Recombinant microorganism for producing 1,3-propanediol and method for producing 1,3-propanediol using same

Abstract

The present invention relates to a recombinant microorganism for producing 1,3-propanediol and a method for producing 1,3-propanediol using same. More specifically, the present invention provides a recombinant microorganism having an improved ability to produce 1,3-propanediol through overexpression of a transaldolase-encoding gene (tal), a succinyl-CoA synthetase-encoding gene (sucCD), a cystathionine γ-synthase-encoding gene (metB), an aspartate ammonia lyase-encoding gene(aspA), an aspartate aminotransferase-encoding gene (aspB), or a functional fragment thereof, and a method for producing 1,3-propanediol using same.

Description

Recombinant microorganism for 1,3-propanediol production and method for producing 1,3-propanediol using the same

[0001] This application claims priority based on Korean No. 10-2024-0198710 filed on December 27, 2024, and all contents disclosed in the specification and drawings of said application are incorporated into this application.

[0002] The present invention relates to a recombinant microorganism for producing 1,3-propanediol and a method for producing 1,3-propanediol using the same. More specifically, the invention provides a recombinant microorganism with enhanced 1,3-propanediol production ability through the overexpression of a gene coding for transaldolase (tal), a gene coding for succinyl-CoA synthetase (sucCD), a gene coding for cystathionine γ-synthase (metB), a gene coding for aspartate ammonia lyase (aspA) or a gene coding for aspartate aminotransferase (aspB) or a functional fragment thereof, and a method for producing 1,3-propanediol using the same.

[0003] 1,3-propanediol (1,3-PDO) is a chemical used as a monomer in the synthesis of polymers such as polyethers, polyurethanes, and polytrimethylene terephthalate (PTT). Conventional production methods for 1,3-PDO have primarily utilized chemical synthesis, employing methods such as the hydration of acrolein, the hydroformylation of ethylene oxide in the presence of phosphine, or the enzymatic conversion of glycerol. However, these chemical production methods have limitations due to high costs and environmentally harmful production processes (Non-patent Literature 1 and Patent Literature 1).

[0004] Biological methods for producing 1,3-PDO include using microorganisms, primarily using Klebsiella, Clostridia, Enterobacter, Citrobacter, and Lactobacilli. These all directly convert glycerol into 1,3-PDO through two consecutive metabolic pathways, in which glycerol is converted to 3-hydroxypropionaldehyde (3-HPA) by glycerol dehydratase, and then 3-HPA is reduced to 1,3-PDO by 1,3-propanediol oxidoreductase. DuPont has already succeeded in commercializing 1,3-PDO by introducing the above metabolic pathway into E. coli. However, the reality is that most of the aforementioned microorganisms, including E. coli that biosynthesize 1,3-PDO, still have low production capacity compared to chemical processes. Therefore, it is necessary to identify target genes that can assist in the biosynthesis of 1,3-PDO within microorganisms.

[0005] Accordingly, the inventors made diligent efforts to produce 1,3-PDO more efficiently through biological pathways. As a result, for the purpose of increasing 1,3-PDO productivity in producing 1,3-PDO from a Corynebacterium glutamicum strain, they selected five overexpression target genes predicted to be effective for increasing 1,3-PDO production through in silico simulation, and introduced these genes into a microorganism along with a gene encoding glycerol dehydratase, a gene encoding glycerol reactivase, and a gene encoding 1,3-PDO oxidoreductase, thereby completing the present invention by producing a recombinant microorganism with improved 1,3-PDO production efficiency.

[0006] [Prior Art Literature]

[0007] [Patent Literature]

[0008] (Patent Document 1) U.S. Registered Patent No. 8,236,994

[0009] [Non-patent literature]

[0010] (Non-patent Document 1) Lee, CS, et al. “A review: conversion of bioglycerol into 1, 3-propanediol via biological and chemical method.” Renewable and Sustainable Energy Reviews 42 (2015): 963-972.

[0011] Accordingly, the present invention aims to provide a recombinant vector containing a gene capable of improving 1,3-PDO production capacity compared to a wild-type strain through overexpression.

[0012] Another objective of the present invention is to provide a recombinant microorganism in which a specific gene is overexpressed and the 1,3-PDO production capacity is improved compared to a wild-type strain.

[0013] Another objective of the present invention is to provide a method for producing 1,3-PDO using the recombinant microorganism.

[0014] Another objective of the present invention is to provide a kit for producing 1,3-PDO comprising the recombinant microorganism and culture medium composition, and a method for producing 1,3-PDO using the same.

[0015] To solve the above-mentioned problem, the present invention provides a composition for improving the production capacity of 1,3-propanediol (1,3-propanediol) comprising a recombinant vector comprising one or more selected from the group consisting of (a) to (e) below:

[0016] (a) a gene (tal) encoding transaldolase or a functional fragment thereof;

[0017] (b) a gene (sucCD) encoding succinyl-CoA synthetase or a functional fragment thereof;

[0018] (c) a gene (metB) encoding cystathionine γ-synthase or a functional fragment thereof;

[0019] (d) a gene (aspA) encoding aspartate ammonia lyase or a functional fragment thereof; and

[0020] (e) The gene (aspB) encoding aspartate aminotransferase or a functional fragment thereof.

[0021] In addition, the present invention provides a recombinant vector comprising one or more selected from the group consisting of (a) to (e) for use in the production of a recombinant microorganism for 1,3-propanediol production, and a use of said recombinant vector.

[0022] In the present invention, the tal, sucCD, metB, aspa, and aspaB genes may be derived from Corynebacterium.

[0023] In the present invention, the tal gene may include the nucleic acid sequence of SEQ ID NO. 1, the sucCD gene may include the nucleic acid sequence of SEQ ID NO. 2, the metB gene may include the nucleic acid sequence of SEQ ID NO. 3, the aspa gene may include the nucleic acid sequence of SEQ ID NO. 4, and the aspaB gene may include the nucleic acid sequence of SEQ ID NO. 5.

[0024] In the present invention, the genes may be selected through in silico simulation comprising the following steps (a) to (f):

[0025] (a) A step of determining the maximum and minimum values ​​of the 1,3-PDO production rate of microorganisms;

[0026] (b) a step of dividing the range between the minimum and maximum values ​​of the determined 1,3-PDO production rate into 10 equal steps and calculating the maximum value of the cell growth rate for each 1,3-PDO production rate;

[0027] (c) A step of setting a reference to a metabolic flow in which the cell growth rate has a maximum value while 1,3-PDO is not produced;

[0028] (d) A step of calculating an internal metabolic flow that minimizes the difference from the metabolic flow set as a reference in step (c), while satisfying the maximum value of the cell growth rate according to the 1,3-PDO production rate calculated in step (b) as a limiting condition;

[0029] (e) a step of analyzing the linear positive relationship between internal metabolic flow and 1,3-PDO production rate; and

[0030] (f) A step of selecting a response exhibiting a positive linear relationship as an overexpression target.

[0031] A composition for improving 1,3-PDO production capacity, wherein the vector further comprises a strong promoter selected from the group consisting of tac, trc, H36, and tuf.

[0032] In addition, the present invention provides a recombinant microorganism having enhanced 1,3-PDO production ability in which a gene encoding transaldolase (tal), a gene encoding succinyl-CoA synthetase (sucCD), a gene encoding cystathionine γ-synthase (metB), a gene encoding aspartate ammonia lyase (aspA) or a gene encoding aspartate aminotransferase (aspB) is overexpressed in a microorganism having 1,3-propanediol biosynthetizing ability.

[0033] In the present invention, the tal gene may include the nucleic acid sequence of SEQ ID NO. 1, the sucCD gene may include the nucleic acid sequence of SEQ ID NO. 2, the metB gene may include the nucleic acid sequence of SEQ ID NO. 3, the aspa gene may include the nucleic acid sequence of SEQ ID NO. 4, and the aspaB gene may include the nucleic acid sequence of SEQ ID NO. 5.

[0034] In the present invention, the microorganism having the 1,3-PDO biosynthetic ability may be one in which (i) a gene encoding glycerol dehydratase, (ii) a gene encoding glycerol reactivase, and (iii) a gene encoding 1,3-propanediol oxidoreductase have been introduced.

[0035] In the present invention, the gene encoding glycerol dihydratase and the gene encoding glycerol reactivase may be a pduCDEGH cluster containing the nucleic acid sequence of SEQ ID NO. 19, and the gene encoding 1,3-propanediol oxidoreductase may be a yqhD containing the nucleic acid sequence of SEQ ID NO. 20.

[0036] In the present invention, the genes may be overexpressed by a strong promoter selected from the group consisting of H36 promoter, H30 promoter, lac promoter, trp promoter, trc promoter, tac promoter, tuf promoter, sod promoter, lambda phage PR promoter, PL promoter, T7 promoter, and tet promoter.

[0037] In the present invention, the microorganism having 1,3-PDO biosynthesis may be selected from the group consisting of Corynebacterium, Klebsiella, Clostridia, Enterobacter, Citrobacter, and Lactobacilli.

[0038] In the present invention, the microorganism having 1,3-PDO biosynthesis may be Corynebacterium.

[0039] Additionally, the present invention provides a method for producing 1,3-PDO comprising the following steps (a) and (b):

[0040] (a) a step of producing 1,3-PDO by culturing the aforementioned recombinant microorganism; and

[0041] (b) Step of recovering the produced 1,3-PDO.

[0042] In the present invention, the recombinant microorganism of step (a) may be cultured in a medium containing glycerol.

[0043] In addition, the present invention provides a kit for producing 1,3-propanediol comprising the following (a) and (b):

[0044] (a) the aforementioned composition for improving 1,3-PDO production capacity or the aforementioned recombinant microorganism with improved 1,3-PDO production capacity; and

[0045] (b) Culture medium composition.

[0046] Furthermore, the present invention provides a method for producing 1,3-propanediol using the aforementioned kit.

[0047] The recombinant microorganism according to the present invention has enhanced 1,3-PDO production capacity compared to wild-type strains, as gene overexpression is performed through metabolic engineering methods. In addition, genes for converting glycerol to 1,3-PDO are introduced, enabling high-efficiency production of 1,3-PDO using low-cost glycerol as a carbon source.

[0048] Figure 1 is a schematic diagram showing the in silico simulation process for selecting target genes for overexpression that are predicted to be effective for 1,3-PDO production.

[0049] Figure 2 shows the results of 1,3-PDO production when glucose and glycerol are simultaneously utilized as carbon sources by introducing target genes for overexpression, which were predicted to be effective for 1,3-PDO production through in silico simulation, into Corynebacterium glutamicum strains using a plasmid-based overexpression system.

[0050] The present invention will be described in more detail below.

[0051] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention.

[0052] All numbers expressing the size, quantity, and physical properties of a feature used in this specification and claims should be understood as being modified by the term "approximately" in all cases. Accordingly, unless otherwise indicated, the numerical parameters disclosed in this specification and claims are approximations that may vary depending on the desired properties to be obtained by a person skilled in the art using the teachings disclosed in this specification.

[0053] As described above, since the production capacity of 1,3-PDO using microorganisms is still low compared to chemical processes, it is necessary to identify target genes that can aid in the biosynthesis of 1,3-PDO within microorganisms. Accordingly, the inventors sought a solution to the aforementioned problem by selecting genes capable of enhancing 1,3-PDO biosynthesis through Flux Balance Analysis (FBA), a method that mathematically models metabolic networks within organisms to optimize the flux of metabolic reactions, and by overexpressing these genes within microorganisms to produce recombinant microorganisms with improved 1,3-PDO production capacity compared to wild-type strains.

[0054] Accordingly, the first aspect of the present invention relates to a composition for improving the production capacity of 1,3-propanediol (1,3-propanediol) comprising a recombinant vector comprising one or more selected from the group consisting of (a) to (e) below:

[0055] (a) a gene (tal) encoding transaldolase or a functional fragment thereof;

[0056] (b) a gene (sucCD) encoding succinyl-CoA synthetase or a functional fragment thereof;

[0057] (c) a gene (metB) encoding cystathionine γ-synthase or a functional fragment thereof;

[0058] (d) a gene (aspA) encoding aspartate ammonia lyase or a functional fragment thereof; and

[0059] (e) The gene (aspB) encoding aspartate aminotransferase or a functional fragment thereof.

[0060] In relation to the first aspect above, the present invention also provides a recombinant vector comprising one or more selected from the group consisting of (a) to (e) for use in the production of a recombinant microorganism for 1,3-propanediol production.

[0061] Additionally, the present invention provides a use of a recombinant vector comprising one or more selected from the group consisting of (a) to (e) for use in the production of a recombinant microorganism for 1,3-propanediol production.

[0062] In a specific embodiment of the present invention, a target gene for overexpression predicted to be effective in increasing 1,3-PDO production was selected through in silico simulation using a Corynebacterium glutamicum metabolic model (1,243 metabolites, 1,775 reaction schemes).

[0063] Various methods for predicting target genes to be manipulated for enhanced production of specific biological substances within microorganisms based on genome-scale metabolic models (GEMs) are known. Examples of such methods include, but are not limited to, OptStrain, OptForce, k-OptForce, FSEOF (flux scanning based on enforced objective flux), FVSEOF (flux variability scanning based on enforced objective flux), CosMos (constraint-based strain design using continuous modifications), optimal cofactor swapping, and iBRIDGE.

[0064] Specifically, the aforementioned overexpression target gene can be selected through a process as illustrated in FIG. 1, which is represented step by step as follows:

[0065] (a) A step of determining the maximum and minimum values ​​of the 1,3-PDO production rate of microorganisms;

[0066] (b) a step of dividing the range between the minimum and maximum values ​​of the determined 1,3-PDO production rate into 10 equal steps and calculating the maximum value of the cell growth rate for each 1,3-PDO production rate;

[0067] (c) A step of setting a reference to a metabolic flow in which the cell growth rate has a maximum value while 1,3-PDO is not produced;

[0068] (d) A step of calculating an internal metabolic flow that minimizes the difference from the metabolic flow set as a reference in step (c), while satisfying the maximum value of the cell growth rate according to the 1,3-PDO production rate calculated in step (b) as a limiting condition;

[0069] (e) a step of analyzing the linear positive relationship between internal metabolic flow and 1,3-PDO production rate; and

[0070] (f) A step of selecting a response exhibiting a positive linear relationship as an overexpression target.

[0071] In the present invention, step (d) can be calculated as a slope. The slope can be calculated through linear regression between the 1,3-PDO production rate and the rate of the model's internal reaction equation. Specifically, the slope can be calculated by using the maximum value of the cell growth rate for each 1,3-PDO production rate calculated in step (b) as a constraint, and sequentially applying Least Absolute Deviation (LAD) regression to calculate an internal metabolic flow that satisfies the constraint while undergoing minimal change from the reference flux (x: flow value of the internal reaction equations calculated by applying LAD, y: 1,3-PDO production rate).

[0072] In the present invention, the linear positive relationship of step (e) can be analyzed using the slope calculated in step (d), and the higher the positive value of the calculated slope, the more likely it is to be selected as an overexpression priority target.

[0073] In a specific embodiment of the present invention, the slope was calculated, the maximum value was set to 1 through min-max normalization, and targets with a slope of 0.2 or greater were selected (normalized slope > 0.2).

[0074] Through the process described above, five types of overexpression target genes—tal, sucCD, metB, aspa, and aspa—were selected.

[0075] In the present invention, the term "tal" refers to a gene encoding transaldolase. Preferably, "tal" may be Corynebacterium, more preferably Corynebacterium glutamicum. For example, the tal gene may include or be composed of the nucleic acid sequence of SEQ ID NO. 1, but is not limited thereto.

[0076] In the present invention, the term "sucCD" refers to a gene encoding succinyl-CoA synthetase. Preferably, "sucCD" may be Corynebacterium, more preferably Corynebacterium glutamicum. For example, the sucCD gene may include or be composed of the nucleic acid sequence of SEQ ID NO. 2, but is not limited thereto.

[0077] In the present invention, the term "metB" refers to a gene encoding cystathionine γ-synthase. Preferably, "metB" may be Corynebacterium, and more preferably, Corynebacterium glutamicum. For example, the metB gene may include or be composed of the nucleic acid sequence of SEQ ID NO. 3, but is not limited thereto.

[0078] In the present invention, the term "aspA" refers to a gene encoding aspartate ammonia lyase. Preferably, "aspA" may be Corynebacterium, more preferably Corynebacterium glutamicum. For example, the aspa gene may include or be composed of the nucleic acid sequence of SEQ ID NO. 4, but is not limited thereto.

[0079] In the present invention, the term "aspB" refers to a gene encoding aspartate aminotransferase. Preferably, "aspB" may be Corynebacterium, and more preferably, Corynebacterium glutamicum. For example, the aspB gene may include or be composed of the nucleic acid sequence of SEQ ID NO. 5, but is not limited thereto.

[0080] The "functional fragment" above refers to a gene fragment that is composed of a part of the nucleic acid sequence of SEQ ID NOs 1, 2, 3, 4, or 5, but codes for a fragment that can function substantially identically or similarly to the enzyme encoded by the tal, sucCD, metB, aspa, and aspaB genes, respectively.

[0081] In addition, the nucleic acid sequence may include a gene that can function substantially identically or similarly to the tal, sucCD, metB, aspa, and aspa, respectively, as a nucleic acid sequence exhibiting at least 70%, preferably 80%, more preferably 90%, even more preferably 95%, and most preferably 98% homology with the above nucleic acid sequence. It is obvious that the aforementioned functional fragment is also included within the scope of the present invention.

[0082] The above nucleic acid sequence may be codon-optimized for a type of host cell, and such codon optimization techniques based on host cells are well known in the field (Fuglsang et al., Protein Expr. Purif. 31(2): 247-249, 2003).

[0083] In the present invention, the term "homology" refers to the degree of identity or correspondence between given nucleic acid sequences that may or may not share a common evolutionary origin and may be expressed as a percentage. In this specification, a homologous sequence having the same or similar activity as a given nucleic acid sequence is indicated as "% homology." For example, this can be verified by using standard software, specifically BLAST 2.0, to calculate parameters such as score, identity, and similarity, or by comparing sequences by Southern hybridization experiments under defined strict conditions, and the defined appropriate hybridization conditions can be determined by methods well known to those skilled in the art.

[0084] The gene or functional fragment thereof of the present invention can be produced by extraction from nature, synthesis, or by a genetic recombination method based on a DNA sequence.

[0085] The above gene or its functional fragment may be inserted into a vector, preferably with a known strong promoter.

[0086] In the present invention, the term "vector" refers to a nucleic acid product containing a nucleic acid sequence operably linked to a suitable expression control sequence capable of expressing a gene within a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. When transformed into a suitable host, the vector may replicate and function independently of the host genome, or in some cases, be incorporated into the genome itself. Since plasmids are the most commonly used form of vector currently, "plasmid" and "vector" are sometimes used interchangeably in the specification of the present invention. However, the present invention also includes other forms of vectors having functions equivalent to those known or to be known in the art. Numerous conventional vectors available for use in prokaryotic cells are well known to those skilled in the art, and a suitable vector can be selected and used. Conventional vectors available for use in the present invention include, but are not limited to, pCES208, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pUC19, λgtㆍ4λB, λ-charon, λΔz1, and M13.

[0087] Protein expression vectors used in Escherichia coli include the pET, pCDF, pRSF, pACYC, and pCOLA series from Novagen (USA); the pBAD series from Invitrogen (USA); pHCE or pCOLD from Takara (Japan); the pACE series from Genofocus (South Korea); the pTac15K, pTrc99A, pTacCDFS, and pTrcCDFS series from KAIST (South Korea); and the pBBR1MCS series, which is applicable across a wide range of strains. In Bacillus subtilis, protein expression can be achieved by inserting a target gene into a specific region of the genome, or by using vectors such as the pHT series from MoBiTech (Germany). Protein expression is also possible in fungi and yeasts using genome insertion or self-replication vectors. Plant protein expression vectors can be used by utilizing T-DNA systems such as Agrobacterium tumefaciens or Agrobacterium rhizogenes. Typical expression vectors for mammalian cell culture expression are based, for example, pRK5 (EP 307,247), pSV16B (WO 91 / 08291), and pVL1392 (Pharmingen).

[0088] The term "expression control sequence" refers to a DNA sequence essential for the expression of a coding sequence operably linked in a specific host organism. Such control sequences include a promoter for carrying out transcription, an optional operator sequence for regulating such transcription, a sequence coding for a suitable mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. For example, a control sequence suitable for prokaryotes includes a promoter, optionally an operator sequence, and a ribosome binding site. For eukaryotes, it includes a promoter, a polyadenylation signal, and an enhancer. The factor that most influences the expression level of a gene in a plasmid is the promoter. For high expression, SRα promoters and cytomegalovirus-derived promoters are preferably used.

[0089] Any of the wide variety of expression regulatory sequences may be used in a vector to express the gene or a functional fragment thereof of the present invention. Examples of useful expression regulatory sequences include, in addition to the promoters described above, early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, T3 and T7 promoters, the major operator and promoter regions of phage lambda, the regulatory regions of fd code proteins, promoters for 3-phosphoglycerate kinase or other glycolases, promoters of said phosphatase, e.g., Pho5, promoters of the yeast alpha-mating system, and other sequences of configuration and induction known to regulate the expression of genes in prokaryotic or eukaryotic cells or viruses thereof, and various combinations thereof. The T7 RNA polymerase promoter Φ can be usefully used to express proteins in E. coli.

[0090] Of course, it must be understood that not all vectors and expression regulatory sequences function equally in expressing the DNA sequence of the present invention. Likewise, not all hosts function equally for the same expression system. However, those skilled in the art can make appropriate selections among various vectors, expression regulatory sequences, and hosts without departing from the scope of the present invention and without excessive experimental burden. For example, when selecting a vector, the host must be considered, as the vector must be replicated within it. The copy number of the vector, the ability to regulate the copy number, and the expression of other proteins encoded by said vector, such as antibiotic markers, must also be considered. When selecting an expression regulatory sequence, various factors must also be considered. For example, the relative strength of the sequence, its modulation capabilities, and compatibility with the DNA sequence of the present invention, particularly in relation to potential secondary structures, must be taken into account. A unicellular host must be selected by considering factors such as the selected vector, the toxicity and secretion characteristics of the product encoded by the DNA sequence of the present invention, the ability to accurately fold the protein, culture and fermentation requirements, and the ease of purifying the product encoded by the DNA sequence of the present invention from the host. Within the range of these variables, a person skilled in the art may select various vector / expression control sequence / host combinations capable of expressing the DNA sequence of the present invention in fermentation or large-scale animal culture. Binding methods, panning methods, film emulsion methods, etc., may be applied as screening methods when attempting to clone cDNA by expression cloning.

[0091] In the above vector, nucleic acids are "operably linked" when positioned in a functional relationship with other nucleic acid sequences. This may be a gene and regulatory sequence(s) linked in such a way that a suitable molecule (e.g., a transcription-activating protein) enables gene expression when it binds to the regulatory sequence(s). For example, DNA for a pre-sequence or secretion leader is operably linked to DNA for a polypeptide when expressed as a pre-sequence protein participating in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence when it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence when it influences the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence when positioned to facilitate translation. Generally, "operably linked" means that the linked DNA sequences are in contact, and in the case of a secretion leader, they are in contact and exist within the reading frame. However, an enhancer does not need to be in contact. The linkage of these sequences is performed by ligation at a convenient restriction enzyme site. If such a site is not present, a synthetic oligonucleotide adaptor or linker is used according to conventional methods.

[0092] The above-mentioned recombinant vector may be introduced into a host cell by means such as transformation or transfection. As used herein, the term "transformation" means the introduction of DNA into a host so that the DNA becomes replicable as an extrachromosomal factor or through the completion of chromosomal integration. As used herein, the term "transfection" means the acceptance of an expression vector by a host cell, regardless of whether any coding sequence is actually expressed.

[0093] Transformation of host cells can be performed by numerous methods known in the art. For example, when using prokaryotic cells as host cells, the CaCl2 method, the Hanson method (Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9:2110-2114(1973); and Hanahan, D., J. Mol. Biol., 166:557-580(1983)) and electroporation can be used for transformation.

[0094] As is well known in the art, in order to increase the expression level of a transfected gene in recombinant cells, the gene must be operably linked to transcription and translation expression regulatory sequences that function within a selected expression host.

[0095] Preferably, the expression regulatory sequence and the corresponding gene are included within a single expression vector that also contains a bacterial selection marker and a replication origin. If the expression host is a eukaryotic cell, the expression vector may further include expression markers useful within the eukaryotic expression host.

[0096] In one embodiment of the present invention, the vector may be transformed or transfected into host cells for the overexpression of a specific gene. In a specific embodiment, the vector may be transformed according to standard protocols such as the Gibson assembly protocol (Gibson et al., Nat. Methods 6, 343-345. (2009); Green et al., 4 th A vector can be manufactured in a form in which the desired gene and expression regulatory sequence are linked so as to be operable, but is not limited thereto, and a person skilled in the art can manufacture the vector through conventionally known methods.

[0097] In the present invention, the recombinant vector may additionally include a gene encoding aspartate 1-decarboxylase (ADC).

[0098] The term "panD" in the present invention refers to the gene encoding aspartate 1-decarboxylase (ADC). In one embodiment of the present invention, the panD gene may be inserted into a vector, preferably together with a known strong promoter. The panD gene may be overexpressed by transforming the vector into a microorganism having 1,3-PDO biosynthetic ability. The above panD gene may be, for example, a panD gene derived from Corynebacterium glutamicum, Bacillus subtilis, Rhodococcus opacus, Streptomyces griseus, Bacillus thuringiensis, and Saccharopolyspora erythraea, but is not limited thereto. In a specific embodiment of the present invention, a panD gene derived from Corynebacterium glutamicum, comprising or composed of the nucleic acid sequence of SEQ ID NO. 25, was used.

[0099]

[0100] In the present invention, the recombinant vector may further include a nucleic acid molecule encoding the NCgl2647 protein. The NCgl2647 protein plays a role in inducing the secretion of 1,3-PDO from within the cell to outside the cell for production, and may be composed of the amino acid sequence of SEQ ID NO. 26 or a partial amino acid sequence thereof.

[0101]

[0102] In addition, the nucleic acid molecule encoding the NCgl2647 protein may be composed of the nucleic acid sequence of SEQ ID NO. 27 or a part thereof, but any nucleic acid sequence encoding the NCgl2647 protein or a functional fragment thereof may be used without limitation.

[0103]

[0104] A vector containing each of the five genes of the present invention or a functional fragment thereof can be transformed into a microorganism having 1,3-PDO biosynthetic ability to produce recombinant microorganisms in which each of the five genes is overexpressed. The recombinant microorganism may exhibit improved 1,3-PDO production ability compared to a wild-type strain.

[0105] Accordingly, the second aspect of the present invention relates to a recombinant microorganism having 1,3-PDO biosynthetic ability in which the genes tal, sucCD, metB, aspa, or aspaB are overexpressed, thereby enhancing the 1,3-PDO production ability.

[0106] In the recombinant microorganism of the present invention, the description of the five types of genes is the same as that described in the first aspect above, so the description is omitted.

[0107] The term "recombinant microorganism" in the present invention refers to a microorganism that has been transformed by genetically recombinant DNA introduced through genetic recombination technology. The genetically recombinant DNA refers to DNA produced by combining DNA capable of replication within a cell (carrier) with heterologous DNA in a test tube using enzymes, etc. In the present invention, the recombinant microorganism may have enhanced 1,3-PDO production capacity through genetic recombination technology. In certain embodiments, the recombinant microorganism may be a microorganism into which specific exogenous / endogenous genes are introduced or in which specific exogenous / endogenous genes are overexpressed, but is not limited thereto.

[0108] The term "genetic manipulation" in the present invention refers to an act of altering traits by modifying the function of a gene possessed by an organism, and includes gene overexpression, inhibition or reduction of gene expression, blocking of gene expression, and substitution of a specific gene with another gene. In the present invention, the genetic manipulation may be performed independently, or multiple genetic manipulations may be performed in combination.

[0109] The term "overexpression" of a gene in this invention refers to an increase in gene expression compared to non-modified microorganisms, resulting in an increase in the intracellular concentration of ribonucleic acid, proteins, or enzymes compared to non-modified microorganisms. The above term "overexpression" may be achieved through methods such as replacing the intrinsic promoter of a specific gene with a potent promoter or introducing an external gene, but is not limited thereto.

[0110] Conventionally known techniques for overexpression include, for example,

[0111] (1) Increasing the copy number of a gene in a microorganism; the gene is coded either on a chromosome or extrachromosomally. If the gene is located on a chromosome, multiple copies of the gene may be introduced onto the chromosome by recombination methods (including gene replacement) known to experts in the relevant art. If the gene is located extrachromosomally, it may be retained in the cell by different types of plasmids with different replication origins and consequently different copy numbers. These plasmids are present in the microorganism in 1 to 5 copies, or about 20 copies, or up to 500 copies, depending on the nature of the plasmid: low-copy number plasmids with dense replication (pSC101, RK2), low-copy number plasmids (pACYC, pRSF1010), or high-copy number plasmids (pSK bluescript II).

[0112] (2) Introducing an external gene that can enhance the function of the gene in question;

[0113] (3) Use of a promoter that leads to high levels of gene expression; for example, strong promoters such as the H36 promoter, H30 promoter, lac promoter, trp promoter, trc promoter, tac promoter, tuf promoter, sod promoter, lambda phage PR promoter, PL promoter, T7 promoter and tet promoter may be widely used, but are not limited thereto. These promoters may be "inducible" by specific compounds or by specific external conditions such as temperature or light. These promoters may be homologous or heterologous.

[0114] (4) Attenuating the activity or expression of specific or non-specific transcriptional repressors of genes;

[0115] (5) Using a corresponding messenger RNA stabilizing element (Carrier and Keasling, 1999) or a protein stabilizing element (e.g., GST tag, GE Healthcare);

[0116] (6) There are methods such as changing the sequence of the 5' untranslated region (5' UTR), but are not limited to this.

[0117] The term "gene introduction" above refers to the new introduction of a target gene or a vector containing the same into a target cell or microorganism. In one embodiment of the present invention, the five genes were each inserted into a plasmid vector along with a promoter that increases their expression and then introduced into Corynebacterium glutamicum (C. glutamicum); however, the gene introduction method is not limited thereto and can be introduced into microorganisms through various conventionally known methods. In the present invention, it is preferable to introduce the gene into the microorganism using a vector, but it is not limited thereto. In the present invention, the gene may be directly introduced into the genome of a host cell and exist as a chromosomal factor. It will be obvious to those skilled in the art to which the present invention pertains that inserting the gene into the genomic chromosome of a host cell will produce the same effect as introducing a recombinant vector into the host cell.

[0118] The recombinant microorganism according to the present invention may be modified such that the tal, sucCD, metB, aspa, or aspaB genes are overexpressed, and the activity of the enzyme encoded by each of these genes is enhanced compared to the intrinsic activity of the corresponding enzyme.

[0119] The term "intrinsic activity" as used in this invention refers to the active state of an enzyme that a microorganism possesses in its original, unmodified state, and the meaning of "modified to be enhanced relative to intrinsic activity" means that the said activity is newly introduced or further improved when compared to the enzyme activity in the state prior to modification.

[0120] In the present invention, "enhancement of enzyme activity" includes not only the introduction or increase of the enzyme's own activity to derive an effect beyond its original function, but also an increase in activity caused by an increase in intrinsic gene activity, intrinsic gene amplification from internal or external factors, deletion of a regulatory factor that inhibits said gene expression, an increase in gene copy number, introduction of a gene from the outside, modification of an expression regulatory sequence, particularly a replacement or modification of a promoter, and an increase in enzyme activity caused by an intragenetic mutation.

[0121] In the present invention, "modified to be enhanced relative to intrinsic activity" means a state in which the activity of a microorganism after manipulation is increased compared to the activity of the microorganism before manipulation, such as the introduction of an activity-exhibiting gene, an increase in the copy number of said gene, the deletion of a regulatory factor that inhibits said gene expression, a modification of an expression regulatory sequence, or the use of an improved promoter.

[0122] The term "microorganism having 1,3-PDO biosynthetic ability" in the present invention refers to a microorganism capable of producing 1,3-PDO within a living organism, and may include both microorganisms that do not inherently have 1,3-PDO biosynthetic ability but are endowed with 1,3-PDO biosynthetic ability, and microorganisms that inherently have 1,3-PDO biosynthetic ability. 1,3-PDO biosynthetic ability may be endowed or enhanced by species improvement.

[0123] In the present invention, the microorganism having the 1,3-PDO biosynthetic ability may be selected from the group consisting of Corynebacterium, Klebsiella, Clostridia, Enterobacter, Citrobacter, and Lactobacilli, and preferably may be Corynebacterium, but is not limited thereto. The above Corynebacterium may include Corynebacterium glutamicum, Corynebacterium ammoniagenes, or Corynebacterium thermoaminogenes, but may also include other known microorganisms of the genus Corynebacterium with 1,3-PDO biosynthetic ability without limitation.

[0124] In the present invention, the microorganism having the 1,3-PDO biosynthetic ability may additionally have (i) a gene encoding glycerol dehydratase, (ii) a gene encoding glycerol reactivase, and (iii) a gene encoding 1,3-propanediol oxidoreductase introduced.

[0125] In the present invention, the genes of (i) to (iii) code for enzymes that perform the role of converting glycerol into 1,3-PDO, and the microorganism into which these genes are additionally introduced can produce 1,3-PDO with high efficiency using glycerol as a carbon source.

[0126] The genes of (i) to (iii) above may be derived from Klebsiella pneumoniae, for example, from Klebsiella pneumoniae DSMZ2026, but are not limited thereto. In this case, the gene encoding glycerol dihydratase and the gene encoding glycerol reactivate are gene cluster units dhaB1234gdrAB or pduCDEGH, and the gene encoding 1,3-PDO oxidoreductase may be yqhD or dhaT, or alternatively, the gene encoding 1,3-PDO oxidoreductase derived from E. coli may be yqhD.

[0127] In a specific embodiment of the present invention, the pEKEx1 vector was used to introduce the genes of (i) to (iii) into a microorganism. The pEKEx1 vector may include the pduCDEGH gene derived from Klebsiella pneumoniae and the yqhD gene derived from Escherichia coli. In this case, the nucleic acid sequence of each gene may be a wild-type nucleic acid sequence or a nucleic acid sequence codon-optimized for Corynebacterium glutamicum.

[0128] The nucleic acid sequences of the pduCDEGH gene derived from Klebsiella pneumoniae and the yqhD gene derived from E. coli that is codon-optimized for Corynebacterium glutamicum, used in the present invention, are shown in the following sequence numbers 19 and 20, respectively.

[0129]

[0130]

[0131]

[0132]

[0133] In the present invention, each of the five types of genes may be overexpressed by a strong promoter. Examples of known strong promoters include, but are not limited to, the H36 promoter, H30 promoter, lac promoter, trp promoter, trc promoter, tac promoter, tuf promoter, sod promoter, lambda phage PR promoter, PL promoter, T7 promoter, and tet promoter.

[0134] In a specific embodiment of the present invention, a recombinant vector containing the pduCDEGH gene derived from Klebsiella pneumoniae and the yqhD gene derived from Escherichia coli, which are codon-optimized for Corynebacterium glutamicum, and a vector containing any one of five selected genes were transformed into Corynebacterium glutamicum (C. glutamicum) SC97, the transformed strain was inoculated into a medium containing glycerol and cultured for 48 hours, and the 1,3-PDO production amount was measured. As a result, as confirmed in Figure 2, the production of 1,3-PDO increased most significantly in the transformed strains in which the aspa and sucCD genes were overexpressed, and the production of 1,3-PDO also increased in the transformed strains in which the remaining three genes, namely tal, metB, and aspaB genes, were overexpressed, respectively, compared to the control group (strat transformed with both the pCES208s-Exp-panD vector and the pEK-pduyE vector).

[0135] Accordingly, a recombinant microorganism that overexpresses each of the five selected genes according to the present invention can exhibit a 1,3-PDO production capacity improved by at least about 15 to 60% compared to the same strain in which the corresponding gene is not overexpressed. Preferably, depending on the overexpressed target gene, it can exhibit a 1,3-PDO production capacity improved by 15 to 20%, 40 to 50%, 50 to 56%, 50 to 58%, or 50 to 60%.

[0136] A third aspect of the present invention relates to a method for producing 1,3-PDO, comprising the step of culturing a recombinant microorganism.

[0137] In the present invention, the term "culture" means growing the microorganism under appropriately controlled environmental conditions. The culture process of the present invention may be carried out according to suitable media and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art depending on the selected strain.

[0138] Specifically, the step of culturing the microorganisms described above may be performed by known batch culture methods, continuous culture methods, fed-batch culture methods, etc., although not specifically limited thereto. At this time, the culture conditions may be adjusted to an appropriate pH (e.g., pH 5 to 9, specifically pH 6 to 8, most specifically pH 6.8 to 7.0) using a basic compound (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound (e.g., phosphoric acid or sulfuric acid), although not specifically limited thereto. Additionally, during cultivation, bubble formation may be suppressed by using an antifoaming agent such as a fatty acid polyglycol ester, and in addition, oxygen or an oxygen-containing gas may be injected into the culture to maintain an aerobic state, or nitrogen, hydrogen, or carbon dioxide gas may be injected without gas injection to maintain an anaerobic and microaerobic state. The culture temperature may be maintained at 20 to 35 ℃, specifically 25 to 30 ℃, and the culture period may continue until a production amount of 1,3-PDO is obtained, for example, about 10 to 160 hours, but is not limited thereto. The 1,3-PDO produced by the above culture may be secreted outside the cell and contained in the medium.

[0139] In the present invention, the culture medium used in the method may use sugars and carbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose), oils and fats (e.g., soybean oil, sunflower oil, peanut oil, and coconut oil), fatty acids (e.g., palmitic acid, stearic acid, and linoleic acid), alcohols (e.g., glycerol and ethanol), and organic acids (e.g., acetic acid) individually or in combination as carbon sources, but is not limited thereto. As nitrogen sources, nitrogen-containing organic compounds (e.g., peptone, yeast extract, meat juice, malt extract, corn steep liquid, soybean meal, and urea), or inorganic compounds (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate) individually or in combination may be used, but is not limited thereto. Potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and equivalent sodium-containing salts may be used individually or in combination as phosphorus sources, but are not limited thereto. Additionally, the medium may contain other metal salts (e.g., magnesium sulfate or iron sulfate), amino acids, and essential growth-promoting substances such as vitamins.

[0140] A microorganism of the genus Corynebacterium that produces 1,3-PDO according to the present invention may be further transformed into a recombinant vector in which a gene encoding glycerol dehydratase, a gene encoding glycerol reactivase, and a gene encoding 1,3-PDO oxidoreducductase are operably linked to convert glycerol to 1,3-PDO, and the culture medium preferably contains glycerol as a carbon source.

[0141] In the present invention, the 1,3-PDO produced in the culture step can be recovered from the culture medium without cell lysis using a suitable method known in the art, depending on the culture method. For example, centrifugation, filtration, anion exchange chromatography, crystallization, and HPLC may be used, and the desired 1,3-PDO can be recovered from the medium or microorganisms using a suitable method known in the art. In addition, the recovery step may include a purification process.

[0142] A fourth aspect of the present invention relates to a kit for producing 1,3-PDO and a method for producing 1,3-PDO using the same.

[0143] According to a specific embodiment of the present invention, the kit according to the present invention may include the following (a) and (b):

[0144] (a) a composition for improving 1,3-PDO production capacity according to the first aspect above or a recombinant microorganism with improved 1,3-PDO production capacity according to the second aspect above; and

[0145] (b) Culture medium composition.

[0146] In the kit according to the present invention, the description of the composition for improving 1,3-PDO production capacity and the recombinant microorganism with improved 1,3-PDO production capacity of (a) is the same as described in the first and second aspects, respectively, so the description is omitted.

[0147] A kit according to one embodiment of the present invention may include a recombinant vector and a culture medium composition included in the composition for improving 1,3-PDO production capacity.

[0148] As used herein, the term "culture medium composition" refers to a medium that enables the recombinant microorganism of the present invention to produce and secrete 1,3-PDO. The composition may include general culture components necessary for the growth of host cells, namely carbon sources, nitrogen sources, inorganic compounds, amino acids and / or vitamins, and may further include components that can induce or promote the recombinant microorganism of the present invention to produce 1,3-PDO at high levels. Such a culture medium composition is a concept that encompasses all modified or applied forms of media conventionally used for the cultivation of microorganisms of the genus Corynebacterium.

[0149] According to a specific embodiment of the present invention, the culture medium composition may include the medium components described in the third aspect.

[0150] According to another specific embodiment of the present invention, the genus Corynebacterium microorganism producing 1,3-PDO of the present invention may be further transformed into a recombinant vector in which a gene encoding glycerol dehydratase, a gene encoding glycerol reactivase, and a gene encoding 1,3-PDO oxidoreducductase are operably linked to convert glycerol to 1,3-PDO, and the culture medium composition preferably includes glycerol as a carbon source.

[0151] A specific embodiment of the method for producing 1,3-PDO using the kit of the present invention may include the following steps:

[0152] (a) a step of producing 1,3-PDO by culturing a recombinant microorganism transformed with a recombinant vector included in the composition for improving 1,3-PDO production capacity according to the first aspect, or a recombinant microorganism with improved 1,3-PDO production capacity according to the second aspect; and

[0153] (b) Step of recovering the produced 1,3-PDO.

[0154] In the method for producing 1,3-PDO according to the present invention, the specific description of steps (a) and (b) is the same as that described in the third aspect above, so the description is omitted.

[0155] The present invention will be explained in more detail below through examples. However, as the present invention is susceptible to various modifications and may take various forms, the specific examples and descriptions provided below are intended only to aid in understanding the invention and are not intended to limit the invention to the specific disclosed forms. The scope of the present invention should be understood to include all modifications, equivalents, and substitutions that fall within the spirit and technical scope of the invention.

[0156] [Example 1]

[0157] Selection of target genes for overexpression and construction of vectors for the overexpression of target genes for 1,3-PDO production via in silico simulation

[0158] 1-1. Selection of Overexpressed Genes for 1,3-PDO Production Increase via In Silico Simulation

[0159] A simulation was performed as shown in Figure 1 using a Corynebacterium glutamicum metabolic model (1,243 metabolites, 1,775 reaction equations).

[0160] (1) Maximize the 1,3-PDO production rate (Fig. 1A). (2) Calculate the maximum value of the cell growth rate for each 1,3-PDO production rate by dividing the 1,3-PDO production rate into 10 steps from the minimum value of 0 to the maximum value (Fig. 1B). (3) Set a reference metabolic flow where no 1,3-PDO is produced and the cell growth rate has the maximum value (Fig. 1C). (4) Calculate an internal metabolic flow that minimizes the difference from the reference metabolic flow while satisfying the cell growth rate value for each 1,3-PDO production rate calculated in (2) as a constraint (Fig. 1D). Calculate the correlation between the metabolic flow of the model internal reaction equations obtained through simulation and the 1,3-PDO production rate, and select a reaction equation with a high positive correlation as the overexpression target.

[0161] The genes selected through the above in silico simulation are the tal, sucCD, metB, aspa, and aspaB genes, and the nucleic acid sequences of each of these genes are shown in Table 1.

[0162] [Table 1]

[0163]

[0164]

[0165]

[0166]

[0167] 1-2. Construction of a vector for the overexpression of a selected target gene

[0168] First, using a Corynebacterium glutamicum ATCC 13032 colony as a template, the tal, sucCD, metB, aspa, and aspa genes (sequence numbers 1 to 5, respectively) were obtained by polymerase chain reaction (PCR) using primers tal-F and tal-R, sucCD-F and sucCD-R, metB-F and metB-R, aspA-F and aspA-R, and aspB-F and aspB-R with sequences listed in Table 2 below. Subsequently, the H36 promoter sequence (sequence number 6) was obtained by polymerase chain reaction using primers H36-F and H36-R with sequences listed in Table 2. Then, using primers H36-F and tal-R, sucCD-R, metB-R, aspA-R, and aspB-R of the sequences listed in Table 2, polymerase chain reaction (PCR) was performed to obtain overexpression target gene cassettes in which each overexpression target gene was expressed as an H36 promoter.

[0169] Subsequently, the pCES208s-Exp-panD vector listed in Table 3 below was cleaved with restriction enzyme SalI, and Gibson assembly was performed with the polymerase chain reaction product (H36 promoter and overexpression target gene cassette) to construct the pCES208s-Exp-panD-tal, pCES208s-Exp-panD-sucCD, pCES208s-Exp-panD-metB, pCES208s-Exp-panD-aspA, and pCES208s-Exp-panD-aspB vectors, respectively.

[0170] [Table 2]

[0171]

[0172] [Table 3]

[0173]

[0174] [Example 2]

[0175] Preparation of microorganisms introduced with an overexpression system of a target gene for increased 1,3-PDO production and verification of 1,3-PDO production capacity

[0176] 2-1. Preparation of a microorganism equipped with an overexpression system of a target gene for increased 1,3-PDO production

[0177] First, we intended to confer the ability to produce 1,3-PDO by introducing genes coding for glycerol dihydratase, glycerol reactivase, and 1,3-propanediol oxidoreductase into a Corynebacterium glutamicum strain.

[0178] To this end, the yqhD gene (Sequence No. 20) was obtained by polymerase chain reaction (PCR) using primers yqhD-F and yqhD-R listed in Table 4, with the gene synthesized through codon optimization using the nucleic acid sequence of E. coli yqhD obtained from KEGG Reference Sequence: b3011 serving as a template. Subsequently, the polymerase chain reaction product was cleaved with restriction enzyme DraI and ligated into the pEKEx1 vector listed in Table 3 to construct the pEKEx1-yqhD vector. Next, the pduCDEGH gene (Sequence No. 19) was obtained by PCR using Klebsiella pneumoniae DSM 2026 (DSMZ) colonies as a template, with primers pduCDEGH-F and pduCDEGH-R listed in Table 4. Subsequently, the pEK-pduyE vector was constructed by performing Gibson assembly with the pEKEx1-yqhD vector cleaved with restriction enzymes BamHI and PstI (Gibson et al. (2009) Nat. Method. 6:343-345).

[0179] [Table 4]

[0180]

[0181] Next, the five target gene overexpression vectors constructed in Examples 1-2 were each transformed into Corynebacterium glutamicum (C. glutamicum) SC97 [Depository: Korea Biotechnology Research Institute Biological Resource Center (KCTC), Accession No.: KCTC 16182BP, Date of Deposit: 2024.12.09] together with the pEK-pduyE vector. For comparison, Corynebacterium glutamicum (C. glutamicum) SC97 was transformed into the pCES208s-Exp-panD vector together with the pEK-pduyE vector. Subsequently, they were selected on BHIS agar plates supplemented with 25 μg / L kanamycin and 200 μg / L spectinomycin (composition: 37 g / L Brain Heart Infusion (BHI), 91 g / L sorbitol, 15 g / L agar).

[0182] The above six transformed mutant microorganisms were inoculated into a test tube containing 10 mL of BHIS medium (composition: 37 g / L BHI, 91 g / L sorbitol) and pre-cultured at 30 ℃ for 18 hours. Then, 1 mL of the pre-cultured medium was inoculated into 25 mL of CGXII medium (Table 5) in a 250 mL baffle flask and cultured. The initial glycerol concentration was set to 40 g / L, 10 g / L of yeast extract was added to the medium, and triple-double flask culture was carried out for 48 hours.

[0183] [Table 5]

[0184] Composition of CGXII medium used for Corynebacterium glutamicum culture

[0185]

[0186] 2-2. Verification of 1,3-PDO Production Capacity

[0187] For the measurement of 1,3-PDO, a Waters 1515 high-performance liquid chromatograph (Waters 1 Co., Milford, MA, USA) was used, and a Waters 2414 refractive index detector and an A MetaCarb 87H column (300 by 7.8 mm; Agilent) were used as the detector and column, respectively. Measurements were performed using 0.01 N H2SO4 as the buffer at a flow rate of 0.5 mL / min under conditions of 35°C.

[0188] As a result, as shown in Figure 2 and Table 6, the strains of Corynebacterium glutamicum SC97 transformed with the pCES208s-Exp-panD-sucCD vector and the pEK-pduyE vector, and the strains transformed with the pCES208s-Exp-panD-aspA vector and the pEK-pduyE vector, showed the best improvement in 1,3-PDO production, with 1,3-PDO production increasing by 54.1% and 54.6%, respectively, compared to the control group (Ctrl). In addition, it was confirmed that the production of 1,3-PDO increased by 17.6%, 47.9%, and 45.3% compared to the control (Ctrl) in strains transformed with the pEK-pduyE vector along with pCES208s-Exp-panD-tal, pCES208s-Exp-panD-metB, and pCES208s-Exp-panD-aspB, respectively, within the Corynebacterium glutamicum SC97 strain.

[0189] [Table 6]

[0190]

[0191] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A composition for improving the production capacity of 1,3-propanediol, comprising a recombinant vector comprising a gene coding for transaldolase (tal), a gene coding for succinyl-CoA synthetase (sucCD), a gene coding for cystathionine γ-synthase (metB), a gene coding for aspartate ammonia lyase (aspA), a gene coding for aspartate aminotransferase (aspB), or a functional fragment thereof.

2. A composition for improving 1,3-propanediol production capacity according to claim 1, wherein the tal, sucCD, metB, aspa, and aspaB genes are derived from Corynebacterium.

3. A composition for improving 1,3-propanediol production capacity according to claim 2, wherein the tal gene comprises the nucleic acid sequence of SEQ ID NO. 1, the sucCD gene comprises the nucleic acid sequence of SEQ ID NO. 2, the metB gene comprises the nucleic acid sequence of SEQ ID NO. 3, the aspa gene comprises the nucleic acid sequence of SEQ ID NO. 4, and the aspaB gene comprises the nucleic acid sequence of SEQ ID NO.

5.

4. A composition for enhancing 1,3-propanediol production capacity, wherein the genes are selected through in silico simulation comprising the following steps (a) to (f): (a) A step of determining the maximum and minimum values ​​of the 1,3-propanediol production rate of microorganisms; (b) a step of dividing the range between the minimum and maximum values ​​of the determined 1,3-propanediol production rate into 10 equal steps and calculating the maximum value of the cell growth rate for each 1,3-propanediol production rate; (c) A step of setting a reference to a metabolic flow in which the cell growth rate has a maximum value while 1,3-propanediol is not produced; (d) A step of calculating an internal metabolic flow that minimizes the difference from the metabolic flow set as a reference in step (c), while satisfying the maximum value of the cell growth rate according to the 1,3-propanediol production rate calculated in step (b) as a limiting condition; (e) a step of analyzing the linear positive relationship between internal metabolic flow and 1,3-propanediol production rate; and (f) A step of selecting a response exhibiting a positive linear relationship as an overexpression target.

5. A composition for improving 1,3-propanediol production capacity, wherein the vector further comprises a strong promoter selected from the group consisting of H36 promoter, H30 promoter, lac promoter, trp promoter, trc promoter, tac promoter, tuf promoter, sod promoter, lambda phage PR promoter, PL promoter, T7 promoter, and tet promoter.

6. A recombinant microorganism having 1,3-propanediol biosynthetic ability in which a gene encoding transaldolase (tal), a gene encoding succinyl-CoA synthetase (sucCD), a gene encoding cystathionine γ-synthase (metB), a gene encoding aspartate ammonia lyase (aspA) or a gene encoding aspartate aminotransferase (aspB) is overexpressed, thereby enhancing 1,3-propanediol production ability.

7. A recombinant microorganism according to claim 6, wherein the tal gene comprises the nucleic acid sequence of SEQ ID NO. 1, the sucCD gene comprises the nucleic acid sequence of SEQ ID NO. 2, the metB gene comprises the nucleic acid sequence of SEQ ID NO. 3, the aspa gene comprises the nucleic acid sequence of SEQ ID NO. 4, and the aspaB gene comprises the nucleic acid sequence of SEQ ID NO.

5.

8. In claim 6, the microorganism having the 1,3-propanediol biosynthetic ability is a recombinant microorganism to which (i) a gene encoding glycerol dehydratase, (ii) a gene encoding glycerol reactivase, and (iii) a gene encoding 1,3-propanediol oxidoreductase have been additionally introduced.

9. A recombinant microorganism according to claim 8, wherein the gene encoding glycerol dihydratase and the gene encoding glycerol reactivase are a pduCDEGH cluster comprising the nucleic acid sequence of SEQ ID NO. 19, and the gene encoding 1,3-propanediol oxidoreductase is a yqhD comprising the nucleic acid sequence of SEQ ID NO.

20.

10. A recombinant microorganism according to claim 6, wherein the genes are overexpressed by a strong promoter selected from the group consisting of the H36 promoter, lac promoter, trp promoter, trc promoter, tac promoter, tuf promoter, lambda phage PR promoter, PL promoter, T7 promoter, and tet promoter.

11. In claim 9, the recombinant microorganism having 1,3-propanediol biosynthesis is selected from the group consisting of Corynebacterium, Klebsiella, Clostridia, Enterobacter, Citrobacter, and Lactobacilli.

12. A recombinant microorganism according to claim 11, wherein the microorganism having 1,3-propanediol biosynthesis is Corynebacterium.

13. A method for producing 1,3-propanediol comprising the following steps (a) and (b): (a) a step of producing 1,3-propanediol by culturing a recombinant microorganism according to any one of claims 6 to 12; and (b) A step of recovering the produced 1,3-propanediol.

14. A method for producing 1,3-propanediol according to claim 13, wherein the recombinant microorganism of step (a) is cultured in a medium containing glycerol.

15. A kit for the production of 1,3-propanediol comprising the following (a) and (b): (a) a composition according to any one of claims 1 to 5 or a recombinant microorganism according to any one of claims 6 to 12; and (b) Culture medium composition.

16. Method for producing 1,3-propanediol using the kit of claim 15. 17.1 Use of a recombinant vector comprising one or more selected from the group consisting of (a) to (e) below for use in the production of a recombinant microorganism for the production of 3-propanediol: (a) a gene (tal) encoding transaldolase or a functional fragment thereof; (b) a gene (sucCD) encoding succinyl-CoA synthetase or a functional fragment thereof; (c) a gene (metB) encoding cystathionine γ-synthase or a functional fragment thereof; (d) a gene (aspA) encoding aspartate ammonia lyase or a functional fragment thereof; and (e) The gene (aspB) encoding aspartate aminotransferase or a functional fragment thereof.

18. In paragraph 17, the above-mentioned tal, sucCD, metB, aspa, and aspaB genes are derived from Corynebacterium.

19. In claim 18, the above tal gene comprises the nucleic acid sequence of SEQ ID NO. 1, the above sucCD gene comprises the nucleic acid sequence of SEQ ID NO. 2, the above metB gene comprises the nucleic acid sequence of SEQ ID NO. 3, the above aspa gene comprises the nucleic acid sequence of SEQ ID NO. 4, and the above aspaB gene comprises the nucleic acid sequence of SEQ ID NO.

5.

20. In claim 17, the above genes are selected through in silico simulation comprising the following steps (a) to (f), for use: (a) A step of determining the maximum and minimum values ​​of the 1,3-propanediol production rate of microorganisms; (b) a step of dividing the range between the minimum and maximum values ​​of the determined 1,3-propanediol production rate into 10 equal steps and calculating the maximum value of the cell growth rate for each 1,3-propanediol production rate; (c) A step of setting a reference to a metabolic flow in which the cell growth rate has a maximum value while 1,3-propanediol is not produced; (d) A step of calculating an internal metabolic flow that minimizes the difference from the metabolic flow set as a reference in step (c), while satisfying the maximum value of the cell growth rate according to the 1,3-propanediol production rate calculated in step (b) as a limiting condition; (e) a step of analyzing the linear positive relationship between internal metabolic flow and 1,3-propanediol production rate; and (f) A step of selecting a response exhibiting a positive linear relationship as an overexpression target.

21. In paragraph 17, the vector further comprises a strong promoter selected from the group consisting of H36 promoter, H30 promoter, lac promoter, trp promoter, trc promoter, tac promoter, tuf promoter, sod promoter, lambda phage PR promoter, PL promoter, T7 promoter, and tet promoter. 22.

1. A recombinant vector comprising one or more selected from the group consisting of (a) to (e) below for use in the production of a recombinant microorganism for the production of 3-propanediol: (a) a gene (tal) encoding transaldolase or a functional fragment thereof; (b) a gene (sucCD) encoding succinyl-CoA synthetase or a functional fragment thereof; (c) a gene (metB) encoding cystathionine γ-synthase or a functional fragment thereof; (d) a gene (aspA) encoding aspartate ammonia lyase or a functional fragment thereof; and (e) The gene (aspB) encoding aspartate aminotransferase or a functional fragment thereof.

23. A recombinant vector in which the tal, sucCD, metB, aspa, and aspaB genes are derived from Corynebacterium, in paragraph 22.

24. A recombinant vector according to claim 23, wherein the tal gene comprises the nucleic acid sequence of SEQ ID NO. 1, the sucCD gene comprises the nucleic acid sequence of SEQ ID NO. 2, the metB gene comprises the nucleic acid sequence of SEQ ID NO. 3, the aspa gene comprises the nucleic acid sequence of SEQ ID NO. 4, and the aspaB gene comprises the nucleic acid sequence of SEQ ID NO.

5.

25. In paragraph 22, the recombinant vector wherein the genes are selected through in silico simulation comprising the following steps (a) to (f): (a) A step of determining the maximum and minimum values ​​of the 1,3-propanediol production rate of microorganisms; (b) a step of dividing the range between the minimum and maximum values ​​of the determined 1,3-propanediol production rate into 10 equal steps and calculating the maximum value of the cell growth rate for each 1,3-propanediol production rate; (c) A step of setting a reference to a metabolic flow in which the cell growth rate has a maximum value while 1,3-propanediol is not produced; (d) A step of calculating an internal metabolic flow that minimizes the difference from the metabolic flow set as a reference in step (c), while satisfying the maximum value of the cell growth rate according to the 1,3-propanediol production rate calculated in step (b) as a limiting condition; (e) a step of analyzing the linear positive relationship between internal metabolic flow and 1,3-propanediol production rate; and (f) A step of selecting a response exhibiting a positive linear relationship as an overexpression target.

26. The recombinant vector according to claim 22, wherein the vector further comprises a strong promoter selected from the group consisting of H36 promoter, H30 promoter, lac promoter, trp promoter, trc promoter, tac promoter, tuf promoter, sod promoter, lambda phage PR promoter, PL promoter, T7 promoter, and tet promoter.

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