Genetically engineered bacteria for producing 1,3-butanediol and application thereof

Abstract

The present application relates to a kind of 1,3-butanediol genetically engineered bacteria.The bacteria include the 5' end untranslated region (UTR) sequence that is artificially modified, which is the UTR sequence of the gene gltA encoding citrate synthase, and a constitutive promoter sequence is added before the artificially modified UTR sequence, which can enhance the accumulation of substrate acetyl-coa, reduce the acetyl-coa into the tricarboxylic acid cycle, and improve the yield of 1,3-butanediol.And by adding other genes to strengthen the synthesis of precursors, the chassis organism is further modified, and finally the obtained strain is a high-yield 1,3-butanediol genetically engineered bacteria.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a genetically engineered bacterium that produces 1,3-butanediol and its application, specifically to a genetically engineered bacterium that produces 1,3-butanediol and its application in the production of 1,3-butanediol. Background Technology

[0002] 1,3-Butanediol has the molecular formula C4H 10 O2, with a molecular weight of 90.121, is a tetracarbon diol. It can be used as a valuable pharmaceutical intermediate, such as in antibiotics and pheromones, and also as a food additive, such as in livestock feed. Furthermore, it is a commonly used moisturizer in cosmetics, possessing certain antibacterial properties and excellent biocompatibility. In summary, 1,3-Butanediol has significant application value in the pharmaceutical, food, and cosmetic fields.

[0003] Currently, 1,3-butanediol is mainly produced through chemical synthesis. However, chemical synthesis uses acetaldehyde as a raw material, resulting in numerous byproducts and environmental pollution.

[0004] Therefore, the current problem is that we need to research and develop a 1,3-butanediol biosynthesis technology that has a high conversion rate, good economic efficiency, and is easy to industrialize. Summary of the Invention

[0005] One of the objectives of this invention is to provide a genetically engineered bacterium that produces 1,3-butanediol, which is a high-yield 1,3-butanediol-producing bacterium. The production of 1,3-butanediol using this genetically engineered bacterium has a high conversion rate, good economic efficiency, and is easy to industrialize.

[0006] The second objective of this invention is to provide the application of the above-mentioned genetically engineered bacteria that produce 1,3-butanediol in the production of 1,3-butanediol.

[0007] Therefore, the first aspect of the present invention provides a genetically engineered bacterium that produces 1,3-butanediol.

[0008] According to some embodiments of the present invention, the genetically engineered bacterium producing 1,3-butanediol is a recombinant Escherichia coli containing a UTR sequence of the 5' untranslated region of the gene gltA encoding citrate synthase, which has been artificially modified.

[0009] According to the present invention, the artificial modification is to replace the UTR sequence of the 5' untranslated region of the gene gltA encoding citrate synthase, and to add a constitutive promoter sequence before the UTR sequence; preferably, the sequence of the gene gltA encoding citrate synthase is shown in SEQ ID NO. 36, and the UTR sequence is shown in SEQ ID NO. 1.

[0010] In this invention, the gene gltA encoding citrate synthase is an endogenous gene of Escherichia coli; preferably, the modification is a change in the genome of Escherichia coli; more preferably, the promoters used in the genetically engineered bacteria are all strong promoters.

[0011] According to other embodiments of the present invention, the genetically engineered bacteria are 1,3-butanediol-producing genetically engineered bacteria modified with chassis microorganisms; preferably, the chassis microorganism modification includes strengthening the 1,3-butanediol synthesis pathway, knocking out or weakening genes related to competitive metabolic pathways, and expressing cofactor metabolic regulatory genes.

[0012] In this invention, the enhanced 1,3-butanediol synthesis pathway includes overexpressing in recombinant *E. coli* a mutant gene Bld-L273T encoding heterologous acetyl-CoA thiolytic enzyme PhaA, a codon-optimized gene encoding heterologous acetyl-CoA dehydrogenase PhaB, a codon-optimized gene encoding butyraldehyde dehydrogenase Bld, and overexpressing in recombinant *E. coli* a codon-optimized endogenous gene encoding alcohol dehydrogenase yqhd.

[0013] In some embodiments of the present invention, the heterologous acetyl-CoA thiolytic enzyme gene PhaA is derived from hookworm copper-loving bacteria, and the sequence of the gene PhaA encoding heterologous acetyl-CoA thiolytic enzyme after codon optimization is shown in SEQ ID NO. 2.

[0014] In some embodiments of the present invention, the gene PhaB encoding heterologous acetyl-CoA dehydrogenase is derived from hookworm Copper-loving bacteria, and the sequence of the gene PhaB encoding heterologous acetyl-CoA dehydrogenase after codon optimization is shown in SEQ ID NO. 3.

[0015] In some embodiments of the present invention, the gene yqhd encoding alcohol dehydrogenase is derived from Escherichia coli, and the sequence of the gene yqhd encoding alcohol dehydrogenase after codon optimization is shown in SEQ ID NO. 4.

[0016] In some embodiments of the present invention, the gene encoding butyraldehyde dehydrogenase Bld is derived from Clostridium sucralose. The mutant gene Bld-L273T encoding butyraldehyde dehydrogenase Bld is a gene encoding butyraldehyde dehydrogenase Bld in which leucine at position 273 is mutated to threonine. The sequence of the mutant gene Bld-L273T encoding butyraldehyde dehydrogenase Bld after codon optimization is shown in SEQ ID NO. 5.

[0017] In this invention, the cofactor metabolic regulatory genes include one or more of the following: pntA, which encodes the α subunit of the cofactor regulatory protein NAD(P) transhydrogenase; pntB, which encodes the β subunit of the cofactor regulatory protein NAD(P) transhydrogenase; nadk, which encodes the NAD kinase; and fdh1, which encodes formate dehydrogenase.

[0018] In some embodiments of the present invention, the gene pntA encoding the α subunit of the cofactor regulatory protein NAD(P) transhydrogenase is derived from Escherichia coli, and its sequence is shown in SEQ ID NO. 6.

[0019] In some embodiments of the present invention, the gene pntB encoding the cofactor regulatory protein NAD(P) transhydrogenase β subunit is derived from Escherichia coli, and its sequence is shown in SEQ ID NO. 7.

[0020] In some embodiments of the present invention, the gene nadk encoding NAD kinase is derived from Escherichia coli, and its sequence is shown in SEQ ID NO. 12.

[0021] In some embodiments of the present invention, the gene fdh1, which heterologously encodes formate dehydrogenase, is derived from Saccharomyces cerevisiae, and its sequence is shown in SEQ ID NO. 8.

[0022] In this invention, the competitive metabolic pathway-related genes include tricarboxylic acid cycle-related genes; preferably, the tricarboxylic acid cycle-related genes include the gene gltA encoding citrate synthase; more preferably, the sequence of the gene gltA encoding citrate synthase is shown in SEQ ID NO. 36.

[0023] The second aspect of the present invention provides the application of the genetically engineered bacteria as described in the first aspect of the present invention in the production of 1,3-butanediol.

[0024] According to the present invention, the application includes inoculating a genetically engineered bacterium that produces 1,3-butanediol into a fermentation medium, carrying out fermentation culture, and then separating and purifying the obtained fermentation culture broth to obtain 1,3-butanediol.

[0025] In some embodiments of the present invention, the fermentation culture conditions are as follows: fermentation temperature is 30-37℃, fermentation culture time is 72h, and IPTG induction concentration is 0.05-1.2mM.

[0026] In some embodiments of the present invention, the separation and purification of the obtained fermentation broth includes:

[0027] Step S1: Centrifuge the fermentation broth to obtain the supernatant;

[0028] Step S2: Filter the supernatant using a 0.22 μm aqueous filter membrane to obtain 1,3-butanediol.

[0029] The inventors used *Escherichia coli* as the host cell and introduced genes encoding acetyl-CoA thiolase, acetyl-CoA dehydrogenase, butyraldehyde dehydrogenase, alcohol dehydrogenase, NAD(P) transhydrogenase α subunit, NAD(P) transhydrogenase β subunit, NAD kinase, and formate dehydrogenase. By knocking out or weakening genes related to competing metabolic pathways, the inventors modified the chassis microorganisms of *E. coli* to construct and obtain a genetically engineered bacterium that produces 1,3-butanediol. This bacterium is a high-yield 1,3-butanediol-producing genetically engineered bacterium. Using this genetically engineered bacterium to produce 1,3-butanediol results in high conversion rate, good economics, and ease of industrial production. Attached Figure Description

[0030] The present invention will now be described in further detail with reference to the accompanying drawings:

[0031] Figure 1 This demonstrates the reaction mechanism for the biosynthesis of 1,3-butanediol;

[0032] Figure 2 The graph shows the 1,3-butanediol yield after the original strain was introduced into the pathway plasmid.

[0033] Figure 3 The graph shows the 1,3-butanediol yield after the original strain was introduced with the pathway plasmid and cofactor plasmid.

[0034] Figure 4 This figure shows the yield of the gene gltA, which encodes citrate synthase, in different UTR sequences of the dominant pathway plasmid and the cofactor plasmid. Detailed Implementation

[0035] To facilitate understanding of the present invention, it will be described in detail below with reference to the accompanying drawings. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0036] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials similar to or equivalent to those described herein may also be used in the practice or testing of this invention, preferred methods and materials are now described.

[0037] I. Terminology

[0038] The term "UTR sequence" as used in this invention refers to a single mRNA strand containing multiple coding regions. The 5' end, 3' end, and the spaces between each coding region are untranslated regions, which have the function of regulating gene transcription levels and influencing polymerase binding capacity.

[0039] The term "chassis microorganisms," also known as "chassis microbial cells," as used in this invention refers to the use of microbial cells as a platform into which functionalized biological systems are placed, enabling these cells to possess the functions required by humans for biosynthesis. Just as a car needs a chassis for its foundation, upon which various vehicle bodies can be manufactured and functional components installed, chassis microbial cells need to have simplified functions themselves, but must possess the most basic self-replication and metabolic capabilities, thus becoming a blank platform on which functions can be continuously added.

[0040] In this invention, the term "genetically engineered bacteria" refers to bacteria into which a target gene is introduced into a host organism (i.e., host cell, chassis microorganism, or bacterial body) to express it, or to chassis microorganisms that are modified to produce the desired protein, including enhancing precursor synthesis pathways and knocking out or weakening genes related to competitive metabolic pathways. Examples include *Escherichia coli*. The core technology of genetic engineering is DNA recombination technology; therefore, in this invention, genetically engineered bacteria are also referred to as recombinant microorganisms.

[0041] The term "recombination" as used in this invention refers to the process of using the genetic material of a donor organism or artificially synthesized genes, cutting them in vitro or in vitro with restriction enzymes, and then linking them with a suitable vector to form recombinant DNA molecules. These recombinant DNA molecules are then introduced into recipient cells or recipient organisms to construct transgenic organisms, which can then exhibit certain traits of another organism according to a pre-designed blueprint.

[0042] The symbol “*” used in this invention represents a mutant gene.

[0043] II. Implementation Plan

[0044] To address the shortcomings of current biosynthetic methods for 1,3-butanediol, such as low conversion rates and poor economic efficiency, which hinder industrialization, this invention presents a comprehensive study of the biosynthetic process technology for 1,3-butanediol. To achieve the aforementioned goal of biosynthesizing 1,3-butanediol, the inventors used *Escherichia coli* as the host cell, replacing the artificially modified 5' untranslated region (UTR) sequence with the native UTR sequence encoding the citrate synthase gene *gltA*, and adding a constitutive promoter sequence before this modified UTR sequence to the recombinant host bacterium. Based on this host bacterium, by introducing genes encoding acetyl-CoA thiolase, acetyl-CoA dehydrogenase, butyraldehyde dehydrogenase, alcohol dehydrogenase, NAD(P) transhydrogenase α subunit, NAD(P) transhydrogenase β subunit, NAD kinase, and formate dehydrogenase, and by knocking out or weakening genes related to competing metabolic pathways, the chassis microorganisms of *E. coli* were modified, and a genetically engineered bacterium producing 1,3-butanediol was successfully constructed and obtained. The production of 1,3-butanediol using this genetically engineered bacterium exhibits high conversion rate, good economic efficiency, and is easily scalable for industrial production. This invention is thus derived.

[0045] Therefore, the first aspect of the present invention provides a novel route for the synthesis of 1,3-butanediol, which achieves efficient synthesis of 1,3-butanediol using glucose as a precursor through a genetically engineered bacterium that produces high yields of 1,3-butanediol.

[0046] To achieve the above technical solution, the present invention provides a genetically engineered bacterium capable of producing 1,3-butanediol.

[0047] According to some embodiments of the present invention, the genetically engineered bacterium producing 1,3-butanediol is a recombinant Escherichia coli containing a UTR sequence of the 5' untranslated region of the gene gltA encoding citrate synthase, which has been artificially modified.

[0048] According to the present invention, the artificial modification is to replace the UTR sequence of the 5' untranslated region of the gene gltA encoding citrate synthase, and to add a constitutive promoter sequence before the UTR sequence.

[0049] In this invention, the nucleotide sequence of the gene gltA (GenBank: AAA23892.1) encoding citrate synthase is shown in SEQ ID NO. 36, and the natural UTR sequence of the gene gltA (GenBank: AAA23892.1) encoding citrate synthase is shown in SEQ No. 37. The UTR sequence was obtained through screening, and its sequence is shown in SEQ ID NO. 1.

[0050] In some specific preferred embodiments of the present invention, the sequence of the constitutive promoter is shown in SEQ ID NO. 13.

[0051] In some further preferred embodiments of the present invention, a promoter sequence and the complete sequence of the gene gltA encoding citrate synthase, which replaces the selected UTR sequence, are added as shown in SEQ ID NO. 14.

[0052] The genetically engineered bacteria producing 1,3-butanediol in this invention are characterized by:

[0053] (1) The gene gltA encoding citrate synthase is an endogenous gene of Escherichia coli;

[0054] (2) The modifications made were changes to the genome of Escherichia coli.

[0055] (3) The promoters used in the genetically engineered bacteria are all strong promoters, including the tac promoter, T7 promoter, etc.

[0056] (4) The Escherichia coli includes Escherichia coli K12 or Escherichia coli JM109, etc.

[0057] In some specific embodiments of the present invention, an artificially modified 5' untranslated region (UTR) sequence is inserted into the natural UTR sequence of the gene gltA encoding citrate synthase in the host bacterium (Escherichia coli), and a constitutive promoter sequence is added before the artificially modified UTR sequence. By screening the UTR sequence, a strain capable of accumulating the precursor substrate acetyl-CoA is obtained, i.e., a strain that produces high levels of 1,3-butanediol.

[0058] According to other embodiments of the present invention, the genetically engineered bacteria are 1,3-butanediol-producing genetically engineered bacteria that have undergone chassis microbial modification, wherein the chassis microbial modification includes strengthening the 1,3-butanediol synthesis pathway, knocking out or weakening genes related to competitive metabolic pathways, and expressing cofactor metabolic regulatory genes.

[0059] In this invention, the enhancement of the precursor synthesis pathway includes overexpressing key genes of the precursor synthesis pathway in genetically engineered bacteria, which enhances the accumulation of precursor acetyl-CoA, thereby achieving efficient synthesis of 1,3-butanediol from simple carbon sources such as glucose or xylose.

[0060] In this invention, the enhanced 1,3-butanediol synthesis pathway includes overexpressing in recombinant *E. coli* a mutant gene Bld-L273T encoding heterologous acetyl-CoA thiolytic enzyme PhaA, a codon-optimized gene encoding heterologous acetyl-CoA dehydrogenase PhaB, a codon-optimized gene encoding butyraldehyde dehydrogenase Bld, and overexpressing in recombinant *E. coli* a codon-optimized endogenous gene encoding alcohol dehydrogenase yqhd.

[0061] Based on the above easy understanding, the genetically engineered bacteria that produce 1,3-butanediol involved in this invention are the mutant gene Bld-L273T that expresses the codon-optimized gene PhaA encoding heterologous acetyl-CoA thiolytic enzyme, the codon-optimized gene PhaB encoding heterologous acetyl-CoA dehydrogenase, the codon-optimized gene Bld encoding butyraldehyde dehydrogenase, and the recombinant host bacteria that overexpress the codon-optimized endogenous gene yqhd encoding alcohol dehydrogenase in recombinant Escherichia coli.

[0062] In some embodiments of the present invention, the heterologous acetyl-CoA thiolytic gene PhaA is derived from Cupriavidusnecator ATCC 17699.

[0063] The nucleotide sequence of the gene PhaA (GenBank: AAA21972.1) encoding acetyl-CoA thiolytic enzyme, derived from Cupriavidusnecator ATCC 17699, is shown in SEQ ID NO. 9.

[0064] The sequence of PhaA, the gene encoding heterologous acetyl-CoA thiolytic enzyme, after codon optimization, is shown in SEQ ID NO. 2.

[0065] In some embodiments of the present invention, the gene PhaB encoding heterologous acetyl-CoA dehydrogenase is derived from Cupriavidusnecator ATCC 17699. The nucleotide sequence of the gene PhaB (GenBank: AAA21973.1) encoding acetyl-CoA dehydrogenase derived from Cupriavidusnecator ATCC 17699 is shown in SEQ ID NO. 10; the sequence of the codon-optimized gene PhaB encoding heterologous acetyl-CoA dehydrogenase is shown in SEQ ID NO. 3.

[0066] In some embodiments of the present invention, the gene yqhd encoding alcohol dehydrogenase is derived from Escherichia coli (strain K12), and the sequence of the gene yqhd encoding alcohol dehydrogenase after codon optimization is shown in SEQ ID NO. 4.

[0067] In some embodiments of the present invention, the gene encoding butyraldehyde dehydrogenase Bld is derived from *Clostridium saccharoper butylacetonicum* N1-4 (HMT), and the nucleotide sequence of the gene encoding butyraldehyde dehydrogenase Bld (GenBank: AAP42563.1) derived from *Clostridium saccharoper butylacetonicum* is shown in SEQ ID NO. 11; the mutant gene Bld* encoding butyraldehyde dehydrogenase Bld is a gene encoding butyraldehyde dehydrogenase with a leucine residue at position 273 mutated to threonine, and the sequence of the mutant gene Bld* encoding butyraldehyde dehydrogenase Bld after codon optimization is shown in SEQ ID NO. 5.

[0068] In this invention, the cofactor metabolic regulatory genes include one or more of the following: pntA, which encodes the α subunit of the cofactor regulatory protein NAD(P) transhydrogenase; pntB, which encodes the β subunit of the cofactor regulatory protein NAD(P) transhydrogenase; nadk, which encodes the NAD kinase; and fdh1, which encodes formate dehydrogenase.

[0069] In some embodiments of the present invention, the gene pntA encoding the cofactor regulatory protein NAD(P) transhydrogenase α subunit is derived from Escherichia coli, and the nucleotide sequence of the gene pntA (GenBank: CAB37089.1) encoding the NAD(P) transhydrogenase α subunit derived from Escherichia coli is shown in SEQ ID NO. 6.

[0070] In some embodiments of the present invention, the gene pntB encoding the cofactor regulatory protein NAD(P) transhydrogenase β subunit is derived from Escherichia coli, and the nucleotide sequence of the gene pntB (GenBank: CAB37090.1) encoding the NAD(P) transhydrogenase β subunit derived from Escherichia coli is shown in SEQ ID NO. 7.

[0071] In some embodiments of the present invention, the gene nadk encoding NAD kinase is derived from Escherichia coli, and the nucleotide sequence of the gene nadk encoding NAD kinase derived from Escherichia coli (GenBank: AAA79785.1) is shown in SEQ ID NO. 12.

[0072] In some embodiments of the present invention, the heterologous gene fdh1 encoding formate dehydrogenase is derived from Saccharomyces cerevisiae, and the nucleotide sequence of the gene fdh1 (GenBank:CAA99720.1) encoding formate dehydrogenase derived from Saccharomyces cerevisiae is shown in SEQ ID NO. 8.

[0073] According to the present invention, chassis microorganisms are modified by knocking out or weakening genes related to competitive metabolic pathways, thereby regulating the direction of carbon metabolism and directing more metabolic flux toward the synthesis of 1,3-butanediol, thereby directing more substrates toward the synthesis of 1,3-butanediol, and thus obtaining Escherichia coli genetically engineered bacteria that produce high levels of 1,3-butanediol.

[0074] In this invention, the competitive metabolic pathway-related genes include tricarboxylic acid cycle-related genes; preferably, the tricarboxylic acid cycle-related genes include the gene gltA encoding citrate synthase; more preferably, the sequence of the gene gltA encoding citrate synthase is shown in SEQ ID NO. 36.

[0075] This invention uses *Escherichia coli* as the host cell and modifies the chassis microorganisms of *E. coli* by introducing genes encoding acetyl-CoA thiolysis, acetyl-CoA dehydrogenase, butyraldehyde dehydrogenase, alcohol dehydrogenase, NAD(P) transhydrogenase α subunit, NAD(P) transhydrogenase β subunit, NAD kinase and formate dehydrogenase, as well as genes related to enhancing precursor synthesis pathways and knocking out or weakening competitive metabolic pathways. This successfully constructed and obtained a genetically engineered bacterium with high 1,3-butanediol content.

[0076] In some specific embodiments of the present invention, genes related to enzymes in the 1,3-butanediol synthesis pathway are efficiently expressed in host bacteria (e.g., original or modified *Escherichia coli* K12, JM109, etc.). Preferably, the gene encoding acetyl-CoA thiolytic enzyme PhaA (GenBank: AAA21972.1) from *Cupriavidusnecator* ATCC 17699, after codon optimization, or the gene encoding acetyl-CoA dehydrogenase PhaB (GenBank: AAA21973.1) from *Cupriavidusnecator* ATCC 17699, after codon optimization, or the gene from *Clostridium saccharoperbutylacetonicum*, after expression, is used. The gene encoding butyraldehyde dehydrogenase Bld (GenBank: AAP42563.1) of N1-4(HMT) is a codon-optimized gene. In this implementation, the mutant Bld*, namely Bld-L273T, of the gene encoding butyraldehyde dehydrogenase Bld, is used. The gene yqhd (GenBank: AAA69178.1) encoding alcohol dehydrogenase is a codon-optimized gene derived from Escherichia coli (strain K12). The gene pntA (GenBank: CAB37089.1) encoding the α subunit of NAD(P) transhydrogenase is also a codon-optimized gene derived from Escherichia coli (strain K12). The gene pntB (GenBank: CAB37090.1) encoding the β subunit of NAD(P) transhydrogenase, derived from K12, is a codon-optimized gene; the gene nadk (GenBank: AAA79785.1) encoding NAD kinase, derived from Escherichia coli (strain K12), is a codon-optimized gene; and the gene fdh1 (Saccharomyces cerevisiae S288c) encoding formate dehydrogenase, derived from Saccharomyces cerevisiae S288c, is a codon-optimized gene.

[0077] In this invention, Escherichia coli, which produces high levels of 1,3-butanediol, is used as a host to produce 1,3-butanediol. By artificially modifying the 5' untranslated region of the gene gltA encoding citrate synthase, the accumulation of acetyl-CoA can be enhanced, the amount of acetyl-CoA entering the tricarboxylic acid cycle can be reduced, and the yield of 1,3-butanediol can be increased.

[0078] In this invention, there are no special requirements for the type of expression plasmid. Adjustments can be made according to the choice of host. It can be assumed that the construction method for expressing the target gene in Escherichia coli can adopt various methods commonly used in the field, such as ligating the target gene and expression vector after enzyme digestion. Further details will not be elaborated hereafter.

[0079] In some specific preferred embodiments, Escherichia coli strain Trans10 (purchased from Beijing TransGen Biotech Co., Ltd.) was used for vector construction, while Escherichia coli JM109(DE3) (Shanghai Weidi Biotechnology Co., Ltd.) was used as the fermentation strain.

[0080] In some examples, the UTR sequence of the gene gltA encoding citrate synthase was replaced by homologous recombination. Primers used to construct different UTR sequences are shown in Table 1, and the corresponding sequences are shown in SEQ No. 15-19.

[0081] Table 1. Primers for constructing recombinant UTR sequences

[0082]

[0083] Note: Underlined sequences are UTR sequences, italicized sequences are starter sequences, and bold sequences are selected sequences.

[0084] In some examples, for instance, genetically engineered bacteria can be constructed using genome alteration protocols, and / or using genes PhaA, PhaB, Bld*, yqhd, pntA, pntB, nadk, fdh1. The primers used for constructing recombinant plasmids are shown in Table 1, and the corresponding sequences are shown in SEQ Nos. 20-35.

[0085] Table 2. Primers for constructing recombinant plasmids (genes PhaA, PhaB, Bld*, yqhd, pntA, pntB, nadk, and fdh1)

[0086]

[0087]

[0088] Note: The underlined part is the ribosome binding site sequence.

[0089] The application of the genetically engineered bacteria described in the first aspect of the present invention in the production of 1,3-butanediol, as described in the second aspect of the present invention, can be understood as a method for producing 1,3-butanediol using the genetically engineered bacteria described in the first aspect of the present invention.

[0090] According to the present invention, the application includes inoculating a genetically engineered bacterium that produces 1,3-butanediol into a fermentation medium, carrying out fermentation culture, and then separating and purifying the obtained fermentation culture broth to obtain 1,3-butanediol.

[0091] In some embodiments of the present invention, the inoculation of the 1,3-butanediol-producing genetically engineered bacteria into the fermentation medium for fermentation culture includes: inoculating the 1,3-butanediol-producing genetically engineered bacteria into the fermentation medium, culturing at 30-37°C, preferably 30°C, at 200 rpm for 72 hours to obtain a fermentation culture broth; when the OD600 = 0.6, adding IPTG at a final concentration of 0.05-1.2 mM, preferably 0.1 mM, for induction.

[0092] In other embodiments of the present invention, the separation and purification of the obtained fermentation broth includes:

[0093] Step S1: Centrifuge the fermentation broth at 12000 rpm to obtain the supernatant;

[0094] Step S2: Filter the supernatant using a 0.22 μm aqueous filter membrane to obtain 1,3-butanediol.

[0095] The fermentation medium is not particularly limited in this invention, as long as it is a fermentation medium used for the production of 1,3-butanediol. Preferably, the fermentation medium is M9 medium, with the following formula: glucose 18g / L, yeast extract 3g / L, Na2PO4·7H2O 12.8g / L, KH2PO4 3.0g / L, NaCl 0.5g / L, NH4Cl 1.0g / L, MgSO4·7H2O 0.25g / L, CaCl2 0.11g / L, biotin 1mM, thiamine 1mM, and 10mL of trace element solution. The trace element formula is as follows: FeCl3·6H2O 0.83g / L, ZnCl2 84mg / L, CuCl2·2H2O 13mg / L, CoCl2·2H2O 10mg / L, H3BO4 10mg / L, MnCl2·4H2O 1.6mg / L.

[0096] III. Examples

[0097] The present invention will be specifically described below through specific embodiments. Unless otherwise specified, the experimental methods described below are standard laboratory methods. Unless otherwise specified, the experimental materials described below are commercially available.

[0098] Example 1: Replacement of UTR sequence

[0099] The primers used in this embodiment are shown in Table 1 above.

[0100] The UTR sequence of the gene gltA encoding citrate synthase was replaced using Red homologous recombination. Homologous recombination was performed by introducing the target fragment, during which the UTR sequence of gltA was replaced by a designed UTR sequence. The target fragment was the PCR product, designed using the F-terminal primers and universal R-terminal primers listed in Table 2.

[0101] Example 2: Construction of Recombinant Plasmids

[0102] The primers and ribosome binding sites used in this embodiment are shown in Table 2 above.

[0103] BGI Genomics commissioned the following gene sequences to be analyzed: PhaA (GenBank: AAA21972.1, codon-optimized nucleotide sequence shown in SEQ No. 2), encoding acetyl-CoA dehydrogenase, derived from *Cupriavidusnecator* ATCC 17699; PhaB (GenBank: AAA21973.1, codon-optimized nucleotide sequence shown in SEQ No. 3), encoding acetyl-CoA dehydrogenase, derived from *Cupriavidusnecator* ATCC 17699; and Bld (GenBank: AAA21973.1, codon-optimized nucleotide sequence shown in SEQ No. 3), encoding butyraldehyde dehydrogenase, derived from *Clostridium saccharoperbutylacetonicum* N1-4(HMT); after a specific sequence mutation (L273T), the codon-optimized nucleotide sequence is shown in SEQ No. 3. The entire genome was synthesized (as shown in No. 5) to obtain pUC57 plasmids (pUC57-PhaA, pUC57-PhaB, pUC57-Bld*) carrying the PhaA, PhaB, and Bld* genes. Using PhaA-F / PhaA-R, PhaB-F / PhaB-R, and Bld*-F / Bld*-R as primers, and pUC57-PhaA, pUC57-PhaB, and pUC57-Bld* as templates, the target genes PhaA, PhaB, and Bld* were amplified.

[0104] The primers and ribosome binding sites used in this embodiment are shown in Table 2 above. Except for the genes synthesized by BGI Genomics, the remaining genes were obtained by PCR amplification using the genome as a template.

[0105] The nucleotide sequence of the gene encoding alcohol dehydrogenase yqhd (GenBank: AAA69178.1) is shown in SEQ No. 4. The nucleotide sequence of the gene encoding the α subunit of NAD(P) transhydrogenase (GenBank: CAB37089.1) is shown in SEQ ID NO. 6. The nucleotide sequence of the gene encoding the β subunit of NAD(P) transhydrogenase (GenBank: CAB37090.1) is shown in SEQ ID NO. 7. The nucleotide sequence of the gene encoding NAD kinase (GenBank: AAA79785.1) is shown in SEQ ID NO. 12. The genes yqhd, pntA, pntB, and nadk are derived from *Escherichia coli* (strain K12).

[0106] The nucleotide sequence of the gene fdh1 (GenBank: CAA99720.1) encoding formate dehydrogenase is shown in SEQ ID NO. 8. The gene fdh1 encoding formate dehydrogenase is derived from Saccharomyces cerevisiae S288c.

[0107] Amplification was performed using the genome as a template, respectively, using yqhd-F / yqhd-R, pntA-F / pntA-R, pntB-F / pntB-R, nadk-F / nadk-R, and fdh1-F / fdh1-R.

[0108] All the amplified genes were first recovered and then ligated using Gibson ligase to connect the target genes to plasmids digested with restriction endonucleases. The genes PhaA, PhaB, Bld*, and yqhd were inserted into plasmid pCDF-tac to obtain plasmid pCDF-tbytp. The genes fdh1, nadk, pntA, and pntB were inserted into plasmid pACYC-Duet-1 to obtain plasmid pACYC-fnp.

[0109] Example 2: Preparation of recombinant strains

[0110] Competent cells of *E. coli* were prepared and aliquoted into 1.5 mL EP tubes (100 μL each) for electroporation. The constructed plasmid pCDF-tbytp (100-200 ng) was added to a 1.5 mL centrifuge tube containing 100 μL of competent cells, mixed thoroughly, and incubated on ice for 5-10 min. The plasmid was then electroporated into the competent cells. After electroporation, LB medium [10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, sterilized at 116 °C for 25 min. For the corresponding solid medium, 1.8%-2% agar was added] was rapidly added, and the mixture was transferred to 1.5 mL centrifuge tubes and incubated at 37 °C for 1-1.5 h. The bacterial culture was then plated onto plates containing the appropriate antibiotic and incubated at 37 °C for 12-16 h. This resulted in the preparation of strain VT01, which produces 1,3-butanediol (see Table 3).

[0111] Competent cells of *E. coli* were prepared and aliquoted into 100 μL 1.5 mL EP tubes for electroporation. 100-200 ng of the constructed plasmid pCDF-tbytp and pACYC-fnp were added to a 1.5 mL centrifuge tube containing 100 μL of competent cells, mixed thoroughly, and incubated on ice for 5-10 min. The plasmid was then electroporated into the competent cells using an electroporator. After electroporation, LB medium [10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, sterilized at 116 °C for 25 min. For the corresponding solid medium, 1.8%-2% agar was added] was quickly added, and the mixture was transferred to 1.5 mL centrifuge tubes and incubated at 37 °C for 1-1.5 h on a shaker. The bacterial culture was then plated onto plates containing the appropriate antibiotics and incubated at 37 °C for 12-16 h. This yielded strain VT02, which produces 1,3-butanediol (see Table 3).

[0112] Competent cells of a recombinant strain encoding the modified UTR sequence of the citrate synthase gene gltA were prepared and aliquoted into 100 μL of each cells into 1.5 mL EP tubes for electroporation. 100-200 ng of the constructed plasmid pCDF-tbytp and pACYC-fnp were added to a 1.5 mL centrifuge tube containing 100 μL of competent cells, mixed thoroughly, and incubated on ice for 5-10 min. The plasmid was then electroporated into the competent cells. After electroporation, LB medium [10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, sterilized at 116 °C for 25 min. For the corresponding solid medium, 1.8%-2% agar was added] was rapidly added, and the mixture was transferred to 1.5 mL centrifuge tubes and incubated at 37 °C for 1-1.5 h. The bacterial culture was then plated onto plates containing the appropriate antibiotics and incubated at 37 °C for 12-16 h. The strain VT03, which produces 1,3-butanediol, was prepared (see Table 3).

[0113] Table 3 Plasmids and strains

[0114]

[0115] In Table 3, pCDF-tac and pACYC-Duet-1 are laboratory preservation vectors.

[0116] Example 3: Fermentation of a strain producing 1,3-butanediol

[0117] (1) Shake flask culture of 1,3-butanediol-producing genetically engineered strain

[0118] Single colonies were picked from plates of strains VT01 / VT02 / VT03 that produce 1,3-butanediol and inoculated into 4 mL of resistant liquid LB. The culture was then incubated at 37°C for 12-16 h. The culture was then transferred to 30 mL of M9 medium (containing 18 g / L glucose, 3 g / L yeast extract, 12.8 g / L Na₂PO₄·7H₂O, 3.0 g / L KH₂PO₄, 0.5 g / L NaCl, 1.0 g / L NH₄Cl, 0.25 g / L MgSO₄·7H₂O, 0.11 g / L CaCl₂, 1 mM biotin, 1 mM thiamine, and 10 mL of trace element solution. The trace element solution was: 0.83 g / L FeCl₃·6H₂O, 84 mg / L ZnCl₂, and 0.25 mg / L CuCl₂·2H₂O). The mixture contained 13 mg / L of volatile organic compounds (VOCs), 10 mg / L of CoCl₂·2H₂O, 10 mg / L of H₃BO₄, and 1.6 mg / L of MnCl₂·4H₂O. Fermentation was carried out at 37°C and 200 rpm. The OD value was... 600 When the concentration of 1,3-butanediol reached approximately 0.6-0.8, IPTG was added to a final concentration of 0.1 mM for induction. The fermentation conditions were then changed to 37°C, 200 rpm, and cultured for 72 hours. Samples were taken and the concentration of 1,3-butanediol was determined using high-performance liquid chromatography. The final yield of strain cgN was as follows: Figure 2 and Figure 3 and Figure 4 As shown, the final yield of the strain is as follows Figure 4 As shown.

[0119] (2) Biomass determination

[0120] Dilute the fermentation broth with an appropriate amount of sterile distilled water to an OD value of [missing value]. 600 = Between 0.2 and 0.8, take 1 mL of the diluted fermentation broth into a cuvette and measure the absorbance at a wavelength of 600 nm using a spectrophotometer.

[0121] (3) Sample processing and testing

[0122] The fermentation broth was centrifuged at 4°C and 12,000 rpm for 10 min. The supernatant was filtered through a 0.22 μm aqueous membrane. The product was then analyzed qualitatively and quantitatively using high-performance liquid chromatography (HPLC).

[0123] It should be noted that the embodiments described above are only for explaining the present invention and do not constitute any limitation on the present invention. The present invention has been described with reference to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory terms, not limiting terms. Modifications can be made to the present invention within the scope of the claims, and revisions can be made to the present invention without departing from the scope and spirit of the present invention. Although the present invention described herein relates to specific methods, materials, and embodiments, it does not mean that the present invention is limited to the specific examples disclosed herein; on the contrary, the present invention can be extended to all other methods and applications with the same function. sequence list <110> Beijing University of Chemical Technology <120> A genetically engineered bacterium producing 1,3-butanediol and its applications <130> RB2102341-FF <160> 37 <170> SIPOSequenceListing 1.0 <210> 1 <211> 25 <212> DNA <213> (A man-designed UTR sequence encoding citrate synthase) <400> 1 tgctgatgct cctttgagct attgc 25 <210> 2 <211> 1182 <212> DNA <213> (PhaA gene encoding acetyl-CoA thiolase, optimized with codons) <400> 2 atgactgacg ttgttatttgt ttccgccgca cgtactgcgg taggtaagtt cggtggttct 60 ctggcgaaaa tcccagctcc ggaactgggc gcagtcgtca ttaaagctgc gctggaacgt 120 gcaggtgtaa aaccggagca ggtttctgag gtaatcatgg gccaggttct gaccgcgggt 180 tctggccaaa atcctgcacg tcaggctgcc atcaaagctg gcctgcctgc tatggtgcct 240 gcaatgacca tcaacaaggt atgcggttct ggcctgaaag cattaatgct ggctgcaaac 300 gctattatgg ctggtgacgc ggaaatcgtg gtcgctggtg gtcaggaaaa catgtctgct 360 gcacctcacg ttctgccggg ttctcgtgat ggcttccgta tgggcgacgc gaaactggtt 420 gatactatga tcgtcgacgg cctgtgggat gtttacaacc agtaccacat gggtattacc 480 540 ggctcccaaa acaaagcaga agctgcgcag aaagctggca aattcgatga agaatcgtg 600 ccggtactga tcccgcaacg caaaggtgac cctgtggcct tcaaaactga cgaattcgtg 660 cgtcagggcg ctaccctgga ctccatgtct ggtctgaaac cggcattcga taaagccggt 720 actgtgaccg cagcaaacgc tagcggtctg aacgacggcg cagcagctgt tgtcgttatg 780 tctgccgcca aagcgaaaga actgggtctg actccactgg ctacgatcaa gtcctacgct 840 aacgccggtg tcgatcctaa agtcatgggt atgggtccgg ttccggcctc taaacgtgcg 900 ctgagccgtg ctgaatggac cccacaggat ctggacctga tggaaattaa tgaagccttc 960 gctgcacagg ccctggctgt ccatcagcag atgggttggg ataccagcaa agtgaacgtt 1020 aacggtggtg ccatcgcaat tggccaccca atcggcgcgt ctggttgtcg tatcctggta 1080 accctgctgc acgaaatgaa acgtcgtgat gctaaaaaag gtctggcttc tctgtgtatc 1140 ggtggtggta tgggtgttgc actggccgta gagcgtaaat aa 1182 <210> 3 <211> 741 <212> DNA <213> (Codon-optimized gene PhaB encoding acetoacetyl-CoA dehydrogenase) <400> 3 atgacccagc gcattgcgta tgtgaccggc ggcatgggcg gtattggtac cgcgatttgc 60 caacgcctgg cgaaagatgg ctttcgcgtg gtggcgggct gtggccctaa tagccctcgt 120 cgtgaaaaat ggctggaaca gcagaaagcg ctgggctttg attttattgc gagcgaaggc 180 aacgtggcgg attgggatag caccaaaacc gcgtttgata aagtgaaaag cgaagtgggc 240 gaagtggatg tgctgattaa caacgcgggc attacccgcg atgtggtgtt tcgcaaaatg 300 acccgcgcgg attgggatgc ggtgattgat accaacctga ccagcctgtt taacgtgacc 360 aaacaggtga ttgatggcat ggcggatcgc ggctggggcc gtattgttaa cattagcagc 420 gtgaacggcc agaaaggcca gtttggccag accaactata gcaccgcgaa agcgggcctg 480 catggcttta ccatggcgct ggcgcaagaa gtggcgacca aaggcgttac cgtgaacacc 540 gtgagccctg gctatattgc gaccgatatg gtgaaagcga ttcgccagga tgtgctggat 600 aaaattgtgg cgaccattcc ggtgaaacgc ctgggcttac ctgaagaaat tgcgagcatt 660 tgcgcgtggc tgagcagcga agaaagcggc tttagcaccg gcgcggattt tagcctgaac 720 ggcggcttac atatgggcta a 741 <210> 4 <211> 1164 <212> DNA <213> (Gene yqhd encoding alcohol dehydrogenase optimized for codons) <400> 4 atgaacaact ttaatctgca caccccaacc cgcattctgt ttggtaaagg cgcaatcgct 609] ggtttacgcg aacaaattcc tcacgatgct cgcgtattga ttacctacgg cggcggcagc 120 gtgaaaaaaa ccggcgttct cgatcaagtt ctggatgccc tgaaaggcat ggacgtgctg 180 gaatttggcg gtattgagcc aaacccggct tatgaaacgc tgatgaacgc cgtgaaactg 240 gttcgcgaac agaaagtgac tttcctgctg gcggttggcg gcggttctgt actggacggc 300 accaaattta tcgccgcagc ggctaactat ccggaaaata tcgatccgtg gcacattctg 360 caaacgggcg gtaaagagat taaaagcgcc atcccgatgg gctgtgtgct gacgctgcca 420 gcaaccggtt cagaatccaa cgcaggcgcg gtgatctccc gtaaaaccac aggcgacaag 480 caggcgttcc attctgccca tgttcagccg gtatttgccg tgctcgatcc ggtttatacc 540 tacaccctgc cgccgcgtca ggtggctaac ggcgtagtgg acgcctttgt acacaccgtg 600 gaacagtatg ttaccaaacc ggttgatgcc aaaattcagg accgtttcgc agaaggcatt 660 ttgctgacgc taatcgaaga tggtccgaaa gccctgaaag agccagaaaa ctacgatgtg 720 cgcgccaacg tcatgtgggc ggcgactcag gcgctgaacg gtttgattgg cgctggcgta 780 ccgcaggact gggcaacgca tatgctgggc cacgaactga ctgcgatgca cggtctggat 840 cacgcgcaaa cactggctat cgtcctgcct gcactgtgga atgaaaaacg cgataccaag 900 cgcgctaagc tgctgcaata tgctgaacgc gtctggaaca tcactgaagg ttccgatgat 960 gagcgtattg acgccgcgat tgccgcaacc cgcaatttct ttgagcaatt aggcgtgccg 1020 acccacctct ccgactacgg tctggacggc agctccatcc cggctttgct gaaaaaactg 1080 gaagagcacg gcatgaccca actgggcgaa aatcatgaca ttacgttgga tgtcagccgc 1140 cgtatatacg aagccgcccg ctaa 1164 <210> 5 <211> 1408 <212> DNA <213> (Bld*, i.e., Bld-L273T, is a mutant gene of Bld that encodes butyraldehyde dehydrogenase after codon optimization.) <400> 5 atgattaaag ataccctggt tagcatcacg aaagacctga aactgaaaac taatgtggaa 60 aacgctaacc tgaaaaacta taaagatgat tccagctgtt tcggcgtttt cgagaacgtt 120 gaaaacgcga tcagcaatgc agtgcacgcc cagaaaatcc tgtctctgca ctacacgaag 180 gaacaacgtg aaaaaatcat caccgaaatc cgtaaagctg ctctggaaaa taaagaaatc 240 ctggccacta tgatcctgga ggaaacccat atgggtcgct acgaagataa aattctgaaa 300 cacgaactgg ttgccaaata cactccgggc acggaagatc tgacgaccac tgcgtggtct 360 ggtgataacg gtctgaccgt agttgaaatg agcccgtacg gcgtaattgg cgctatcact 420 ccgagcacca accctacgga aaccgttatc tgcaacagca tcggcatgat cgctgcgggt 480 aacaccgtgg tgttcaacgg tcacccgggc gctaaaaaat gtgtggcgtt cgcagttgaa 540 atgatcaaca aggcgatcat ctcctgtggc ggtccggaaa acctggtaac cactattaag 600 aacccaacca tggactccct ggacgctatc atcaaacacc cgtccattaa actgctgtgt 660 ggtactggcg gcccaggtat ggtgaaaact ctgctgaact ccggtaaaaa agcgattggc 720 gctggtgcag gtaaccctcc ggtaattgtt gacgataccg cagacattga aaaggctggt 780 aagtctatca tcgagggttg ttccttcgat aataataccc cgtgcatcgc tgaaaaggaa 840 gtatttgtat tcgaaaacgt cgctgacgac ctgatctcca acatgctgaa aaacaacgct 900 gtaatcatta acgaagatca agttagcaaa ctgatcgatc tggtgctgca aaaaaacaac 960 gaaacccagg agtactccat tataaaaaa tgggtaggta aagatgcgaa actgttcctg 1020 gacgaaattg acgtcgaatc tccttcttcc gtaaaatgta ttatctgtga agtttctgcc 1080 cgccacccat tcgttatgac tgaactgatg atgccgatcc tgccaatcgt gcgtgtaaaa 1140 gacatcgatg aagcaattga atacgctaaa atcgctgaac agaaccgtaa acattctgcg 1200 tacatttaca gcaaaaacat cgacaacctg aatcgttttg aacgtgaaat cgacactacc 1260 attttcgtta aaaacgctaa atcctttgcg ggcgttggct acgaggcaga gggttttacc 1320 actttcacga tcgcaggtag caccggcgaa ggtattactt ctgcacgtaa cttcactcgt 1380 cagcgccgct gcgtactggc tggttaaa 1408 <210> 6 <211> 1533 <212> DNA <213> (Gene pntA encoding the α subunit of NAD(P) transhydrogenase) <400> 6 atgcgaattg gcataccaag agaacggtta accaatgaaa cccgtgttgc agcaacgcca 60 aaaacagtgg aacagctgct gaaactgggt tttaccgtcg cggtagagag cggcgcgggt 120 caactggcaa gttttgacga taaagcgttt gtgcaagcgg gcgctgaaat tgtagaaggg 180 aatagcgtct ggcagtcaga gatcattctg aaggtcaatg cgccgttaga tgatgaaatt 240 gcgttactga atcctgggac aacgctggtg agttttatct ggcctgcgca gaatccggaa 300 ttaatgcaaa aacttgcgga acgtaacgtg accgtgatgg cgatggactc tgtgccgcgt 360 atctcacgcg cacaatcgct ggacgcacta agctcgatgg cgaacatcgc cggttatcgc 420 gccattgttg aagcggcaca tgaatttggg cgcttcttta ccgggcaaat tactgcggcc 480 gggaaagtgc caccggcaaa agtgatggtg attggtgcgg gtgttgcagg tctggccgcc 540 attggcgcag caaacagtct cggcgcgatt gtgcgtgcat tcgacacccg cccggaagtg 600 aaagaacaag ttcaaagtat gggcgcggaa ttcctcgagc tggattttaa agaggaagct 660 ggcagcggcg atggctatgc caaagtgatg tcggacgcgt tcatcaaagc ggaaatggaa 720 ctctttgccg cccaggcaaa agaggtcgat atcattgtca ccaccgcgct tattccaggc 780 aaaccagcgc cgaagctaat tacccgtgaa atggttgact ccatgaaggc gggcagtgtg 840 attgtcgacc tggcagccca aaacggcggc aactgtgaat acaccgtgcc gggtgaaatc 900 ttcactacgg aaaatggtgt caaagtgatt ggttataccg atcttccggg ccgtctgccg 960 acgcaatcct cacagcttta cggcacaaac ctcgttaatc tgctgaaact gttgtgcaaa 1020 gagaaagacg gcaatatcac tgttgatttt gatgatgtgg tgattcgcgg cgtgaccgtg 1080 atccgtgcgg gcgaaattac ctggccggca ccgccgattc aggtatcagc tcagccgcag 1140 gcggcacaaa aagcggcacc ggaagtgaaa actgaggaaa aatgtacctg ctcaccgtgg 1200 cgtaaatacg cgttgatggc gctggcaatc attctttttg gctggatggc aagcgttgcg 1260 ccgaaagaat tccttgggca cttcaccgtt ttcgcgctga cctgcgttgt cggttattac 1320 gtggtgtgga atgtatcgca cgcgctgcat acaccgttga tgtcggtcac caacgcgatt 1380 tcagggatta ttgttgtcgg agcactgttg cagattggcc agggcggctg ggttagcttc 1440 cttagtttta tcgcggtgct tatagccagc attaatattt tcggtggctt caccgtgact 1500 cagcgcatgc tgaaaatgtt ccgcaaaaat taa 1533 <210> 7 <211> 1389 <212> DNA <213> (Gene pntB encoding the β subunit of NAD(P) transhydrogenase) <400> 7 atgtctggag gattagttac agctgcatac attgttgccg cgatcctgtt tatcttcagt 60 ctggccggtc tttcgaaaca tgaaacgtct cgccagggta acaacttcgg tatcgccggg 120 atggcgattg cgttaatcgc aaccattttt ggaccggata cgggtaatgt tggctggatc 180 ttgctggcga tggtcattgg tggggcaatt ggtatccgtc tggcgaagaa agttgaaatg 240 accgaaatgc cagaactggt ggcgatcctg catagcttcg tgggtctggc ggcagtgctg 300 gttggcttta acagctatct gcatcatgac gcgggaatgg caccgattct ggtcaatatt 360 cacctgacgg aagtgttcct cggtatcttc atcggggcgg taacgttcac gggttcggtg 420 gtggcgttcg gcaaactgtg tggcaagatt tcgtctaaac cattgatgct gccaaaccgt 480 cacaaaatga acctggcggc tctggtcgtt tccttcctgc tgctgattgt atttgttcgc 540 acggacagcg tcggcctgca agtgctggca ttgctgataa tgaccgcaat tgcgctggta 600 ttcggctggc atttagtcgc ctccatcggt ggtgcagata tgccagtggt ggtgtcgatg 660 ctgaactcgt actccggctg ggcggctgcg gctgcgggct ttatgctcag caacgacctg 720 ctgattgtga ccggtgcgct ggtcggttct tcgggggcta tcctttctta cattatgtgt 780 aaggcgatga accgttcctt tatcagcgtt attgcgggtg gtttcggcac cgacggctct 840 tctactggcg atgatcagga agtgggtgag caccgcgaaa tcaccgcaga agagacagcg 900 gaactgctga aaaactccca ttcagtgatc attactccgg ggtacggcat ggcagtcgcg 960 caggcgcaat atcctgtcgc tgaaattact gagaaattgc gcgctcgtgg tattaatgtg 1020 cgtttcggta tccacccggt cgcggggcgt ttgcctggac atatgaacgt attgctggct 1080 gaagcaaaag taccgtatga catcgtgctg gaaatggacg agatcaatga tgactttgct 1140 gataccgata ccgtactggt gattggtgct aacgatacgg ttaacccggc ggcgcaggat 1200 gatccgaaga gtccgattgc tggtatgcct gtgctggaag tgtggaaagc gcagaacgtg 1260 attgtcttta aacgttcgat gaacactggc tatgctggtg tgcaaaaccc gctgttcttc 1320 aaggaaaaca cccacatgct gtttggtgac gccaaagcca gcgtggatgc aatcctgaaa 1380 gctctgtaa 1389 <210> 8 <211> {{1131}} <212> DNA <213> (Gene fhd1 encoding formate dehydrogenase) <400> 8 atgtcgaagg gaaaggtttt gctggttctt tacgaaggtg gtaagcatgc tgaagagcag 60 gaaaagttat tggggtgtat tgaaaatgaa cttggtatca gaaatttcat tgaagaacag 120 ggatacgagt tggttactac cattgacaag gaccctgagc caacctcaac ggtagacagg 180 gagttgaaag acgctgaaat tgtcattact acgccctttt tccccgccta catctcgaga 240 aacaggattg cagaagctcc taacctgaag ctctgtgtaa ccgctggcgt cggttcagac 300 catgtcgatt tagaagctgc aaatgaacgg aaaatcacgg tcaccgaagt tactggttct 360 aacgtcgttt ctgtcgcaga gcacgttatg gccacaattt tggttttgat aagaaactat 420 aatggtggtc atcaacaagc aattaatggt gagtgggata ttgccggcgt ggctaaaaat 480 gagtatgatc tggaagacaa aataatttca acggtaggtg ccggtagaat tggatatagg 540 gttctggaaa gattggtcgc attaatccg aagaagttac tgtactacga ctaccaggaa 600 ctacctgcgg aagcaatcaa tagattgaac gaggccagca agcttttcaa tggcagaggt 660 gatattgttc agagagtaga gaaattggag gatatggttg ctcagtcaga tgttgttacc 720 atcaactgtc cattgcacaa ggactcaagg ggtttattca ataaaaagct tatttcccac 780 atgaaagatg gtgcatactt ggtgaatacc gctagaggtg ctatttgtgt cgcagaagat 840 gttgccgagg cagtcaagtc tggtaaattg gctggctatg gtggtgatgt ctgggataag 900 caaccagcac caaaagacca tccctggagg actatggaca ataaggacca cgtgggaaac 960 gcaatgactg ttcatatcag tggcacatct ctggatgctc aaaagaggta cgctcaggga 1020 gtaaagaaca tcctaaatag ttacttttcc aaaaagtttg attaccgtcc acaggatatt 1080 attgtgcaga atggttctta tgccaccaga gcttatggac agaagaaata a 1131 <210> 9 <211> 1182 <212> DNA <213> (Gene PhaA encoding acetoacetyl-CoA thiolase) <400> 9 atgactgacg ttgtcatcgt atccgccgcc cgcaccgcgg tcggcaagtt tggcggctcg 60 ctggccaaga tcccggcacc ggaactgggt gccgtggtca tcaaggccgc gctggagcgc 120 gccggcgtca agccggagca ggtgagcgaa gtcatcatgg gccaggtgct gaccgccggt 180 tcgggccaga accccgcacg ccaggccgcg atcaaggccg gcctgccggc gatggtgccg 240 gccatgacca tcaacaaggt gtgcggctcg ggcctgaagg ccgtgatgct ggccgccaac 300 gcgatcatgg cgggcgacgc cgagatcgtg gtggccggcg gccaggaaaa catgagcgcc 360 gccccgcacg tgctgccggg ctcgcgcgat ggtttccgca tgggcgatgc caagctggtc 420 gacaccatga tcgtcgacgg cctgtgggac gtgtacaacc agtaccacat gggcatcacc 480 gccgagaacg tggccaagga atacggcatc acacgcgagg cgcaggatga gttcgccgtc 540 ggctcgcaga acaaggccga agccgcgcag aaggccggca agtttgacga agagatcgtc 600 ccggtgctga tcccgcagcg caagggcgac ccggtggcct tcaagaccga cgagttcgtg 660 cgccagggcg ccacgctgga cagcatgtcc ggcctcaagc ccgccttcga caaggccggc 720 acggtgaccg cggccaacgc ctcgggcctg aacgacggcg ccgccgcggt ggtggtgatg 780 tcggcggcca aggccaagga actgggcctg accccgctgg ccacgatcaa gagctatgcc 840 aacgccggtg tcgatcccaa ggtgatgggc atgggcccgg tgccggcctc caagcgcgcc 900 ctgtcgcgcg ccgagtggac cccgcaagac ctggacctga tggagatcaa cgaggccttt 960 gccgcgcagg cgctggcggt gcaccagcag atgggctggg acacctccaa ggtcaatgtg 1020 aacggcggcg ccatcgccat cggccacccg atcggcgcgt cgggctgccg tatcctggtg 1080 acgctgctgc acgagatgaa gcgccgtgac gcgaagaagg gcctggcctc gctgtgcatc 1140 ggcggcggca tgggcgtggc gctggcagtc gagcgcaaat aa 1182 <210> 10 <211> 741 <212> DNA <213> (Gene PhaB encoding acetoacetyl-CoA dehydrogenase) <400> 10 atgactcagc gcattgcgta tgtgaccggc ggcatgggtg gtatcggaac cgccatttgc 60 cagcggctgg ccaaggatgg ctttcgtgtg gtggccggtt gcggccccaa ctcgccgcgc 120 cgcgaaaagt ggctggagca gcagaaggcc ctgggcttcg atttcattgc ctcggaaggc 180 aatgtggctg actgggactc gaccaagacc gcattcgaca aggtcaagtc cgaggtcggc 240 gaggttgatg tgctgatcaa caacgccggt atcacccgcg acgtggtgtt ccgcaagatg 300 acccgcgccg actgggatgc ggtgatcgac accaacctga cctcgctgtt caacgtcacc 360 aagcaggtga tcgacggcat ggccgaccgt ggctggggcc gcatcgtcaa catctcgtcg 420 gtgaacgggc agaagggcca gttcggccag accaactact ccaccgccaa ggccggcctg 480 catggcttca ccatggcact ggcgcaggaa gtggcgacca agggcgtgac cgtcaacacg 540 gtctctccgg gctatatcgc caccgacatg gtcaaggcga tccgccagga cgtgctcgac 600 aagatcgtcg cgacgatccc ggtcaagcgc ctgggcctgc cggaagagat cgcctcgatc 660 tgcgcctggt tgtcgcgga ggagtccggt ttctcgaccg gcgccgactt ctcgctcaac 720 ggcggcctgc atatgggctg a 741 <210> 11 <211> 1407 <212> DNA <213> (Bld <400> 11 atgattaaag acacgctagt ttctataaca aaagatttaa attaaaaac aaatgttgaa 60 aatgccaatc taaagaacta caaggatgat tcttcatgtt tcggagtttt cgaaaatgtt 120 gaaaatgcta taagcaatgc cgtacacgca caaaagatat tatcccttca ttatacaaaa 180 gaacaaagag aaaaaatcat aactgagata agaaaggccg cattagaaaa taaagagatt 240 ctagctacaa tgattcttga agaacacat atgggagat atgagaata atattaag 300 catgaattag tagctaata cactcctggg acagagatt taactac tgcttggtca 360 ggagataacg gcttacagt tgtagaatg tctccatatg gcgttatagg tgcaatact 420 ccttctacga atccactga aactgtaata tgtaatagta taggcatgat agctgctgga 480 atactgtgg tattaacgg acatccaggc gctaaaaaat gtgttgcttt tgctgtcgaa 540 atgataaata aagcttat ttcatgtggt ggtcctgaga atttagtaac aacttaaaa 600 aatccaacta tggactct agatgcaatt atttaagcacc cttcataaa actactttgc 660 ggaactggag ggccaggaat ggtaaaaacc ctcttaaatt ctggtaagaa agctataggt 720 gctggtgctg gaaatccacc agttattgta gatgatactg ctgatataga aaggctggt 780 aagagtatca ttgaggctg ttctttgat ataatttac cttgtattgc agaaaaagaa 840 gtatttgttt ttgagaacgt tgcagatgat ttaatatcta acatgctaaa aaataatgct 900 gttattaata atgagatca agtatcaag ttatagatt tgttattaca aaaaataat 960 gaactcaag atactctat aaataaaa tgggtcggaa aagatgcaaa attattctta 1020 gatgaaatag atgttgagtc tccttcaagt gttaaatgca taatctgcga agtaagtgca 1080 aggcatccat ttgttatgac agaactcatg atgccaatt taccaattgt aagagttaaa 1140 gatatagag aagctattga atatgcaaa atagcagaac aaatagaaa acatagtgcc 1200 tattattt caaaaatat agacaaccta ataggtttg aagagaat cgatactact 1260 atctttgtaa agaatgctaa atctttgcc ggtgttggtt atgagcaga aggctttaca 1320 actttcacta ttgctggatc cactggtgaa ggaataactt ctgcaagaaa ttttacaga 1380 chaaacht gtgtactcgc cggttaa 1407 <210> 12 <211> 879 <212> DNA <213> (NAD is not nadk) <400> 12 atgaataatc atttcaagtg tattggcatt gtgggacacc cacggcaccc cactgcactg 60 acacacatg aaatgctcta ccgctggctg tgcacaaag gttacgaggt catcgttgag 120 caacaatcg ctcacgaact gcaacgaag atgtgaaa ctggcacgct cgcggagatt 180 gggcaactag ctgatctcgc ggtagtcgtt ggtggcgacg gtaatatgct gggcgcggca 240 cgcacactcg cccgttacga tattaaagtt attggaatca accgtggcaa cctgggtttc 300 ctgactgacc ttgaccccga taacgcccag caacagttag ccgatgtgct ggaaggccac 360 tacatcagcg agaaacgttt tttgctggaa gcgcaagtct gtcagcaaga ttgccagaaa 420 cgcatcagca ccgcgataaa tgaagtggtg cttcatccag gcaaagtggc gcatatgatt 480 gagttcgaag tgtatatcga cgagatcttt gcgttttctc agcgatctga tggactaatt 540 atttcgacgc caacaggctc caccgcctat tccctctctg caggcggtcc tattctgacc 600 ccctctctgg atgcgattac cctggtgccc atgttcccgc atacgttgtc agcacgacca 660 ctggtcataa acagcagcag cacgatccgt ctgcgttttt cgcatcgccg taacgacctg 720 gaaatcagtt gcgacagcca gatagcactg ccgattcagg aaggtgaaga tgtcctgatt 780 cgtcgctgtg attaccatct gaatctgatt catccgaaag attacagtta tttcaacaca 840 ttaagcacca agctcggctg gtcaaaaaaa ttattctaa 879 <210> 13 <211> 35 <212> DNA <213> (Promoter sequence of the UTR sequence of the artificially encoded citrate synthase gene) <400> 13 ctgacagcta gctcagtcct aggtataatg ctagc 35 <210> 14 <211> 1344 <212> DNA <213> (Promoter sequence, UTR sequence, and gltA gene, which is artificially encoded citrate synthase) <400> 14 ctgacagcta gctcagtcct aggtataatg ctagctgctg atgctccttt gagctattgc 60 atggctgata caaaagcaaa actcaccctc aacggggata cagctgttga actggatgtg 120 ctgaaaggca cgctgggtca agatgttat gatatccgta ctctcggttc aaaaggtgtg 180 ttcacctttg acccaggctt cacttcaacc gcatcctgcg aatctaaaat tacttttatt 240 gatggtgatg aaggtatttt gctgcaccgc ggtttcccga tcgatcagct ggcgaccgat 300 tctaactacc tggaagtttg ttacatcctg ctgaatggtg aaaaaccgac tcaggaacag 360 tatgacgaat ttaaaactac ggtgacccgt cataccatga tccacgagca gattacccgt 420 ctgttccatg ctttccgtcg cgactcgcat ccaatggcag tcatgtgtgg tattaccggc 480 gcgctggcgg cgttctatca cgactcgctg gatgttaaca atcctcgtca ccgtgaaatt 540 gccgcgttcc gcctgctgtc gaaaatgccg accatggccg cgatgtgtta caagtattcc 600 attggtcagc catttgttta cccgcgcaac gatctctcct acgccggtaa cttcctgaat 660 atgatgttct ccacgccgtg cgaaccgtat gaagttaatc cgattctgga acgtgctatg 720 gaccgtattc tgatcctgca cgctgaccat gaacagaacg cctctacctc caccgtgcgt 780 accgctggct cttcgggtgc gaacccgttt gcctgtatcg cagcaggtat tgcttcactg 840 tggggacctg cgcacggcgg tgctaacgaa gcggcgctga aaatgctgga agaaatcagc 900 tccgttaaac acattccgga atttgttcgt cgtgcgaaag acaaaaatga ttctttccgc 960 ctgatgggct tcggtcaccg cgtgtacaaa aattacgacc cgcgcgccac cgtaatgcgt 1020 gaaacctgcc atgaagtgct gaaagagctg ggcacgaagg atgacctgct ggaagtggct 1080 atggagctgg aaaacatcgc gctgaacgac ccgtacttta tcgagaagaa actgtacccg 1140 aacgtcgatt tctactctgg tatcatcctg aaagcgatgg gtattccgtc ttccatgttc 1200 accgtcattt tcgcaatggc acgtaccgtt ggctggatcg cccactggag cgaaatgcac 1260 agtgacggta tgaagattgc ccgtccgcgt cagctgtata caggatatga aaaacgcgac 1320 tttaaaagcg atatcaagcg ttaa 1344 <210> 15 <211> 84 <212> DNA <213> (Primer U1) <400> 15 ctgacagcta gctcagtcct aggtataatg ctagcataca gcggaaaagg agcatcttcg 60 atggctgata caaaagcaaa actc 84 <210> 16 <211> 85 <212> DNA <213> (Primer U2) <400> 16 ctgacagcta gctcagtcct aggtataatg ctagcgcccg atgctccttt aattgacagg 60 atggctgata caaaagcaaa actca 85 <210> 17 <211> 88 <212> DNA <213> (Primer U3) <400> 17 ctgacagcta gctcagtcct aggtataatg ctagcgaccg atgctccttt gtctctagcg 60 atggctgata caaaagcaaa actcaccc 88 <210> 18 <211> 88 <212> DNA<00006​​<400> 18 ctgacagcta gctcagtcct aggtataatg ctagcttagg atgctccttt cagacacccg atggctgata caaaagcaaa actcaccc <210> 19 <211> 88 <212> DNA <213> (Figure U5) <400> 19 ctgacagcta gctcagtcct aggtataatg ctagctgctg atgctccttt gagctattgc atggctgata caaaagcaaa actcaccc <210> 20 <211> 59 <212> DNA <213> (Signs PhaA‐F) <400> 20 59. cggcggctta catatgggct aaggagata taccatgact gacgttgtta ttgtttccg <210> 21 <211> 48 <212> DNA <213> (Signs PhaA‐R) <400> 21 ttgcactggc cgtagcgt grandfather ttcgagctcc gtcgaca <210> 22 <211> 48 <212> DNA <213> (pronounced PhaB‐F) <400> 22 ttgtttaact ttaggaga ggatatacat gacccagcgc attgcgta <210> 23 <211> 22 <212> DNA <213> (Primer PhaB-R) <400> 23 ttagcccata tgtaagccgc cg 22 <210> 24 <211> 55 <212> DNA <213> (Primer Bld*-F) <400> 24 gtttaacttt aaggagaagg atatacatga ttaaagatac cctggttagc atcac 55 <210> 25 <211> 21 <212> DNA <213> (Primer Bld*-R) <400> 25 tttaaccagc cagtacgcag c 21 <210> 26 <211> 59 <212> DNA <213> (Primer yqhd-F) <400> 26 gctgcgtact ggctggttaa aaggagatat accatgaaca actttaatct gcacacccc 59 <210> 27 <211> 50 <212> DNA <213> (Primer yqhd-R) <400> 27 gatgattaat tgtcaaattt cctaatgcag gagttagcgg gcggcttcgt 50 <210> 28 <211> 55 <212> DNA <213> (Primer fdh1-F) <400> 28 ttgtttaact ttaataagga gatataccat ggatgaccaa agttctggca gttct 55 <210> 29 <211> 49 <212> DNA <213> (Primer fdh1-R) <400> 29 cgcgccgagc tcgaattcgg atccggatcc ttatttttct gcttcaccg 49 <210> 30 <211> 53 <212> DNA <213> (Primer nadk-F) <400> 30 atatacatat ggcagatctc aattgatgaa taatcatttc aagtgtattg gca 53 <210> 31 <211> 37 <212> DNA <213> (Primer nadk-R) <400> 31 ttattaaagt tttagaataa tttttttgac cagccga 37 <210> 32 <211> 54 <212> DNA <213> (Primers pntA-F) <400> 32 aattattcta aaactttaat aaggagatat aatgcgaatt ggcataccaa gaga 54 <210> 33 <211> 49 <212> DNA <213> (Primer pntA-R) <400> 33 ctaatcctcc agacatatgt taccccttaa tttttgcgga acattttca 49 <210> 34 <211> 28 <212> DNA <213> (Primer pntB-F) <400> 34 ggggtaacat atgtctggag gattagtt 28 <210> 35 <211> 49 <212> DNA <213> (Primer pntB-R) <400> 35 tgctcagcgg tggcagcagc ctaggttaca gagctttcag gattgcatc 49 <210> 36 <211> 1284 <212> DNA <213> (Gene gltA encoding citrate synthase) <400> 36 atggctgata caaaagcaaa actcaccctc aacggggata cagctgttga actggatgtg 60 ctgaaaggca cgctgggtca agatgttatt gatatccgta ctctcggttc aaaaggtgtg 120 ttcacctttg acccaggctt cacttcaacc gcatcctgcg aatctaaaat tacttttatt 180 gatggtgatg aaggtatttt gctgcaccgc ggtttcccga tcgatcagct ggcgaccgat 240 tctaactacc tggaagtttg ttacatcctg ctgaatggtg aaaaaccgac tcaggaacag 300 tatgacgaat ttaaaactac ggtgacccgt cataccatga tccacgagca gattacccgt 360 ctgttccatg ctttccgtcg cgactcgcat ccaatggcag tcatgtgtgg tattaccggc 420 gcgctggcgg cgttctatca cgactcgctg gatgttaaca atcctcgtca ccgtgaaatt 480 gccgcgttcc gcctgctgtc gaaaatgccg accatggccg cgatgtgtta caagtattcc 540 attggtcagc catttgttta cccgcgcaac gatctctcct acgccggtaa cttcctgaat 600 atgatgttct ccacgccgtg cgaaccgtat gaagttaatc cgattctgga acgtgctatg 660 gaccgtattc tgatcctgca cgctgaccat gaacagaacg cctctacctc caccgtgcgt 720 accgctggct cttcgggtgc gaacccgttt gcctgtatcg cagcaggtat tgcttcactg 780 tggggacctg cgcacggcgg tgctaacgaa gcggcgctga aaatgctgga agaaatcagc 840 tccgttaaac acattccgga atttgttcgt cgtgcgaaag acaaaaatga ttctttccgc 900 ctgatgggct tcggtcaccg cgtgtacaaa aattacgacc cgcgcgccac cgtaatgcgt 960 gaaacctgcc atgaagtgct gaaagagctg ggcacgaagg atgacctgct ggaagtggct 1020 atggagctgg aaaacatcgc gctgaacgac ccgtacttta tcgagaagaa actgtacccg 1080 aacgtcgatt tctactctgg tatcatcctg aaagcgatgg gtattccgtc ttccatgttc 1140 accgtcattt tcgcaatggc acgtaccgtt ggctggatcg cccactggag cgaaatgcac 1200 agtgacggta tgaagattgc ccgtccgcgt cagctgtata caggatatga aaaacgcgac 1260 tttaaaagcg atatcaagcg ttaa 1284 <210> 37 <211> 50 <212> DNA <213> (Natural UTR sequence of gltA gene encoding citrate synthase) <400> 37 aaatttaagt tccggcagtc ttacgcaata aggcgctaag gagaccttaa 50

Claims

1. A genetically engineered bacterium producing 1,3-butanediol, characterized in that, The genetically engineered bacteria are recombinant Escherichia coli that produce 1,3-butanediol through chassis microbial modification; the chassis microbial modification is to enhance the 1,3-butanediol synthesis pathway, weaken genes related to competitive metabolic pathways, and express cofactor metabolic regulatory genes. The enhanced 1,3-butanediol synthesis pathway involves overexpressing the codon-optimized gene PhaA encoding heterologous acetyl-CoA thiolytic enzyme, the codon-optimized gene PhaB encoding heterologous acetyl-CoA dehydrogenase, the codon-optimized gene Bld encoding butyraldehyde dehydrogenase mutant gene Bld in recombinant Escherichia coli, and overexpressing the codon-optimized endogenous gene yqhd encoding alcohol dehydrogenase in recombinant Escherichia coli. The cofactor metabolism regulatory genes are pntA, which encodes the α subunit of NAD(P) transhydrogenase, an endogenous gene of Escherichia coli; pntB, which encodes the β subunit of NAD(P) transhydrogenase, an endogenous gene of Escherichia coli; nadk, which encodes NAD kinase, an endogenous gene of Escherichia coli; and fdh1, which encodes formate dehydrogenase, a heterologous gene. The gene related to the competitive metabolic pathway is gltA, which encodes citrate synthase. The weakening of the competing metabolic pathway-related genes involves replacing the 5' untranslated region UTR sequence of the gene gltA encoding citrate synthase with a manually designed UTR sequence, and adding a constitutive promoter sequence before the UTR sequence. The nucleotide sequence of the gene gltA encoding citrate synthase is shown in SEQ ID NO.36, the manually designed UTR sequence is shown in SEQ ID NO.1, and the constitutive promoter sequence is CTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGC. The gene gltA encoding citrate synthase is an endogenous gene of Escherichia coli; the modifications made are changes to the genome of Escherichia coli; the promoters used in the genetically engineered bacteria are all strong promoters; The complete nucleotide sequence of the gene gltA encoding citrate synthase, with the promoter sequence added and the UTR sequence replaced, is shown in SEQ ID NO.14; The nucleotide sequence of the codon-optimized gene PhaA encoding heteroacetyl-CoA thiolytic enzyme is shown in SEQ ID NO.2; the nucleotide sequence of the codon-optimized gene PhaB encoding heteroacetyl-CoA dehydrogenase is shown in SEQ ID NO.3; the nucleotide sequence of the codon-optimized gene yqhd encoding alcohol dehydrogenase is shown in SEQ ID NO.4; and the nucleotide sequence of the mutant gene Bld-L273T encoding butyraldehyde dehydrogenase Bld is shown in SEQ ID NO.

5. The nucleotide sequence of the gene pntA encoding the α subunit of the cofactor regulatory protein NAD(P) transhydrogenase is shown in SEQ ID NO.6; the nucleotide sequence of the gene pntB encoding the β subunit of the cofactor regulatory protein NAD(P) transhydrogenase is shown in SEQ ID NO.7; the nucleotide sequence of the gene nadk encoding NAD kinase is shown in SEQ ID NO.12; and the nucleotide sequence of the gene fdh1 heterologously encoding formate dehydrogenase is shown in SEQ ID NO.

8.

2. The application of the genetically engineered bacteria as described in claim 1 in the production of 1,3-butanediol.

3. The application according to claim 2, characterized in that, The application involves inoculating a genetically engineered bacterium that produces 1,3-butanediol into a fermentation medium, fermenting the culture, and then separating and purifying the obtained fermentation broth to obtain 1,3-butanediol.

4. The application according to claim 3, characterized in that, The fermentation conditions were as follows: fermentation temperature of 30-37℃, fermentation time of 72h, and IPTG induction concentration of 0.05-1.2mM.

5. The application according to claim 3 or 4, characterized in that, The separation and purification of the obtained fermentation broth includes: Step S1: Centrifuge the fermentation broth to obtain the supernatant; In step S2, the supernatant was filtered through a 0.22 μm aqueous filter membrane to obtain 1,3-butanediol.

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