Recombinant escherichia coli strain for producing 1, 3-butanediol from glucose and method for producing 1, 3-butanediol by using recombinant escherichia coli strain

Abstract

The present invention relates to a recombinant Escherichia coli strain for producing 1, 3-butanediol from glucose and a method for producing 1, 3-butanediol by using the same, in which a recombinant Escherichia coli strain is developed in which a gene of a pathway required for biosynthesis of 1, 3-BDO from acetyl-CoA is cloned, and removing genes interfering with the pathway or a pathway competing with the pathway. Furthermore, a strain of Escherichia coli has been developed in which the pathways and genes involved in the conversion of glucose to acetyl-CoA, the rate of regeneration of NADPH used as a cofactor, and the effective utilization of the TCA cycle pathway are modified and optimized, which are necessary to improve the biosynthesis efficiency of 1, 3-BDO. Therefore, the recombinant Escherichia coli strain of the present invention can produce 1, 3-butanediol from glucose at high concentration, high yield, and high rate.

Description

Technical Field

[0001] The present disclosure relates to a recombinant Escherichia coli (E. coli) strain for producing 1,3-butanediol from glucose and a method for producing 1,3-butanediol using the strain. Background Art

[0002] 1,3-Butanediol (1,3-BDO), also known as butanediol, is a non-natural alcohol used as a building block for the production of industrial chemicals required for cosmetics, food, and pharmaceuticals. In the food industry, it is used as an additive, flavoring agent, solvent or solubilizer for flavoring agents, stabilizer, emulsifier, antimicrobial agent, and preservative. In the pharmaceutical industry, it is also used as a solvent for parenteral drugs, while (R)-1,3-BDO is used as a precursor for the synthesis of azetidinone derivatives, which are the main chiral intermediates for the synthesis of penem and carbapenem β-lactam antibiotics. 1,3-Butanediol has been developed for a variety of uses, especially in the cosmetic industry. It is used as an emollient, humectant, anti-crystallization agent for insoluble ingredients, solubilizer for low water-soluble ingredients (such as fragrances), antimicrobial agent, and preservative. For example, it can be used as a moisturizer in hair sprays and styling lotions, reduce the fragrance loss of essential oils, prevent spoilage caused by microorganisms, and is therefore used as a solvent for parabens. 1,3-Butanediol can be used in a variety of concentrations from less than 0.1% to more than 50%. In cosmetics, it is used in hair and bath products, eye and facial makeup, perfumes, personal hygiene products, and shaving and skin care preparations.

[0003] Due to the increasing demand for 1,3-BDO, several studies have been conducted on production methods that provide high yields and are environmentally friendly. Chemically, 1,3-BDO is mainly produced from petroleum-derived substances, such as narrow precursors (acetaldehyde and 4-hydroxy-2-butanone (4H2B)), and has been commercialized. In this process, some environmentally harmful reagents are used and harsh reaction conditions are involved. In contrast, biocatalytic processes offer the advantages of being renewable, leaving fewer by-products, and no residual metals in the final product. However, due to the low productivity and low economic feasibility of bioprocessing, it has not yet been commercialized.

[0004] 1,3-BDO is a non-natural substance, and its naturally occurring metabolic pathway for production has not been identified. Therefore, the production of 1,3-BDO by biotechnological methods relies on the development of recombinant bacterial strains, including genetic engineering and metabolic engineering. It has been reported that the use of strain and pathway engineering strategies has resulted in the production of 2.4 g / L of R-1,3-BDO from glucose in a bioreactor, accounting for 11% of the maximum theoretical yield. In addition, Kataoka et al. in Japan reported that they produced up to 9.05 g / L (100.4 mM) in 98 hours. Recently, Liu et al. in China produced 13 g / L of 1,3-BDO under aerobic conditions through a pathway that uses 3-hydroxybutyrate as an intermediate. GENOMATICA, which is well known for the biological production of 1,4-BDO, announced that it has produced 54 g / L of 1,3-BDO, but there are currently no technical details to support this. Despite the achievements in research to date, bioprocessing lags far behind chemical processes, and major improvements in titer, productivity and yield are needed for the commercialization of bioprocessing. Summary of the invention Technical issues

[0005] The present disclosure aims to provide a recombinant E. coli strain in which genes of a pathway required for the biosynthesis of 1,3-BDO from acetyl-CoA are cloned, and genes of a pathway that interferes with or competes with the aforementioned pathway are removed from the strain. In addition, a recombinant E. coli strain is provided, which has a modified and optimized pathway and genes involved in the conversion of glucose to acetyl-CoA, which is necessary to improve the biosynthesis efficiency of 1,3-BDO, the regeneration rate of NADPH used as a cofactor, and the efficient utilization of the TCA cycle.

[0006] In addition, another object of the present disclosure is to provide a method for increasing the production of 1,3-BDO, comprising culturing the recombinant Escherichia coli strain. Technical Solution

[0007] To achieve the above objectives, the present disclosure provides a recombinant expression vector, which sequentially comprises a butyraldehyde dehydrogenase (bld) gene, an acetyl-CoA acetyltransferase (phaA) gene, an acetoacetyl-CoA reductase (phaB) gene and a 3-hydroxybutyraldehyde (3-HBA) reductase gene.

[0008] In addition, the present disclosure provides a recombinant Escherichia coli strain with increased 1,3-BDO production, wherein the strain is transformed with the recombinant expression vector.

[0009] In addition, the present disclosure provides a method for increasing 1,3-BDO production, the method comprising culturing the recombinant Escherichia coli strain. Beneficial Effects

[0010] The present disclosure relates to a recombinant Escherichia coli strain for producing 1,3-butanediol from glucose and a method for producing 1,3-butanediol using the strain, wherein a recombinant Escherichia coli strain is developed, in which genes of a pathway required for the biosynthesis of 1,3-BDO from acetyl-CoA are cloned, and genes of a pathway that interferes with or competes with the aforementioned pathway are removed from the strain. An Escherichia coli strain is also developed, the strain having a modified and optimized pathway and genes, the pathway and genes being involved in the conversion of glucose to acetyl-CoA, which is necessary to improve the biosynthesis efficiency of 1,3-BDO, the regeneration rate of NADPH used as a cofactor, and the efficient utilization of the TCA cycle. Therefore, the recombinant Escherichia coli strain of the present disclosure is capable of producing 1,3-butanediol from glucose at a high concentration, high yield, and high rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 The strategy and major metabolic pathways for constructing the recombinant strain producing 1,3-butanediol of the present disclosure are shown.

[0012] Figure 2 Shown are the results of a screening of 3-hydroxybutyryl-CoA (3-HB-CoA) reductases that affect 1,3-BDO production.

[0013] Figure 3 Shown are the results of a screen for 3-hydroxybutyraldehyde (3-HBA) reductase.

[0014] Figure 4 It was shown that with the increase of phaA and phaB gene expression, carbon tended to flow more from the acetyl-CoA node to the production of 1,3-BDO.

[0015] Figure 5 The performance of strains with deleted byproduct production pathways is shown.

[0016] Figure 6 The effect of the volume of fermentation broth on 1,3-butanediol production is shown.

[0017] Figure 7 The effects of changes in acetyl-CoA production and the TCA cycle on 1,3-BDO production are shown.

[0018] Figure 8The effect of deletion of the pdhR or gltA gene on 1,3-BDO production by bioreactor cultivation is shown. The K6-Q10 strain did not undergo deletion of the pdhR gene and the gltA gene. OD, optical density; Gluc, glucose; 1,3-BDO, 1,3-butanediol; 3-HB, 3-hydroxybutyrate; Pyr, pyruvate; Suc, succinate; EtOH, ethanol; Ace, acetic acid.

[0019] Fig. 9 The results showed that 3-HBA was toxic in inhibiting the growth of the strain.

[0020] Fig.10 The performance differences of three different 3-HBA reductases (i.e., YqhD, YgjB, and YahK) in the production of 1,3-BDO by bioreactor experiments are shown. The YqhD enzyme was used in the K8-Q10 strain, the YgjB enzyme was used in the K8-Q25 strain, and the YahK enzyme was used in the K8-Q10 strain. OD, optical density; Gluc, glucose; 1,3-BDO, 1,3-butanediol; 3-HB, 3-hydroxybutyrate; Pyr, pyruvate; Suc, succinate; EtOH, ethanol; Ace, acetic acid.

[0021] Fig.11 The 1,3-BDO productivity of strains with enhanced ED pathways in glucose metabolism by bioreactor experiments is shown. The K8-Q27 strain is a strain with unchanged glycolysis pathway, and the K22-Q27 strain is a strain with Δpgi, zwf ++ , △gntR, and K23-Q27 strains are △pgi, zwf ++ , △gntR, edd ++ and eda ++ OD, optical density; Gluc, glucose; 1,3-BDO, 1,3-butanediol; 3-HB, 3-hydroxybutyrate; Pyr, pyruvate; Suc, succinate; EtOH, ethanol; Ace, acetic acid.

[0022] Fig.12The effect of the expression of carboxylic acid reductase (CAR) on the production of 1,3-butanediol in the bioreactor is shown. The K23-Q27 strain is a strain without the introduction of CAR, the K23-Q31 strain is a strain into which the Mm_CAR derived from Mycobacterium marinum is introduced, and the K23-Q32 strain is a strain into which the Ma_CAR of Mycobacterium abscessus is introduced. OD, optical density; Gluc, glucose; 1,3-BDO, 1,3-butanediol; 3-HB, 3-hydroxybutyric acid; Pyr, pyruvic acid; Suc, succinic acid; EtOH, ethanol; Ace, acetic acid. DETAILED DESCRIPTION

[0023] The present disclosure provides a recombinant expression vector, which sequentially comprises a butyraldehyde dehydrogenase (bld) gene, an acetyl-CoA acetyltransferase (phaA) gene, an acetoacetyl-CoA reductase (phaB) gene, and a 3-hydroxybutyraldehyde (3-HBA) reductase gene.

[0024] Preferably, the bld gene may include the nucleotide sequence shown by SEQ ID NO:7, the phaA gene may include the nucleotide sequence shown by SEQ ID NO:5, and the phaB gene may include the nucleotide sequence shown by SEQ ID NO:6, but are not limited thereto.

[0025] Preferably, the 3-HBA reductase gene may be, but is not limited to, the yqhD gene containing the nucleotide sequence shown by SEQ ID NO:34, the ygjB gene containing the nucleotide sequence shown by SEQ ID NO:35, or the yahK gene containing the nucleotide sequence shown by SEQ ID NO:36.

[0026] More preferably, the ribosome binding site (RBS) in the 5' untranslated region (UTR) of the ygjB gene may comprise the nucleotide sequence shown by SEQ ID NO: 37 or SEQ ID NO: 38, but is not limited thereto.

[0027] Preferably, the recombinant expression vector may further comprise a carboxylic acid reductase (CAR) gene, and more preferably, the CAR gene may comprise the nucleotide sequence shown by SEQ ID NO: 20 or SEQ ID NO: 21, but is not limited thereto.

[0028] As used herein, the term "vector" refers to a self-replicating DNA molecule used to carry a cloned gene (or other fragment of cloned DNA).

[0029] The term "expression vector" as used herein refers to a recombinant DNA molecule comprising a desired coding sequence and an appropriate nucleic acid sequence necessary for expressing an operably linked coding sequence in a specific host organism. The expression vector may preferably include one or more selective markers. The marker is a nucleic acid sequence that is characterized by being selected by chemical means, including any gene that can distinguish transformed cells from untransformed cells. Examples include, but are not limited to, genes that are resistant to antibiotics such as ampicillin, kanamycin, geneticin (G418), bleomycin, hygromycin and chloramphenicol, and those skilled in the art may appropriately select it.

[0030] In addition, the present disclosure provides a recombinant Escherichia coli strain with improved 1,3-butanediol (1,3-BDO) production, wherein the strain is transformed with the recombinant expression vector. The basic Escherichia coli strain used in the present disclosure is K12MG1655, which is a representative strain of the K group among various Escherichia coli and is a generally recognized as safe (GRAS) strain whose safety has been recognized.

[0031] Preferably, the recombinant Escherichia coli strain may be, but is not limited to, a strain obtained by transforming an Escherichia coli strain with the recombinant expression vector, wherein the Escherichia coli strain lacks the alcohol dehydrogenase (adhE) gene, the pyruvate oxidase (poxB) gene, the D-lactate dehydrogenase (ldhA) gene, the phosphate acetyltransferase-acetate kinase (pta-ackA) gene and the acyl-CoA thioesterase (yciA) gene.

[0032] More preferably, the recombinant Escherichia coli strain may be, but is not limited to, a strain obtained by transforming an Escherichia coli strain with the recombinant expression vector, wherein the DNA-binding transcription dual regulator (pdhR) gene is additionally deleted in the Escherichia coli strain.

[0033] More preferably, the recombinant Escherichia coli strain may be a strain obtained by transforming an Escherichia coli strain with the recombinant expression vector, wherein the citrate synthase (gltA) gene is additionally deleted in the Escherichia coli strain; the recombinant Escherichia coli strain may be a strain obtained by transforming an Escherichia coli strain with the recombinant expression vector, wherein the glucose-6-phosphate isomerase (pgi) gene and the DNA-binding transcription inhibitor (gntR) gene are additionally deleted in the Escherichia coli strain, and the NADP+-dependent glucose-6-phosphate dehydrogenase (zwf) gene is additionally overexpressed therein; and the recombinant Escherichia coli strain may be a strain obtained by transforming an Escherichia coli strain with the recombinant expression vector, wherein the Escherichia coli strain additionally overexpresses the phosphogluconate dehydratase (edd) gene and the 2-keto-3-deoxygluconate 6-phosphate aldolase (eda) gene, but is not limited thereto.

[0034] More preferably, the recombinant Escherichia coli strain may be a strain deposited under the deposit number KCTC 15315BP, and the strain deposited under the deposit number KCTC 15315BP is the strain listed as K23-Q32 strain in the present specification.

[0035] The recombinant E. coli strain prepared in the present disclosure is a recombinant microorganism prepared by the inventors by introducing a 1,3-BDO biosynthetic pathway and eliminating a competing pathway or a degradation pathway of an intermediate metabolite on the 1,3-BDO biosynthetic pathway, wherein the strain uses glucose as a raw material. In addition, it is an E. coli strain having a modified and optimized pathway and gene, which is involved in the conversion of glucose to acetyl-CoA, which is necessary to improve the biosynthetic efficiency of 1,3-BDO, the regeneration rate of NADPH used as a cofactor, and the efficient utilization of the TCA cycle. In this process, new genes are introduced, gene expression levels are optimized, and carbon and energy metabolism are regulated and optimized to increase the production rate and yield of 1,3-BDO. E. coli K12 does not have the ability to produce 1,3-BDO from glucose, but the recombinant E. coli prepared in the present disclosure successfully produces 1,3-BDO during growth in the presence of glucose.

[0036] The genetically recombinant E. coli strain developed by the present inventors is capable of producing 1,3-BDO with excellent performance, ensuring a maximum concentration of 80 g / L (883 mM), a glucose yield of 0.74 mol / mol, and a production rate of 1.67 g / L / h.

[0037] In addition, the present disclosure provides a method for increasing 1,3-BDO production, the method comprising culturing the recombinant Escherichia coli strain.

[0038] In the present disclosure, a total of 36 genes can be modified, deleted or up-regulated, and the genes modified, used or deleted in the present disclosure, the names of the proteins encoded by them, and the sequence numbers are shown in Table 1.

[0039] Table 1

[0041] Hereinafter, the present disclosure will be described in more detail by way of examples. For those skilled in the art, it is obvious that these examples are only intended to describe the present disclosure in more detail, and according to the gist of the present disclosure, the scope of the present disclosure is not limited to the following examples. Example

[0042] Hereinafter, the present disclosure will be described in more detail by way of examples. However, the present disclosure is not limited to these examples.

[0044] <Experimental Example>

[0045] According to the present disclosure, the following experimental examples are intended to provide experimental examples generally applied to each embodiment.

[0047] 1. Materials and Methods

[0048] All restriction enzymes, highly reliable DNA polymerases and other DNA modifying enzymes used for gene cloning were purchased from New England Bio-Labs (Beverly, MA, USA). Microplasmid isolation kits, DNA gel extraction kits and genomic DNA isolation kits were purchased from Cosmotech Co. Ltd. (Seoul) and Promega (Madison, Wisconsin, USA), respectively. RNA isolation kits were purchased from Qiagen (Mannheim, Germany). iScript cDNA synthesis kit and SyBr green RT-PCR master mix were purchased from BioRad (Seoul, South Korea). Oligonucleotide synthesis and DNA sequencing were performed by Macrogen Co., Ltd. (Seoul, South Korea). Tryptone and yeast extract were purchased from Difco (Becton Dickinson, Franklin Lakes, NJ, USA). Unless otherwise specified herein, glycerol, glucose and all other chemicals and enzymes were purchased from Sigma-Aldrich (Franklin Lakes, NJ, USA).

[0050] 2. Construction of genetically recombinant strains and plasmids

[0051] Prepare and use the recombinant strains and plasmids in the following Table 2, including existing strains and plasmids. Here, genetic recombination is carried out as follows. In order to delete the overexpression and development of the inducible plasmid, Escherichia coli DH5α was used. Chromosome genetic manipulation is carried out using the deletion / insertion method of in-frame markers. In short, 500bp in the upstream (upstream fragment, A) and downstream (downstream fragment, B) regions of the target gene are amplified by PCR, and they are connected using the overlapping PCR method. Subsequently, fragment AB is cloned into the pKOV vector at NotI and XbaI or at the BamHI restriction site. The resulting plasmid is used to delete the target gene from chromosomal DNA by homologous recombination. Finally, the deletion inducible mutant strain is screened by PCR and observed by sequencing. For the replacement of all target genes, the target gene is first removed, and then the gene of interest is inserted by a similar in-frame marker method.

[0052] The other recombinant strains shown in Table 2 were prepared in the same manner. Therefore, the restriction enzymes and primers used to prepare the deletion-inducible plasmid (pKOV) are shown in Tables 3 and 4.

[0054] Table 2

[0055] In the above Table 2, "△" indicates the deletion of a gene. In addition, in Table 2, "::" indicates the manipulation of the nucleotide sequence of the gene. For example, "lpdA::lpdAE324K" means that, with respect to the gene encoding the LpdA protein gene, the lpdA gene encoding the following protein is engineered, in which the amino acid residue at position 324 of the LpdA protein is modified from E to K, and the original lpdA gene is replaced by the engineered lpdA gene.

[0056] Table 3

[0058] Table 4

[0060] 3. Culture Conditions

[0061] For cultivation in shake flasks, the cells were cultured in agar LB medium for about 10 to 11 hours to prepare a primary inoculum. In the primary inoculation, the cells were transferred to a freshly prepared modified M9 medium and cultured for about 10 hours to prepare a secondary inoculum. The main culture for the production of 1,3-BDO was initiated by inoculating the secondary inoculum into the same modified M9 medium. The components of the modified M9 medium used in the present disclosure were 1.0 g / L yeast extract, 1.0 g / L NH4SO4, and 0.25 g / LMgSO4·7H2O supplemented with 100 mM potassium phosphate buffer (pH 7.0). 50 g / L glucose was used as a carbon source. For recombinant cells, kanamycin (50 μg / mL) was added as needed for plasmid stability. For gltA-deficient strains, 20 g / L sodium glutamate was added. All strains were cultured in 250 mL conical flasks with a medium volume of 20 mL and cultured in a culture shaker at 37°C / 100 rpm. In all experiments, the strain cultures were grown at a starting OD600 of 0.1 and 0.25 mM IPTG was added for expression of the 1,3-BDO pathway. In the case of two-phase cultures, the cultures were stopped at the end of exponential growth and the cells were harvested by centrifugation.

[0062] In the case of two-stage culture, in other words, when an experiment is performed in which the strain culture in the first stage and the 1,3-BDO production in the second stage are separated, LB or modified M9 medium is generally used in the first stage to promote strain growth; and in the second stage, the strain cultured in the first stage is harvested to remove the residual medium components by centrifugation and washing, and the production of 1,3-BDO is promoted in the presence of a phosphate buffer solution (pH 7.0) and glucose in a state where the nitrogen source is very low or insufficient. In the case of the second stage, since there is almost no bacterial growth, this stage proceeds to a state similar to bioconversion.

[0063] Bioreactor experiments were performed in an Effendroff bioreactor (Germany, 1.0 L modified M9 medium, 2.5 L container). The fermentation was prepared in the same manner as described in the flask experiments. The air injection rate was set to 1.0 vvm and the stirring speed was adjusted in the range of 200 rpm-700 rpm to maintain dissolved oxygen (DO) at 10%. Glucose was maintained at 50 mM to 250 mM by intermittent addition. The cell growth and 1,3-BDO production steps were appropriately divided by adjusting the concentration of the nitrogen source or carbon source in the medium without strain acquisition or washing.

[0065] 4. Measurement of Enzyme Activity

[0066] The activity of 3-HB-CoA reductase was measured as described in the literature. The reaction mixture was prepared to contain 50 mM Tris-HCl, 0.15 mM NADH at pH 7.0 and 0.3 mM 3-hydroxybutyryl-CoA as substrate. The purified enzyme was added and the decrease in NADH concentration at 37°C was continuously monitored by a photometer at 340 nm.

[0067] The activity of alcohol dehydrogenase was determined by monitoring the increase / decrease of NADP+ / NADPH by a photometer at 340 nm. The reaction mixture included 50 mM Tris-HCl at pH 7.0. To measure the forward reaction, the subsequent preparation included 0.15 mM NADPH and 5 mM 3-hydroxybutyraldehyde as substrates at 37°C. For the reverse reaction, 2 mM NADP+ and 30 mM 1,3-BDO were used as cofactors and substrates, respectively. For the reduction reaction, 1 unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 μmol of NAD(P)H to NADP+ for one minute. For the oxidation reaction, 1 unit of enzyme activity was defined as the amount of enzyme required to reduce 1 μmol of NAD(P)+ to NAD(P)H for one minute.

[0069] 5. Analytical methods

[0070] Cell concentration was measured using a double beam spectrophotometer (Lambda 20, PerkinElmer, Norwalk, CT, USA) in a 10 mm pathlength cuvette as absorbance at 600 nm (OD600). Protein concentration was measured using the Bradford method on a microtiter plate reader (Synergy H1, Bioteck) with bovine serum albumin (BSA) as a standard. The concentrations of glycerol, glucose, 1,3-PDO and other metabolites were measured using high performance liquid chromatography (HPLC, Agilent 1260infinity II, USA). For liquid chromatography analysis, the supernatant was obtained by centrifuging the culture sample at 10000×g for 10 min, filtered with a Tuffryn membrane (Acrodisc, Pall Life Sciences), and separated by a 300 mm×7.8 mm Aminex HPX-87H column (Bio-Rad, USA) at 65°C using 2.5 mmol / L H2SO4 as the mobile phase.

[0072] <Example 1> Construction of basic strains and screening of 3-hydroxybutyryl-CoA (3-HB-CoA) reductase

[0073] When E. coli grows in glucose, it produces acetyl-CoA. Therefore, in the presence of glucose, the following four enzymes are required to produce acetyl-CoA or 1,3-BDO. (1) β-Ketothiolase (also known as acetyl-CoA acetyltransferase), which condenses / converts two acetyl-CoAs into one acetoacetyl-CoA; (2) acetoacetyl-CoA reductase, which converts acetoacetyl-CoA into β-hydroxybutyryl-CoA by reduction; (3) β-hydroxybutyryl-CoA reductase, which converts β-hydroxybutyryl-CoA into 3-hydroxybutyraldehyde (3-HBA); and (4) 3-HBA reductase, which converts 3-HBA into 1,3-BDO by reduction. Among the four reactions, reactions (2) to (4) require NADPH or NADH as reducing power. In the basic strain, the PhaA enzyme encoded by Cn_phaA of Copperworm was selected as the first enzyme, the PhaB enzyme encoded by Cn_phaB of Copperworm was selected as the second enzyme, and the Bld enzyme encoded by Cs_bld of Clostridium saccharoperbutylacetonicum was selected as the third enzyme. The fourth 3-HBA reductase was not selected, but the aldehyde dehydrogenase possessed by the host cell Escherichia coli was allowed to catalyze the reaction.

[0074] Two recombinant plasmids, pC1 and pQ1, were developed and introduced into E. coli K12. The constructs were made by expressing Cn_phaB and Cn_phaA of Copperophora insecticidalis under the control of the Ptet promoter in pC1 derived from the pCDF plasmid with a medium copy number, and Cs_bld of Clostridium saccharoperbutylacetonicum under the control of the T5 promoter in pQ1 derived from the pQE80L plasmid with a high copy number. The two plasmids were then introduced into wild-type E. coli K12, and the strain was named K1-C1Q1.

[0075] The 1,3-BDO productivity of K1-C1Q1 was studied by two-stage cultivation. First, the strain was cultured in LB medium for about 6 hours, and then bioconversion was carried out in a phosphate buffer solution containing 30 g / L glucose. K1-C1Q1 produced 7.6 mM BDO in 24 hours of bioconversion. As byproducts, 3 mM EtOH and 15 mM acetate were produced. On the other hand, the K1-CQ control strain, into which only the blank plasmid was introduced but not the 1,3-BDO pathway, did not produce 1,3-BDO at all. These results indicate that the 1,3-BDO production pathway is essential for the production of 1,3-BDO, and that the 1,3-BDO biosynthetic pathway developed in the basic strain K1-C1Q1 functions normally.

[0076] The role of 3-HB-CoA reductase in the base strain was examined. This enzyme converts 3-HB-CoA to 3-HBA using NAD(P)H as a reducing force, and there are no reports of native enzymes specific for this reaction. Bld was used to prepare the base strain K1-C1Q1, but this enzyme is known to be sensitive to oxygen.

[0077] Four enzymes were studied, including three PduPs known to be resistant to oxygen and malonyl-CoA reductase MCR-C (derived from C. aurantiacus) (Table 2). These enzymes, but not bld, were cloned in pQE80L plasmid. The three selected PduPs were Se-pduP (K1-C1Q2) derived from Salmonella enterica, Kp_pduP (K1-C1Q3) derived from Klebsiella pneumonia, and Lr_pduP (K1-C1Q4) derived from Lactobacillus reuteri. The Ca_mcrC strain was named K1-C1Q5.

[0078] Among the 3-HB-CoA reductases tested, only Se_PduP (derived from Salmonella enterica) provided comparable 1,3-BDO production (6.2 mM) as Bld. However, Se_PduP produced about 3 times more ethanol than Cs_Bld, suggesting that Se_PduP has undesirable side reaction activity toward acetyl-CoA.

[0079] K1-C1Q1 (Cs_Bld) showed high BDO titer and low ethanol production, so it was selected for the next stage of strain development ( Figure 2 ).

[0081] <Example 2> Screening of 3-hydroxybutyraldehyde (3-HBA) reductase

[0082] To date, the enzyme for converting 3-HBA to BDO, 3-HBA reductase for the fourth step, has not been overexpressed, but BDO has been successfully produced. This suggests that the inherent aldehyde dehydrogenase or alcohol dehydrogenase (ADH) present in the E. coli K12 host is active on 3-HBA.

[0083] For example, it has been reported that ADH of Escherichia coli (such as Ec_YqhD) can reduce a variety of aldehydes to ethanol, thereby detoxifying aldehydes in a nonspecific manner.

[0084] To study the effect of overexpression of 3-HBA reductase on 1,3-BDO production, Ec_YqhD (K1-C1Q7) was cloned into the pQE80L plasmid together with the bld gene to study the production of 1,3-BDO. In addition, overexpression of three genes was also studied, including Kp_DhaT (K1-C1Q8) from Klebsiella pneumoniae, Lr_PduQ (K1-C1Q9) from Lactobacillus reuteri, and Cs_Bdh (K1C1Q6) from Clostridium saccharoperbutylacetonicum. Among them, Kp_DhaT and Lr_PduQ have been widely used to produce 1,3-propanediol from 3-hydroxypropanal, and Cs_bld has been used to produce butanol from butyraldehyde. The genes encoding the four enzymes (Cs_Bdh (pQ6), Ec_YqhD (pQ7), Kp_DhaT (pQ8), and Lr_PduQ (pQ9)) were cloned downstream of Cs_bld in the pQ1 plasmid and their recombinant E. coli were tested for 1,3-BDO production ( Figure 3 ).

[0085] YqhD (K1-C1Q7) produced the highest amount of 1,3-BDO and the lowest amount of acetate. The K1-C1Q7 strain overexpressing YqhD was used for future strain development.

[0087] <Example 3> Increasing the production of 1,3-BDO by overexpressing phaA and phaB

[0088] In all strains developed so far, acetate (up to about 15 mM) always accumulated as the major byproduct. It is speculated that the reason is that the "pull" from the acetyl-CoA node to the 1,3-BDO synthesis pathway is not high enough compared to the pull toward the TCA cycle, which may be caused by the low expression of phaA and phaB. The phaA and phaB genes were cloned into the medium copy plasmid pCDF.

[0089] Both Cn_phaA and Cn_phaB genes were transferred to pQ1 plasmid derived from pQE80L, which is a high copy plasmid that allows the expression of four genes (Cn_phaA, Cn_phaB, Cs_bld, and Ec_yqhD) in a single plasmid designated as pQ10. Here, the Cs_bld gene was placed first in the pQ1 plasmid because the Cs_Bld enzyme is known to have a low kcat value for 3-HB-CoA as a substrate.

[0090] In the K1-Q10 gene recombinant strain in which Cn_phaA and Cn_phaB were transferred to a high-copy plasmid, the acetic acid production was significantly reduced from 8 mM to 4 mM ( Figure 4 ). In addition, the production of BDO increased from 10.7 mM to 25 mM, an increase of about 2.5 times.

[0092] <Example 4> Elimination of by-product generation pathways

[0093] The following seven genes were removed in sequence: adhE, poxB, ldhA, pta-ackA, atoB, tesB and yciA. First, for the purpose of preventing the loss of precursors (PYR, AC-CoA) and cofactors (NADH), the deletion of lactate dehydrogenase (ldhA), alcohol dehydrogenase (adhE) and pyruvate oxidase (poxB) was performed. The production of lactic acid was eliminated from the K2-Q10 strain (ΔadhEΔpoxBΔldhA), but the production of ethanol or acetate was not affected. Ethanol can be produced by the non-specific activity of Bld and / or YqhD for acetyl-CoA and / or acetaldehyde. Acetate can also be produced by acetate phosphatase and kinase (pta-ackA) and / or thioesterase (yciA).

[0094] Deletion of acetate phosphatase and kinase (pta-ackA) from strain K3-Q10 did not reduce acetate production. However, 1,3-BDO production was enhanced by deletion of pta-ackA.

[0095] Acetyl-CoA acetyltransferase (AtoB) in E. coli is known to have a strong reverse cleavage activity on acetoacetyl-CoA, generating two acetyl-CoAs from acetoacetyl-CoA. If this activity is very high, it may form a so-called "futile cycle" in which acetyl-CoA and acetoacetyl-CoA continue to circulate with acetyl-CoA acetyltransferase, which is encoded by phaA and catalyzes the forward reaction. However, no positive effect was observed with the deletion of atoB (K4).

[0096] The tesB and yciA encoding thioesterases remove CoA from CoA compounds and thus may increase the production of 3-HB or acetate. In previous studies, these genes were overexpressed to produce 3-HB. The tesB deletion did not reduce the production of 3-HB or acetate. On the other hand, the yciA deletion (K6-Q10) reduced the production of both acetate and 3-HB, increased the BDO titer to about 42 mM, and increased the yield to about 0.42 mol / mol.

[0097] However, the double deletion of tesB and yciA (K7Q10) did not show better results than the single deletion of yciA (K6-Q10) in reducing 3-HB or acetate. Many thioesterases are known to induce 3-HB production. However, in this study, only the yci deletion was effective in reducing 3-HB production.

[0098] The K6Q10 strain (ΔadhEΔpoxBΔldhAΔpta-ackAΔyciA) was selected for further strain development ( Figure 5 ).

[0100] <Example 5> TCA cycle and genetic modification related to TCA cycle

[0101] Pyruvate is an important metabolic intermediate or precursor through which metabolic pathways such as primary glycolysis, TCA cycle, and 1,3-BDO biosynthesis are connected. In general, the accumulation of pyruvate indicates that the pathways following pyruvate are not functioning properly. When pyruvate begins to accumulate, it in turn blocks the upstream pathway of glucose conversion to pyruvate, and eventually both the intracellular influx and catabolism of the initial glucose are stopped. In the fermentation of the K6Q10 strain using flasks, pyruvate accumulated in the second half of the fermentation when the stirring rate was reduced ( Figure 6 ). Increasing the stirring rate reduced the accumulation of pyruvate, but at this time, the production of BDO decreased due to the high carbon flux entering the TCA cycle. The optimal volume of the fermentation broth was 12 mL.

[0102] Theoretically, if glucose does not enter the TCA cycle through metabolic processes, but all of it enters the 1,3-BDO pathway, then 1 mol of 1,3-BDO and 1 mol of NADH will be produced for each mol of glucose. It is necessary to assume that energetically 1 NADPH is produced from 1 NADH and 1 ATP.

[0103] 1 glucose + 2 ADP + 3 NAD + +2NADPH→1 1,3-BDO+2ATP+3NADH+2NADP + +2CO2

[0105] When acetyl-CoA is metabolized through the TCA cycle after glucose is converted to 1,3-BDO, a large amount of NADH is produced. As a result, redox imbalance occurs. Under anaerobic conditions or oxygen-limited conditions, this redox imbalance may inhibit the activity of the subenzyme LpdA of pyruvate dehydrogenase (PDH), resulting in pyruvate accumulation and blocked acetyl-CoA production.

[0106] In addition, pyruvate accumulation activates pdhR gene expression, and the protein of this gene represses the expression of the pdh operon, thereby further reducing PDH activity.

[0107] To increase PDH activity, K8-Q10 ( Figure 7 ). At flask scale, removal of pdhR reduced pyruvate accumulation and significantly increased the titer and yield of 1,3-BDO (148 mM and 0.52 mol / mol). This indicates that PDH activity is the rate-determining step for BDO production in previous strains such as K6, and suggests that overexpression of PDH and thereby high PDH activity is very important for BDO production.

[0108] To further increase PDH activity, a mutant lpdA encoding a mutant LpdA with reduced sensitivity to NADH was used ( Figure 7 ). When used alone (K9-Q10) or in combination with pdhR deletion (K10-Q10), BDO production was lower than that of K8-Q10. This suggests that the amount of PDH protein is rate-limiting rather than inhibition of activity by NADH at the enzyme level.

[0109] Literature shows that when TCA flux is reduced by reducing the expression of citrate synthase (gltA), the production of both butanol and mevalonate is improved. Both butanol and mevalonate use acetyl-CoA as a precursor.

[0110] The recombinant E. coli (K12-Q10) lacking the gltA mutation showed an improvement in the production rate and yield of 1,3-BDO in the initial stage in the flask ( Figure 7 ). However, in the late stage of bioconversion, it showed a large accumulation of pyruvate. Based on pyruvate, this result was interpreted as showing the importance of downstream, especially TCA cycle flux.

[0111] Removal of frdABCD was attempted to reduce flux in the reductive TCA cycle, while removal of ppc was attempted to reduce the rate of conversion of PEP to oxaloacetate (OAA) and reduce flux in the reductive TCA cycle. Reduction of flux through the reductive TCA cycle by deletion of either frdABCD or ppc (K11-Q11 and K13-Q11) produced pyruvate and reduced BDO titer ( Figure 7 ).

[0112] Deletion of iclR (K14-Q10) has been attempted to increase the activity of the glyoxylate shunt. Deletion of iclR also impairs the production of 1,3-BDO. BDO production is reduced and pyruvate accumulates ( Figure 7 ).

[0113] Bioreactor experiments were conducted on the K8-Q10 strain lacking pdhR and the K12-Q10 strain lacking gltA ( Figure 8 ). The K6-Q10 strain was used as a control. The best results were obtained in the K8-Q10 strain lacking pdhR. After 36 hours of fermentation, a concentration of 410 mM (37.4 g / L) of 1,3-BDO was obtained at a yield of 0.48 mol / mol. In contrast, 200 mM (0.31 mol / mol) was obtained in the control K6-Q10, and 312 mM (0.41 mol / mol) was obtained in the K12-Q10 strain lacking both gltA and pdhR.

[0115] <Example 6> Secondary screening of 3-HBA reductase

[0116] In previous bioreactor experiments, the deletion of pdhR(K8) significantly improved the production of 1,3-BDO to approximately 410 mM. However, 3-hydroxybutyraldehyde (3-HBA) accumulated at high levels (10 mM) in the culture medium. 3-HBA was highly toxic to cell growth at concentrations as low as approximately 6 mM ( Fig. 9 ). Aldehydes are known to inhibit cell growth due to their high reactivity, and 3-HBA has been determined to be more toxic than other aldehydes such as acetaldehyde and propionaldehyde. The accumulation of 3-HBA suggests that the rate of 3-HBA production from 3-HB-CoA is faster than the rate of 3-HBA conversion to 1,3-BDO. In addition, if the conversion of 3-HBA to 1,3-BDO is not rapid, 3-HB may be produced instead of 1,3-BDO. Figure 8 All recombinant strains accumulated approximately 40 mM 3-HB.

[0117] Previously, four enzymes were tested with 3-HBA reductase, such as Cs_bdh, Ec_yqhD, Kp_dhaT, and Lr_pduQ ( Figure 3 ), yqhD, which showed the best performance, was used. However, the continuous accumulation of 3-HBA and 3-HB indicated that these enzymes lacked activity. Four aldehyde-keto reductases (AKRs) were tested to improve the conversion of 3-HBA to 1,3-BDO. Two of the selected AKRs were derived from Pseudomonas denitrificans (P. denitrificans), one was derived from Pseudomonas putida (P. putida), and the other was derived from Pseudomonas aeruginosa (P. aeruginosa). In addition, ygjB and yahK, also known as alcohol dehydrogenase (ADH) from Escherichia coli, were detected. Among them, the enzyme encoded by ygjB was reported to have high activity against a variety of aldehydes, especially butyraldehyde (kcat / Km, 221sec-1 mM -1 ), and the enzyme encoded by yahK was also reported to have high activity towards a variety of aldehydes, especially butyraldehyde (kcat / Km, 19.8sec -1 mM -1 ). Multiple ADH genes were cloned into E. coli, mass produced, isolated and purified, and then the pure enzymes were tested for activity against 3-HBA. The three enzymes Pa_AKR, Ec_YahK and Ec_YgjB showed very high activity, 1.51 U / mg protein, 6.98 U / mg protein and 238 U / mg protein, respectively (Table 5). In contrast, Ec_yqhD, which has been used so far, showed very low activity, only 0.06 U / mg protein.

[0118] Recombinant E. coli with three enzymes, YqhD, YgjB and YahK, were subjected to bioreactor experiments ( Fig.10 Compared with the K8-Q10 strain with YqhD, which produced 410 mM (37.4 g / L) 1,3-BDO, the K8-Q25 strain with YgjB produced 605 mM (56 g / L) after 42 hours of fermentation, while the K8-Q26 strain with YahK produced a high concentration of 570 mM (51.3 g / L) of 1,3-BDO after 42 hours of fermentation. Fig.10 ). This shows that good ADH use is very important for 1,3-BDO production. However, the accumulation of 3-HB did not decrease but increased, and the accumulation of pyruvate occurred in the late fermentation.

[0119] Table 5

[0121] The YgjB enzyme with the best results was selected to study the effect of the expression level of the enzyme on the production of 1,3-BDO. For this purpose, four plasmids including pQ27, pQ28, pQ29, and pQ30 were prepared by weakening the RBS strength (Table 6). The relative strength of RBS was 15% to 189% compared to the control. The newly prepared plasmids were introduced into K8 host cells, and flask experiments were performed using the obtained recombinant strains (Table 7). As a result, the pQ-27 plasmid showed the best results, wherein the UTR of the pQ-27 plasmid was about 50% weaker than the control pQ25. The final concentration of 1,3-BDO was 160mM, and the yield was 0.53mol / mol. The YgjB activity in the cell extract was also studied, and it was 1.3U / mg protein in the pQ27 plasmid, which was 43% of the 2.98U / mg protein of pQ25 as a control. Future experiments involve the use of the pQ27 plasmid.

[0122] Table 6

[0123] Table 7

[0125] <Example 7> Expansion of NADPH supply

[0126] NADPH plays an important role in cell growth and is used as a coenzyme for a variety of enzymes in metabolic processes. Therefore, an adequate supply of NADPH is essential to maintain cell viability and enzyme activity. In redox reactions, the rate of enzymatic reactions within cells is often limited by the availability or regeneration rate of cofactors (NADH and / or NADPH) rather than the amount of enzyme.

[0127] The stoichiometry for the production of 1,3-BDO from glucose is as follows.

[0128] 1 glucose + 2 ADP + 3 NAD + +2NADPH→1 1,3-BDO+2ATP+3NADH+2NADP + +2CO2

[0130] If NADH can be freely converted to NADPH, the conversion can proceed without problems, but if it cannot, 2 moles of NADPH are required for every 1 mole of 1,3-BDO, and NADPH becomes the rate determining factor. In other words, if NADPH is deficient, the conversion of acetyl-CoA to 1,3-BDO becomes rate limiting, and if this slows down the TCA cycle, pyruvate may accumulate due to the lack of NADPH.

[0131] In glucose metabolism by E. coli, NADPH is produced by the following enzymes or pathways:

[0132] 1) Transhydrogenase

[0133] 2) Malic enzyme

[0134] 3) PP and ED pathways

[0135] 4) Isocitrate dehydrogenase in the TCA cycle

[0137] First, the effect of pntAB was studied (Table 8). When pntAB was deleted (K15), the production of 1,3-BDO was significantly reduced by about 50% compared with the wild-type K8. This indicates that NADPH generated by PntAB is very important for the production of BDO.

[0138] Next, for pntAB, the intrinsic promoter was replaced with a stronger trc promoter (K16) to induce overexpression. RT-PCR showed that the transcription of pntAB was enhanced 2-fold. Unfortunately, however, the production of 1,3-BDO was almost unchanged. This may be due to the following reasons: (i) PntAB expressed in the wild type has sufficient activity, or (ii) the enzyme activity of PntAB is not increased after transcriptional upregulation. The experimental results of producing 1,3-BDO by modifying PntAB demonstrated the importance of NADPH supply, but did not show a quantitative relationship.

[0139] Table 8

[0141] To further investigate the role of NADPH, strains K17-K23 were constructed for the role of various pathways / enzymes (Table 8). The second study, following the role of pntAB, was about upregulating the maeB gene, which encodes malic enzyme, which produces NADPH while converting malate to pyruvate (K17). The third study was about blocking the Embden-Meyerhoff-Parnas (EMP) pathway and activating the pentose phosphate (PP) or Entner-Doudoroff (ED) pathways in glycolysis. The third strategy was to perform various gene deletions / upregulations, for example: (1) To block the EMP pathway, the pgi gene encoding phosphoglucose isomerase was removed, which is an enzyme that converts glucose-6-phosphate to fructose-6-phosphate (K18). (2) The zwf gene encoding glucose-6-phosphate dehydrogenase, which converts glucose-6-phosphate to 6-phosphogluconate (6-PG), was upregulated to enhance carbon flux from the glucose-6-phosphate node to the PP pathway and the ED pathway (K19). For this purpose, the intrinsic promoter of the zwf gene was replaced with the stronger trc promoter. (3) To enhance carbon flow from the 6-PG node to the PP pathway, the gnd gene encoding 6-phosphogluconate dehydrogenase, which is an enzyme that converts 6-PG to ribulose-5-phosphate (Ru5P), was upregulated (K20). For this purpose, the intrinsic promoter of the gnd gene was replaced with the stronger trc promoter. (4) To further enhance the PP pathway, the edd gene encoding phosphogluconate dehydratase, which is a key enzyme of the ED pathway and converts 6PG to 2-keto-3-deoxy-6-phosphogluconate (KDPG), was additionally removed. (5) To promote the influx of carbon from 6PG into the ED pathway, the expression of edd and eda genes was increased by removing gntR, a protein involved in the transcriptional regulation of the ED pathway (K22). (6) In addition, edd and eda genes were overexpressed to further enhance ED pathway activity (K23). To this end, the intrinsic promoter of the edd / eda genes was replaced with the stronger trc promoter.

[0142] The pQ27 plasmid was introduced into various variants and the productivity of 1,3-BDO was studied. The K22-Q27 (194 mM and 0.69 mol / mol) and K23-Q27 (178 mM and 0.71 mol / mol) strains with overexpression of the ED pathway showed the best performance. These results indicate that switching the carbon flow from the conventional EMP pathway to the ED pathway significantly increased 1,3-BDO biosynthesis. However, the initial cell growth rate tended to be slightly reduced.

[0143] The strains K22-Q27 and K23-Q27 that showed the best performance in the flask experiments were selected and then subjected to bioreactor experiments ( Fig.11 ). The K8-Q27 strain was used as a control. In the bioreactor, after 48 hours of fermentation, the K22-Q27 strain produced 1,3-BDO at 795 mM (71.2 g / L) and a yield of 0.66 mol / mol. The K23-Q27 strain also produced 1,3-BDO at 760 mM (68.2 g / L) and a yield of 0.62 mol / mol. In contrast, the control strain K8-Q27 produced 1,3-BDO at 620 mM (55.8 g / L) and a yield of 0.60 mol / mol ( Fig.11 ).

[0145] <Example 8> Conversion of 3-HB using carboxylic acid reductase

[0146] In a bioreactor, the K23-Q27 strain produced 1,3-BDO at 760 mM (68.2 g / L) and a yield of 0.62 mol / mol by fermentation for 48 h. However, the strain produced 140 mM of 3-HB as a byproduct. Recently, results of converting 3-HB to 3-HBA using carboxylic acid reductase (CAR) have been reported.

[0147] Since Mm_CAR of Mycobacterium marinum and Ma_CAR of Mycobacterium abscessus were reported to be efficient in producing short-chain diols, two plasmids, pQ31 (containing Mm_CAR) and pQ32 (containing Ma_CAR), were developed and introduced into the K23 strain, respectively, which convert 3-HB into 1,3-BDO produced by additionally expressing these two enzymes.

[0148] After using Ma_CAR enzyme in the bioreactor experiment, 3-HB production was reduced by 60%, BDO titer increased to 883mM, and the yield increased to 0.74mol / mol ( Fig.12 ). In addition, after using the Mm_CAR enzyme, 3-HB production was reduced by 40%, BDO titer increased to 825mM, and the yield increased to 0.70mol / mol. The K23-Q31 and K23-Q32 strains with CAR overexpression showed excellent performance, which solved the problem of 3-HB accumulation in the final stage of 1,3-BDO production. When the dissolved oxygen concentration is high and the NADPH supply is insufficient, 3-HB accumulation will be aggravated, and because the K23-Q31 and K23-Q32 strains minimize the impact of aeration, they become commercially advantageous.

[0150] Although the specific parts of the present disclosure have been described in detail above, it is clear to those skilled in the art that this specific description is only a preferred exemplary embodiment, and the scope of the present disclosure is not limited thereto. Therefore, the essential scope of the present disclosure is defined by the appended claims and their equivalents.

[0151] The scope of the present disclosure is shown by the claims, and the meaning and scope of the claims and all modified or changed forms derived from equivalent concepts should be understood to be included in the scope of the present disclosure. (Translation) Proof of receipt in original depository condition recipient: 51, Pancheongangbyeon-gil, Eonyang-eup, Ulsan, South Korea Same as the original translation above

Claims

1. A recombinant expression vector, which comprises a butyraldehyde dehydrogenase (bld) gene, an acetyl-CoA acetyltransferase (phaA) gene, an acetoacetyl-CoA reductase (phaB) gene and a 3-hydroxybutyraldehyde (3-HBA) reductase gene in sequence.

2. The recombinant expression vector according to claim 1, wherein The bld gene comprises the nucleotide sequence shown by SEQ ID NO:7, the phaA gene comprises the nucleotide sequence shown by SEQ ID NO:5, and the phaB gene comprises the nucleotide sequence shown by SEQ ID NO:

6.

3. The recombinant expression vector according to claim 1, wherein The 3-HBA reductase gene is the yqhD gene containing the nucleotide sequence shown by SEQ ID NO:34, the ygjB gene containing the nucleotide sequence shown by SEQ ID NO:35, or the yahK gene containing the nucleotide sequence shown by SEQ ID NO:

36.

4. The recombinant expression vector according to claim 3, wherein The ribosome binding site (RBS) in the 5' untranslated region (UTR) of the ygjB gene comprises the nucleotide sequence shown by SEQ ID NO:37 or SEQ ID NO:

38.

5. The recombinant expression vector according to claim 1, wherein The recombinant expression vector further comprises a carboxylic acid reductase (CAR) gene.

6. The recombinant expression vector according to claim 5, wherein The CAR gene comprises the nucleotide sequence shown by SEQ ID NO:20 or SEQ ID NO:

21.

7. A recombinant Escherichia coli strain having increased 1,3-butanediol (1,3-BDO) production, wherein the recombinant Escherichia coli strain is transformed with the recombinant expression vector according to any one of claims 1 to 6.

8. The recombinant E. coli strain with increased 1,3-BDO production according to claim 7, wherein: The recombinant Escherichia coli strain is a strain obtained by transforming an Escherichia coli strain in which the following genes are deleted using the recombinant expression vector: alcohol dehydrogenase (adhE) gene, pyruvate oxidase (poxB) gene, D-lactate dehydrogenase (ldhA) gene, phosphate acetyltransferase-acetate kinase (pta-ackA) gene and acyl-CoA thioesterase (yciA) gene.

9. The recombinant E. coli strain with increased 1,3-BDO production according to claim 8, wherein: The recombinant Escherichia coli strain is a strain obtained by transforming an Escherichia coli strain in which the DNA binding transcription double regulatory factor (pdhR) gene is additionally deleted with the recombinant expression vector.

10. The recombinant E. coli strain with increased 1,3-BDO production according to claim 9, wherein The recombinant Escherichia coli strain is a strain obtained by transforming an Escherichia coli strain in which the citrate synthase (gltA) gene is additionally deleted with the recombinant expression vector.

11. The recombinant Escherichia coli strain with increased 1,3-BDO production according to claim 9, wherein: The recombinant Escherichia coli strain is a strain obtained by transforming the following Escherichia coli strain with the recombinant expression vector, in which the glucose-6-phosphate isomerase (pgi) gene and the DNA-binding transcription repressor (gntR) gene are additionally deleted, and the NADP+-dependent glucose-6-phosphate dehydrogenase (zwf) gene is additionally overexpressed.

12. The recombinant Escherichia coli strain with increased 1,3-BDO production according to claim 11, wherein The recombinant Escherichia coli strain is a strain obtained by transforming an Escherichia coli strain in which the following genes are additionally overexpressed: phosphogluconate dehydratase (edd) gene and 2-keto-3-deoxygluconate 6-phosphate aldolase (eda) gene with the recombinant expression vector.

13. The recombinant E. coli strain with increased 1,3-BDO production according to claim 12, wherein: The recombinant Escherichia coli strain is a strain deposited with the deposit number KCTC 15315BP.

14. A method for increasing 1,3-BDO production, the method comprising culturing the recombinant Escherichia coli strain according to claim 7.

Images