehydratase activator; A5) Introduction of an alcohol dehydrogenase gene and / or introduction of a substance that regulates alcohol dehydrogenase gene expression and / or introduction of a substance that regulates alcohol dehydrogenase activity or content.

6. The recombinant bacterium according to any one of claims 1 to 4, characterized in that: The recombinant bacteria is recombinant Escherichia coli, which is prepared by performing the following operations on Escherichia coli as a recipient bacteria: B1) introducing a methanol dehydrogenase gene and / or introducing a substance that regulates the expression of the methanol dehydrogenase gene and / or introducing a substance that regulates the activity or content of methanol dehydrogenase; B2) introducing a 3-hexulose-6-phosphate synthase gene and / or introducing a substance that regulates the expression of 3-hexulose-6-phosphate synthase and / or introducing a substance that regulates the activity or content of 3-hexulose-6-phosphate synthase; B3) knocking out the hydroxymethyl-glutathione dehydrogenase gene of the recipient bacteria; B4) introducing and / or introducing a substance that regulates the expression of the 6-phosphate-3-hexose isomerase gene and / or introducing a substance that regulates the activity or content of 6-phosphate-3-hexose isomerase; B5) knocking out the triose phosphate isomerase gene of the recipient bacterium; B6) introducing a dihydroxyacetone phosphate reductase gene and / or introducing a substance that regulates the expression of the dihydroxyacetone phosphate reductase gene and / or introducing a substance that regulates the activity or content of the dihydroxyacetone phosphate reductase; B7) introducing a 3-phosphoglycerate phosphatase gene and / or introducing a substance that regulates the expression of the 3-phosphoglycerate phosphatase gene and / or introducing a substance that regulates the activity or content of 3-phosphoglycerate phosphatase; B8) introducing a glycerol dehydratase gene and / or introducing a substance that regulates the expression of glycerol dehydratase and / or introducing a substance that regulates the activity or content of glycerol dehydratase; B9) introducing an alcohol dehydrogenase gene and / or introducing a substance that regulates alcohol dehydrogenase gene expression and / or introducing a substance that regulates alcohol dehydrogenase activity or content; B10) introducing a glycerol dehydratase activator gene and / or introducing a substance that regulates the expression of the glycerol dehydratase activator gene and / or introducing a substance that regulates the activity or content of the glycerol dehydratase activator.

7. The recombinant bacterium according to claim 6, characterized in that: The recombinant bacteria also include the following characteristics: (15) containing a promoter that enhances the expression of the transketolase gene or / and enhances the content and / or activity of the transketolase; (16) A promoter that enhances the expression of the ribulose phosphate epimerase gene or / and enhances the content and / or activity of the ribulose phosphate epimerase.

8. An application, characterized in that: The application is the application of the recombinant bacteria according to any one of claim 7 in the preparation of 1,3-propylene glycol.

9. A method for preparing 1,3-propylene glycol, characterized in that: The method comprises using the recombinant bacteria according to any one of claims 1 to 7 as a fermentation strain and methanol as a raw material to prepare 1,3-propylene glycol.

10. A method for constructing the recombinant bacterium according to any one of claims 1 to 7, comprising constructing a metabolic pathway in a recipient bacterium to obtain a recombinant bacterium that synthesizes 1,3-propanediol using methanol as a raw material, The metabolic pathways are (a)-(i): (a) converting methanol into formaldehyde; (b) synthesizing D-arabino-hex-3-ulose 6-phosphate from formaldehyde and D-ribulose 5-phosphate; (c) converting D-arabino-hex-3-ketose 6-phosphate into fructose 6-phosphate; (d) converting fructose 6-phosphate into fructose 1,6-bisphosphate; (e) converting fructose 1,6-diphosphate into dihydroxyacetone phosphate; (f) converting dihydroxyacetone phosphate into glycerol triphosphate; (g) converting glycerol-3-phosphate into glycerol; (h) converting glycerol into 3-hydroxypropionaldehyde; (i) Conversion of 3-hydroxypropanal to 1,3-propanediol.

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