Microorganisms capable of simultaneously producing 1,3-propanediol and 3-hydroxypropionic acid and their uses

Abstract

The present specification relates to a microorganism into which the GPD gene, the GPP gene, the dhaB, the gdrAB, the aldH and / or the yqhD gene have been introduced and / or its use, which enables simultaneous production of 1,3-PDO and 3-HP.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0165039, dated November 30, 2022, and Korean Patent Application No. 10-2022-0165127, dated November 30, 2022, and all contents disclosed in the documents of said Korean patent applications are incorporated herein by reference.

[0002] The present specification relates to a microorganism into which a gene encoding glycerol-3-phosphate dehydrogenase (GPD) (hereinafter referred to as the GPD gene) and / or a gene encoding glycerol-3-phosphate phosphatase (GPP) (hereinafter referred to as the GPP gene), dhaB, gdrAB, aldH, and / or yqhD genes have been introduced, and / or uses thereof. [Background technology]

[0003] The aliphatic diol 1,3-propanediol (1,3-PDO), also known as trimethylene glycol or 1,3-dihydroxypropane, is an organic chemical with a relatively simple structure and is used in a variety of applications, including polymers, cosmetics, and lubricants. Polytrimethylene terephthalate (PTT), which is commercially available after being produced by the polymerization of 1,3-PDO with terephthalic acid, has attracted considerable attention due to its superior properties compared to polyester and nylon in terms of elasticity and optical stability.

[0004] 3-Hydroxypropionic Acid (3-HP) is a platform compound that can be converted into various chemicals such as acrylic acid, methyl acrylate, and acrylate. Since being selected as one of the Top 12 Value-added Biochemicals by the US Department of Energy in 2004, it has been actively researched in both academia and industry.

[0005] Various microorganisms have been developed to simultaneously biosynthesize the high-value-added monomers 1,3-PDO and 3-HP. However, existing strains have low titers, or methods such as mixed culture of two types of microorganisms are used, and there has been insufficient research into the simultaneous biosynthesis of 1,3-PDO and 3-HP from glucose or glycerol, limiting their applicability to actual processes. Summary of the Invention [Problem to be solved by the invention]

[0006] Under these circumstances, the present inventors conducted extensive research on microorganisms for the simultaneous biosynthesis of 1,3-PDO and 3-HP. As a result, they confirmed that a microorganism into which a gene encoding glycerol-3-phosphate dehydrogenase (GPD), a gene encoding glycerol-3-phosphate phosphatase (GPP), and the genes dhaB, gdrAB, aldH, and yqhD were introduced could simultaneously produce 1,3-PDO and 3-HP from glucose and / or glycerol, thereby completing the present invention.

[0007] One example of the present specification provides a microorganism (first recombinant microorganism) containing one or more (two or more, three or more, or four) genes selected from the group consisting of dhaB, gdrAB, aldH, and yqhD. The first recombinant microorganism may have the ability to produce (e.g., simultaneously produce) 1,3-PDO and 3-HP from glycerol.

[0008] The microorganism may additionally contain one or more genes selected from the group consisting of a gene encoding glycerol-3-phosphate dehydrogenase (GPD) (GPD gene) and a gene encoding glycerol-3-phosphate phosphatase (GPP) (GPP gene) (second recombinant microorganism). The second recombinant microorganism may be capable of producing (e.g., simultaneously producing) 1,3-PDO and 3-HP from glucose.

[0009] Another example provides a recombinant vector containing one or more (two or more, three or more, or four) genes selected from the group consisting of dhaB, gdrAB, aldH, and yqhD (first recombinant vector).

[0010] The recombinant vector may additionally contain one or more genes selected from the group consisting of GPD gene and GPP gene (second recombinant vector).

[0011] The first and second recombinant vectors can be used to produce 1,3-PDO and / or 3-HP. In one example, the first recombinant vector can be used to produce 1,3-PDO and / or 3-HP from glycerol. In another example, the second recombinant vector can be used to produce 1,3-PDO and / or 3-HP from glucose.

[0012] Another example provides a composition for producing 1,3-PDO and / or 3-HP, comprising the microorganism and / or recombinant vector. In one example, a composition for producing 1,3-PDO and / or 3-HP from glycerol containing the first recombinant microorganism, the first recombinant vector, or all of these is provided. In another example, a composition for producing 1,3-PDO and / or 3-HP from glucose containing the second recombinant microorganism, the second recombinant vector, or all of these is provided.

[0013] Another example provides a method for producing 1,3-PDO and / or 3-HP, comprising culturing the microorganism.

[0014] Another example provides a fermentation liquid composition containing 1,3-PDO and 3-HP, obtained by fermenting a microorganism having the activity of producing 1,3-PDO and 3-HP. [Means for solving the problem]

[0015] One example of the present specification provides a microorganism comprising one or more (two or more, three or more, or four) genes selected from the group consisting of dhaB, gdrAB, aldH, and yqhD.

[0016] The microorganism may additionally contain one or more genes selected from the group consisting of a gene encoding glycerol-3-phosphate dehydrogenase (GPD) (GPD gene) and a gene encoding glycerol-3-phosphate phosphatase (GPP) (GPP gene).

[0017] The microorganism may contain all of the dhaB, gdrAB, aldH and yqhD genes, but is not limited thereto.

[0018] The microorganism may contain both the GPD gene and the GPP gene, but is not limited thereto.

[0019] The microorganism can produce 1,3-propanediol (1,3-PDO) and / or 3-hydroxypropionic acid (3-HP), and can co-produce 1,3-PDO and 3-HP, but is not limited thereto.

[0020] The microorganism may have the activity of producing 1,3-propanediol (1,3-PDO) and / or 3-hydroxypropionic acid (3-HP), or may have the activity of simultaneously producing 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP), but is not limited thereto.

[0021] The microorganisms provided herein are capable of producing, but are not limited to, 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP) from glucose and / or glycerol.

[0022] Other examples provide compositions for producing 1,3-PDO and / or 3-HP, comprising the microorganisms and / or recombinant vectors provided herein.

[0023] Another example provides a recombinant vector comprising one or more (two or more, three or more, or four) genes selected from the group consisting of dhaB, gdrAB, aldH, and yqhD.

[0024] The recombinant vector may additionally contain one or more genes selected from the group consisting of GPD gene and GPP gene (second recombinant vector).

[0025] The first recombinant vector can contain, but is not limited to, dhaB, gdrAB, aldH and yqhD genes.

[0026] The second recombinant vector may contain, but is not limited to, a GPD gene and a GPP gene.

[0027] Other examples provide methods for producing 1,3-PDO and / or 3-HP, including culturing a microorganism provided herein.

[0028] The present application is described in more detail below.

[0029] As used herein, the term "microorganism" refers to an extremely small organism that is too small to be seen with the naked eye. As used herein, a microorganism may have the ability to synthesize an organic compound, such as the ability to synthesize 1,3-propanediol (1,3-PDO) and / or 3-hydroxypropionic acid (3-HP) (or the ability to produce 1,3-PDO and / or 3-HP). An example of a microorganism may contain genes involved in the production of an organic compound and produce the organic compound itself, or may be a strain genetically engineered to have the ability to produce an organic compound or to enhance the ability to produce an organic compound. The organic compound may be produced within the strain, produced intracellularly and secreted to the outside of the cell, or a combination thereof.

[0030] The microorganism of the present specification may be introduced with one or more genes selected from the group consisting of a gene encoding glycerol-3-phosphate dehydrogenase (GPD) (GPD gene), a gene encoding glycerol-3-phosphate phosphatase (GPP) (GPP gene), dhaB, gdrAB, aldH, and yqhD, to enhance the activity of the introduced gene, but is not limited thereto.

[0031] The microorganism of the present specification may additionally include a feature in which the activity of one or more genes selected from the group consisting of yqhD, glpK, ldhA, ack-pta, gldA, and ptsG is weakened or inactivated, but is not limited thereto.

[0032] The microorganisms herein may additionally include one or more characteristics selected from the group consisting of (a) and (b) below, but are not limited thereto: (a) the activity of one or more genes selected from the group consisting of yqhD, glpK, ldhA, ack-pta, gldA, and ptsG is weakened or inactivated; and (b) The activity of one or more genes selected from the group consisting of galP and glk is enhanced.

[0033] The characteristics (a) and (b) may be characteristics of the microorganism before one or more genes selected from the group consisting of a gene encoding glycerol-3-phosphate dehydrogenase (GPD) (GPD gene), a gene encoding glycerol-3-phosphate phosphatase (GPP) (GPP gene), dhaB, gdrAB, aldH, and yqhD are introduced into the microorganism of the present specification, and these characteristics may or may not be maintained after the genes are introduced, but are not limited to this.

[0034] The microorganisms herein may be, but are not limited to, Escherichia microorganisms (e.g., E. coli, E. albertii, E. fergusonii, E. hermannii, and E. marmotae), Klebsiella microorganisms (e.g., K. pneumoniae, K. aerogenes, K. granulomatis, K. grimontii, K. huaxiensis), and / or Lactobacillus microorganisms (e.g., L. reuteri, L. acetotolerans, L. acidophilus, L. thermophilus).

[0035] The microorganisms provided herein can have, but are not limited to, 1,3-propanediol (1,3-PDO) producing ability and / or 3-HP producing ability.

[0036] As used herein, "containing a gene" can mean that the containing subject (e.g., a microorganism) contains the gene, or that an exogenous gene has been introduced into the subject and is contained therein.

[0037] As used herein, the term "having the ability to produce 1,3-propanediol (1,3-PDO)" can refer to cells and / or microorganisms that naturally have the ability to produce 1,3-PDO, or to microorganisms in which the ability to produce 1,3-PDO has been imparted to a parent strain that does not have the ability to produce 1,3-PDO. It can also refer to a microorganism that has the ability to produce 1,3-PDO by introducing a mutation according to an example, and / or a microorganism that does not have the ability to produce 1,3-PDO by introducing a mutation according to an example. For example, a microorganism of the genus Escherichia that has the ability to produce 1,3-PDO can refer to a naturally occurring microorganism itself, or a microorganism of the genus Escherichia that has been made to have improved 1,3-PDO production ability by inserting an exogenous gene related to the 1,3-PDO production mechanism or by enhancing or inactivating the activity of an endogenous gene.

[0038] As used herein, the term "having the ability to produce 3-HP" can refer to cells and / or microorganisms that naturally have the ability to produce 3-HP, or to microorganisms in which the ability to produce 3-HP has been conferred to a parent strain that does not have the ability to produce 3-HP. It can also refer to a microorganism that has the ability to produce 3-HP as a result of the introduction of a mutation according to an example, and / or a microorganism that does not have the ability to produce 3-HP as a result of the introduction of a mutation according to an example. For example, a microorganism of the genus Escherichia that has the ability to produce 3-HP can refer to a naturally occurring microorganism itself, or to a microorganism of the genus Escherichia that has been given improved 3-HP production ability by inserting an exogenous gene related to the 3-HP production mechanism or by enhancing or inactivating the activity of an endogenous gene.

[0039] In one example, the microorganism may be a strain that has the ability to produce 1,3-PDO and / or 3-HP, or that has been genetically engineered to have the ability to produce 1,3-PDO and / or 3-HP. The 1,3-PDO and / or 3-HP may be produced within the strain, produced intracellularly and secreted to the outside of the cell, or a combination thereof.

[0040] The microorganism (or recombinant cell) may additionally contain a mutation that increases the production of 1,3-PDO and / or 3-HP, and the location of the mutation and / or the type of gene and / or protein to be mutated may be any mutation that increases the production of 1,3-PDO and / or 3-HP. The microorganism may be any cell that can be transformed.

[0041] In the present application, a microorganism before the introduction of a mutation according to an example can be referred to as a host microorganism (or parent strain) in order to distinguish it from a microorganism into which the mutation according to an example has been introduced to increase 1,3-PDO and / or 3-HP production ability or to which the ability to produce 1,3-PDO and / or 3-HP has been imparted by the introduction of a mutation according to an example.

[0042] As used herein, "glycerol-3-phosphate dehydrogenase (GPD)" refers to a polypeptide having the enzyme activity of catalyzing the conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P). Glycerol-3-phosphate dehydrogenase may be NADH-, NADPH-, or FAD-dependent. Regardless of its origin, any glycerol-3-phosphate dehydrogenase having the enzyme activity of catalyzing the conversion of DHAP to G3P ​​is included in the scope of the present application. Examples of genes encoding such glycerol-3-phosphate dehydrogenase include GPD1 and GPD2.

[0043] As used herein, "glycerol-3-phosphate phosphatase (GPP)" refers to a polypeptide having the enzymatic activity of catalyzing the conversion of glycerol-3-phosphate (G3P) to glycerol. Any glycerol-3-phosphate phosphatase having the enzymatic activity of catalyzing the conversion of G3P to glycerol is included in the scope of the present application, regardless of its origin. Examples of genes encoding such glycerol-3-phosphate phosphatases include GPP1 and GPP2.

[0044] As used herein, "glycerol dehydratase" or "diol dehydratase" can refer to a polypeptide having an enzyme activity that catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA). Glycerol dehydratase or diol dehydratase can be coenzyme B12-dependent or -independent. The enzyme may be derived from, but is not limited to, Klebsiella pneumoniae, Citrobacter freundii, Clostridium pasteurianum, Salmonella typhimurium, Klebsiella oxytoca, and / or Clostridium butyricum. Regardless of its origin, the enzyme may be included in the scope of the present application as long as it has the enzymatic activity of catalyzing the conversion of glycerol to 3-hydroxypropionaldehyde (3-HPA). For example, the gene encoding this enzyme may be dhaB.

[0045] As used herein, "glycerol dehydratase reactivase" refers to an enzyme that reactivates glycerol dehydratase, which is irreversibly inactivated during its activity, thereby maintaining its catalytic activity. Any commonly known glycerol dehydratase reactivase can be used, including, but not limited to, those derived from strains of Klebsiella sp. (e.g., Klebsiella pneumoniae), Citrobacter sp., Lactobacillus sp., and Salmonella sp. The glycerol dehydratase reactivase may be DhaFG, GdrAB, DdrAB, or the like, and the genes encoding it may be, for example, dhaFG, gdrAB, and / or ddrAB.

[0046] As used herein, "yqhD" is a gene encoding alcohol dehydrogenase, which encodes an enzyme that converts 3-hydroxypropanal into 1,3-PDO when glycerol is decomposed into 3-hydroxypropanal and water by glycerol dehydratase.

[0047] As used herein, "aldehyde dehydrogenase" refers to an enzyme that catalyzes the oxidation of aldehydes. The enzyme can convert an aldehyde (RC(=O)-H) to a carboxylic acid (RC(=O)-OH). The enzyme may be encoded by the aldH gene, which may be, but is not limited to, the aldH gene (GenBank Accession no. U00096.3; EaldH) derived from Escherichia coli or the E. coli K12 MG1655 18-3 cell strain, the puuC gene derived from Klebsiella pneumoniae, and / or the KGSADH gene derived from Azospirillum brasilense. The aldehyde dehydrogenase protein and the gene encoding it may contain mutations in the gene and / or amino acid sequence as long as the activity for producing 3-HP from 3-HPA is maintained.

[0048] As used herein, "enhanced activity" or "enhanced activity" not only includes the introduction or enhancement of the activity of a protein itself, resulting in an effect greater than its original function, but also refers to a state in which the activity of a microorganism after manipulation is increased compared to the activity of the microorganism before manipulation, such as increased gene expression, increased endogenous gene activity, amplification of an endogenous gene from internal or external factors, deletion of an inhibitory regulator of the gene expression, increased gene copy number, exogenous gene introduction, modification of an expression regulatory sequence, and / or promoter replacement or modification and increased enzyme activity due to intragenic mutation.

[0049] Herein, enhancing the activity of a protein and / or enzyme (e.g., GPD, GPP, dhaB, gdrAB, aldH, and / or yqhD) can be achieved by applying a variety of methods well known in the art. Methods for enhancing or increasing the activity of the protein and / or enzyme can be applied by a variety of methods well known in the art. Examples include, but are not limited to, a method of increasing the copy number of the base sequence encoding the enzyme (or protein) by additionally inserting a polynucleotide containing a base sequence encoding the enzyme (or protein) into a chromosome, a method of introducing the polynucleotide into a vector system, a method of replacing a promoter capable of expressing a polynucleotide with a stronger promoter, a method of introducing a mutation into a promoter, or a method of mutating an enzyme (or protein) with stronger activity by genetic mutation. The polynucleotide (e.g., GPD gene, GPP gene, dhaB, gdrAB, aldH, and / or yqhD) may be inserted into the host cell genetic body (chromosome) using a known method appropriately selected by those skilled in the art, or may be inserted using, for example, but not limited to, one or more of the following: (a) an RNA-guided endonuclease (e.g., Cas9 protein), its encoding gene, or a vector containing said gene; and (b) a guide RNA (e.g., single guide RNA (sgRNA)), its encoding DNA, or a vector containing said DNA (e.g., a mixture of an RNA-guided endonuclease protein and a guide RNA), a complex (e.g., a ribonucleic acid fusion protein (RNP), a recombinant vector (e.g., a vector containing both an RNA-guided endonuclease-encoding gene and a guide RNA-encoding DNA), etc.).

[0050] As used herein, "attenuated activity" refers to a decrease in the activity of a protein and / or enzyme (e.g., GPD, GPP, yqhD, glpK, ldhA, ack-pta, gldA, and / or ptsG) in a cell compared to that of a host cell (or parent strain), such as a wild-type strain or an untransformed strain. The term "attenuated activity" encompasses a combination of a decrease in the activity of the enzyme (or protein) itself compared to that of the enzyme originally possessed by the microorganism, due to a mutation in the gene encoding the enzyme (or protein), and a decrease in the expression level of the enzyme (or protein) due to transcription inhibition and / or translation inhibition of the gene encoding the enzyme, resulting in a decrease in the overall activity of the enzyme (or protein) in a cell compared to that of a wild-type strain or an untransformed strain.

[0051] Furthermore, as used herein, "inactivated" can refer to a state in which the expression of a gene encoding a protein and / or enzyme (e.g., the GPD gene, the GPP gene, yqhD, glpK, ldhA, ack-pta, gldA, and / or ptsG) is not expressed at all, or is expressed but has no activity, compared to a host cell (or parent strain) such as a wild-type strain or an untransformed strain.

[0052] Herein, attenuation or inactivation of the activity of proteins and / or enzymes (e.g., GPD, GPP, yqhD, glpK, ldhA, ack-pta, gldA, and / or ptsG) can be achieved by applying a variety of methods well known in the art. For example, methods include replacing the gene encoding the enzyme on a chromosome with a mutated gene that reduces the activity of the enzyme (or protein), including completely eliminating the activity of the enzyme (or protein); introducing a mutation into the expression regulatory sequence (e.g., promoter) of the gene encoding the enzyme (or protein) on a chromosome; replacing the expression regulatory sequence of the gene encoding the enzyme (or protein) with a sequence that has weaker or no activity; deleting all or part of the gene encoding the enzyme (or protein) on a chromosome; introducing an antisense oligonucleotide (e.g., antisense RNA) that complementarily binds to the transcript of the gene on the chromosome and inhibits translation of the mRNA into the enzyme (or protein); artificially adding a sequence complementary to the SD sequence before the SD sequence of the gene encoding the enzyme (or protein) to form a secondary structure that prevents ribosome attachment; and / or reverse transcription engineering (RTE) methods, in which a promoter is added to the 3' end of the ORF (open reading frame) of the sequence so that it can be reverse-transcribed.

[0053] As used herein, "reduced gene expression" refers to a decrease in the expression level of a gene compared to the expression level in the host cell (parent strain) and / or wild-type strain before mutation. It also includes cases where expression is completely inhibited and no expression occurs. To reduce gene expression in a microorganism, the initiation codon of gene translation can be replaced to reduce its expression, or a previously existing gene can be genetically engineered to reduce its expression. Methods for modifying sequences to regulate expression include introducing mutations into the expression regulatory sequence through deletion, insertion, non-conservative or conservative substitution, or a combination thereof, or replacing a promoter with a weaker promoter. Expression regulatory sequences include, but are not limited to, promoters, operator sequences, sequences encoding ribosome binding sites, and sequences regulating the termination of transcription and translation. If a gene whose expression is to be reduced exists in the microorganism to be mutated, the expression of the gene can be reduced by, for example, replacing the native promoter driving the expression of the gene with a weak promoter or by introducing a mutation. Furthermore, the expression of the gene can be reduced by deleting all or part of an existing gene.

[0054] As used herein, the term "vector" refers to any medium for cloning and / or transferring bases into a host cell. A vector may also be a replication unit to which other DNA fragments can be attached, resulting in replication of the attached fragments. A "replication unit" may refer to any genetic unit (e.g., a plasmid, phage, cosmid, chromosome, or virus) that functions as an independent unit of DNA replication in vivo, i.e., that can replicate under its own control. In the present invention, the vector is not particularly limited as long as it is replicable in the host, and any vector known in the art can be used. The vector used to prepare the recombinant vector may be a naturally occurring or recombinant plasmid, cosmid, virus, and / or bacteriophage. For example, pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII, λZAPII, λgt10, λgt11, Charon4A, and / or Charon21A can be used as phage or cosmid vectors, and pDZ vectors, pBR series, pUC series, pBluescriptII series, pGEM series, pTZ series, pCL series, and / or pET series can be used as plasmid vectors. Usable vectors are not particularly limited, and known expression vectors can be used. For example, the vector may be one in which a gene inserted and transferred into the vector is irreversibly integrated into the genome of the host cell, thereby stably maintaining gene expression within the cell for a long period of time. Such vectors may include transcriptional and coding expression regulatory sequences that enable the gene to be expressed in the selected host. Expression regulatory sequences may include any operator sequence for regulating transcription and / or sequences regulating the termination of transcription and coding. In one example, the initiation and termination codons may generally be considered as part of the nucleic acid sequence encoding the protein of interest, and must be in-frame with the coding sequence to be functional in an individual when the gene construct is administered. In addition, in the case of a replicable expression vector, an origin of replication may be included.Additionally, the vector may appropriately contain an enhancer, a non-translated region at the 3' end of the gene of interest, a selection marker (e.g., antibiotic resistance marker), and / or a replicable unit, etc. The vector may be autonomously replicating or may be integrated into the host genomic DNA.

[0055] By way of example, each component within a vector must be operably linked to one another; linking of these component sequences may be accomplished by ligation at convenient restriction sites or, if such sites do not exist, by the use of synthetic oligonucleotide adaptors or linkers by conventional methods.

[0056] As used herein, "transduction" refers to the phenomenon of bacterial DNA being transferred to other bacteria via bacteriophages, a type of horizontal gene transfer (HGT). Bacteriophages capable of transduction are called "transduction particles" or "transduction phages," and cells that receive DNA from other bacteria are called "transductants."

[0057] As used herein, "transformation" refers to the introduction of a gene into a host cell so that it can be expressed therein. The transformed gene may be inserted into the host cell's chromosome or located extrachromosomally, as long as it can be expressed in the host cell. The gene may include DNA and / or RNA encoding a target protein. The form of introduction of the gene is not limited, as long as it can be introduced into the host microorganism and expressed therein. For example, the gene may be introduced into the host microorganism in the form of an expression cassette, which is a genetic construct containing all elements necessary for its own expression. The expression cassette typically contains expression regulatory elements, such as a promoter, a transcription termination signal, a ribosome binding site, and / or a translation termination signal, operably linked to the gene. The expression cassette may be in the form of an expression vector capable of self-replicating. Alternatively, the gene may be introduced into the host cell in its own form and operably linked to sequences necessary for expression in the host cell.

[0058] As used herein, "promoter" refers to an untranslated nucleic acid sequence upstream of a coding region that contains a binding site for a polymerase and has transcription initiation activity for mRNA of a gene downstream of the promoter, i.e., a DNA region to which a polymerase binds to initiate transcription of a gene, and can be located 5' to the mRNA transcription initiation site.

[0059] In one example, the microorganism provided herein may further comprise a constitutive promoter and operably linked thereto a GPD gene, a GPP gene, dhaB, gdrAB, aldH, and / or yqhD gene, thereby producing 1,3-propanediol (1,3-PDO) and / or 3-hydroxypropionic acid (3-HP), but is not limited thereto. The constitutive promoter can induce expression of the operably linked gene without the need for a separate inducer. For example, the constitutive promoter may be a J23-series promoter (e.g., promoters J23100, J23101, J23102, J23103, J23104, J23105, J23106, J23107, J23109, J23111, J23112, J23113, J23114, J23115, J23116, J23117, and / or J23118), but is not limited thereto. Those skilled in the art can select and use promoters as needed for the desired range of 1,3-PDO and / or 3-HP production. One or more (two or more, three or more, or four) genes selected from the group consisting of the GPD gene, GPP gene, dhaB, gdrAB, aldH, and yqhD genes into which constitutive promoters have been introduced may each be cloned into a vector and introduced into a cell (or microorganism). Alternatively, the four genes may be cloned into a single vector and introduced into a cell (or microorganism).

[0060] As used herein, "productivity" can mean, but is not limited to, the amount of production per hour.

[0061] The microorganisms provided herein may have a 1,3-PDO productivity of 0.1 g / L / hr or more, 0.5 g / L / hr or more, 1 g / L / hr or more, 1.2 g / L / hr or more, 1.3 g / L / hr or more, 1.4 g / L / hr or more, 1.45 g / L / hr or more, 1.6 g / L / hr or more, 1.9 g / L / hr or more, 2 g / L / hr or more, or 2.1 g / L / hr or more, for example, 1.45 g / L / hr or 2.1 g / L / hr. There is no particular upper limit to the 1,3-PDO productivity, and the microorganisms may have a 1,3-PDO productivity of, for example, 100 g / L / hr or less or 10 g / L / hr or less, but are not limited thereto.

[0062] The microorganisms provided herein may have a 3-HP productivity of 0.1 g / L / hr or more, 0.5 g / L / hr or more, 1 g / L / hr or more, 1.2 g / L / hr or more, 1.3 g / L / hr or more, 1.4 g / L / hr or more, 1.45 g / L / hr or more, 1.6 g / L / hr or more, 1.9 g / L / hr or more, or 2 g / L / hr or more, for example, 1.45 g / L / hr or 2 g / L / hr. There is no particular upper limit to the 3-HP productivity, and the microorganisms may have a 3-HP productivity of, for example, 100 g / L / hr or less or 10 g / L / hr or less, but are not limited thereto.

[0063] The present specification provides a recombinant vector comprising one or more genes (two or more, three or more, or four types) selected from the group consisting of dhaB, gdrAB, aldH, and yqhD.

[0064] The present specification provides a recombinant vector (first recombinant vector) comprising one or more (two or more, three or more, or four) genes selected from the group consisting of dhaB, gdrAB, aldH, and yqhD.

[0065] The recombinant vector may additionally contain one or more genes selected from the group consisting of GPD gene and GPP gene (second recombinant vector).

[0066] The microorganism into which the recombinant vector has been introduced can produce 1,3-PDO and / or 3-HP, and can simultaneously produce 1,3-PDO and / or 3-HP, but is not limited thereto.

[0067] The microorganism into which the recombinant vector has been introduced may have the activity of producing 1,3-PDO and / or 3-HP, or may have the activity of co-producing 1,3-PDO and / or 3-HP, but is not limited thereto.

[0068] The present specification provides a method for producing 1,3-PDO and / or 3-HP, which includes a step of culturing the microorganism (specifically, the method includes a step of culturing a microorganism containing one or more (two or more, three or more, or four) genes selected from the group consisting of dhaB, gdrAB, aldH, and yqhD and / or one or more genes selected from the group consisting of a gene encoding glycerol-3-phosphate dehydrogenase (GPD) (GPD gene) and a gene encoding glycerol-3-phosphate phosphatase (GPP) (GPP gene)).

[0069] Another example provides a method for producing 1,3-PDO and / or 3-HP from glycerol, comprising culturing the first recombinant microorganism.Another example provides a method for producing 1,3-PDO and / or 3-HP from glucose, comprising culturing the second recombinant microorganism.

[0070] Culturing the microorganisms (specifically, the first recombinant microorganism and / or the second recombinant microorganism) can include reacting the microorganisms with a substrate, which can be, but is not limited to, glycerol and / or glucose.

[0071] The method for producing 1,3-PDO and / or 3-HP may additionally include, but is not limited to, a step of recovering 1,3-PDO and / or 3-HP from the culture medium or microorganism.

[0072] In one example, the culturing step may include, but is not limited to, a primary culturing step and / or a primary culturing and a secondary culturing step.

[0073] In one example, the culturing step comprises inoculating the strain into LB medium and culturing the strain; inoculating and culturing in M9 medium; and / or The method may include, but is not limited to, the step of inoculating and culturing in MR medium.

[0074] The step of inoculating and culturing in the LB medium may be referred to as primary culture, and the step of inoculating and culturing in the M9 medium and / or MR medium may be referred to as secondary culture, but is not limited thereto.

[0075] The 1,3-PDO productivity of the method for producing 1,3-PDO and / or 3-HP may be 0.1 g / L / hr or more, 0.5 g / L / hr or more, 1 g / L / hr or more, 1.2 g / L / hr or more, 1.3 g / L / hr or more, 1.4 g / L / hr or more, 1.45 g / L / hr or more, 1.6 g / L / hr or more, 1.9 g / L / hr or more, 2 g / L / hr or more, or 2.1 g / L / hr or more, for example, 1.45 g / L / hr or 2.1 g / L / hr. There is no particular upper limit to the 1,3-PDO productivity, and it may be, for example, 100 g / L / hr or less or 10 g / L / hr or less, but is not limited thereto.

[0076] The 3-HP productivity of the method for producing 1,3-PDO and / or 3-HP may be 0.1 g / L / hr or more, 0.5 g / L / hr or more, 1 g / L / hr or more, 1.2 g / L / hr or more, 1.3 g / L / hr or more, 1.4 g / L / hr or more, 1.45 g / L / hr or more, 1.6 g / L / hr or more, 1.9 g / L / hr or more, or 2 g / L / hr or more, for example, 1.45 g / L / hr or more or 2 g / L / hr. There is no particular upper limit to the 3-HP productivity, and it may be, for example, 100 g / L / hr or less or 10 g / L / hr or less, but is not limited thereto.

[0077] The culture may be carried out in a suitable medium and under suitable conditions known in the art, which must be adapted to meet the requirements of the particular strain and can be suitably modified by a person skilled in the art.

[0078] The culture method may include, but is not limited to, for example, batch culture, continuous culture, fed-batch culture, or a combination thereof.

[0079] For example, the medium can contain a variety of carbon sources, nitrogen sources, and trace element components, and the recombinant cells can be cultured in a standard medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc., while adjusting the temperature and / or pH. Usable carbon sources include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and / or cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and / or coconut oil; fatty acids such as palmitic acid, stearic acid, and / or linoleic acid; alcohols such as glycerol and / or ethanol; and organic acids such as acetic acid. These substances can be used individually or in mixtures, but are not limited to these. Usable nitrogen sources include peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean meal, and urea, or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. Nitrogen sources can also be used individually or in mixtures, but are not limited to these. Usable phosphorus sources include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium-containing salts. The medium may also contain, but is not limited to, metal salts necessary for growth, such as magnesium sulfate or iron sulfate. Essential growth substances, such as amino acids and vitamins, may also be included. Appropriate precursors may also be used for the medium. The medium or individual components may be added to the culture solution during the culture process in an appropriate manner, such as batchwise or continuously, but are not limited to these.

[0080] The step of separating and / or recovering 1,3-PDO and / or 3-HP from the cultured microorganisms (or recombinant cells) and / or culture medium may involve collecting the target substance (e.g., 1,3-PDO and / or 3-HP) from the medium, culture solution, or microorganism using a suitable method known in the art, depending on the culture method. Methods that can be used include, but are not limited to, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), and / or chromatography (e.g., ion exchange, affinity, hydrophobic, liquid, and size exclusion). The culture medium may refer to the medium in which the microorganisms (or recombinant cells) are cultured.

[0081] In one example, the method for producing 1,3-PDO and / or 3-HP can additionally include a step of purifying 1,3-PDO and / or 3-HP before, simultaneously with, or after the separation and / or recovery step.

[0082] As used herein, the term "fermentation composition" refers to a composition produced by adding a strain and / or microorganism to a specific substance and subjecting it to a culture and / or fermentation process.

[0083] The present specification provides a fermentation broth composition containing 1,3-PDO and / or 3-HP, which is obtained by fermenting a microorganism having 1,3-PDO and 3-HP-producing activity.

[0084] The fermentation broth composition may contain 1,3-PDO at a concentration of 10 g / L or more, 15 g / L or more, 20 g / L or more, 25 g / L or more, 50 g / L or more, 55 g / L or more, 60 g / L or more, 65 g / L or more, 70 g / L or more, 75 g / L or more, 80 g / L or more, or 81 g / L or more, for example, 26 g / L or 81 g / L. There is no particular upper limit to the 1,3-PDO concentration, and the 1,3-PDO may be contained at a concentration of, for example, 1000 g / L or less or 100 g / L or less, but is not limited thereto.

[0085] The fermentation broth composition may contain 3-HP at a concentration of 10 g / L or more, 15 g / L or more, 20 g / L or more, 25 g / L or more, 50 g / L or more, 55 g / L or more, 60 g / L or more, 65 g / L or more, 70 g / L or more, 75 g / L or more, or 77 g / L or more, for example, 26 g / L or 77 g / L. There is no particular upper limit to the 3-HP concentration, and the 3-HP may be contained at a concentration of, for example, 1000 g / L or less, 100 g / L or less, but is not limited thereto.

[0086] The fermentation liquid composition may contain 1,3-PDO and 3-HP simultaneously, but is not limited thereto.

[0087] The fermentation broth composition can be produced by, but is not limited to, the microorganisms provided herein.

[0088] The fermentation broth composition can be produced by, but is not limited to, the method for producing 1,3-PDO and / or 3-HP provided herein. [Effects of the Invention]

[0089] The present invention relates to a microorganism into which a GPD gene, a GPP gene, a dhaB gene, a gdrAB gene, an aldH gene, and / or a yqhD gene has been introduced, and / or uses thereof, which have the effect of producing 1,3-PDO and / or 3-HP. [Brief explanation of the drawings]

[0090] [Figure 1] 1 is a graph showing the results of confirming the production of 1,3-PDO and 3-HP after culturing a microorganism into which the dhaB, gdrAB, aldH and / or yqhD genes have been introduced. [Figure 2] 1 is a graph showing the results of confirming the production of 1,3-PDO and 3-HP after culturing a microorganism into which the GPD gene, the GPP gene, the dhaB, gdrAB, aldH and / or the yqhD gene have been introduced. DETAILED DESCRIPTION OF THE INVENTION

[0091] The present invention will be described in detail below with reference to examples, but the following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0092] Example 1. Plasmid design for strain recombination Example 1-1. Construction of pCDF-dhaB1, 2, 3-gdrA, B-aldH-yqhD vector A vector containing yqhD was constructed to overexpress the yqhD gene. Specifically, the yqhD gene (SEQ ID NO: 1) was amplified by PCR from the E. coli W3110 strain (deleted yqhD, glpK, ldhA, ack-pta, gldA, and ptsG; overexpressing galP and glk; designated DKALGP::PK strain; KCCM40219) using a primer pair consisting of SEQ ID NOs: 2 and 3 (pre-denaturation at 95°C for 2 minutes, denaturation at 95°C for 1 minute, annealing at 55°C for 30 seconds, and extension at 72°C for 30 seconds, repeated 30 times, with a final extension at 72°C for 2 minutes and storage at 4°C; the final product size was 1.1 kb). The yqhD gene and primer information are listed in Tables 1 and 2 below.

[0093] [Table 1] [Table 2]

[0094] The prepared yqhD gene was cloned into pCDF-dhaB1, 2, 3-gdrA, B-aldH plasmids for producing 3HP.

[0095] Specifically, the pCDF-dhaB1, 2, 3-gdrA, B-aldH plasmid is a vector in which the promoter portion of the pCDFDuet-1 vector (Novagen 71340) has been replaced with the J23101 and J23100 promoters, and the dhaB (dhaB1, dhaB2, dhaB3) and gdrAB genes (gdrA, gdrB) of Klebsiella pneumoniae (K. pneumoniae genomic DNA; KCTC12385) and the aldH gene derived from E. coli K12 MG1655 have been cloned. The dhaB (approximately 2.7 kb; dhaB1, dhaB2, dhaB3) and gdrAB genes (approximately 2.2 kb; gdrA, gdrB) were amplified from the chromosome of Klebsiella pneumoniae using the primers in Table 3 below and NEB (New England Biolabs) Q5 DNA polymerase under the conditions provided by NEB. The promoter information is shown in Table 4 below.

[0096] [Table 3] [Table 4]

[0097] The dhaB123 and gdrA genes are located adjacent to each other on the Klebsiella pneumoniae chromosome and were amplified together. However, because gdrB is located in the opposite direction to dhaB123 and gdrA, gdrB was amplified separately using NEB Q5 DNA polymerase under the conditions provided by NEB. The dhaB123 and gdrA genes were then cloned under the J23101 promoter using the restriction enzymes EcoRI and HindIII, and the gdrB gene was cloned using the restriction enzymes HindIII and AflII to prepare the pCDF-dhaB1,2,3-gdrA,B plasmid (hereafter referred to as pCDF-dhaB-gdrAB vector).

[0098] AldH was amplified from the E. coli K12MG1655 chromosome using the J23100 promoter, using the primers in Table 5 below and NEB Q5 DNA polymerase under conditions provided by NEB.

[0099] [Table 5]

[0100] aldH was cloned into the pCDF-dhaB-gdrAB vector under the J23100 promoter by T4 DNA ligase reaction using the restriction enzymes KpniI and NdeI to produce the pCDF-dhaB1,2,3-gdrA,B-aldH plasmid (hereinafter referred to as pCDF-dhaB-gdrAB-aldH vector).

[0101] The prepared yqhD gene was cloned into the pCDF-dhaB1,2,3-gdrA,B-aldH plasmid prepared as described above by In-Fusion reaction using PacI restriction enzyme to finally prepare the recombinant vector pCDF-dhaB1,2,3-gdrA,B-aldH-yqhD (hereinafter referred to as pCDF-dhaB-gdrAB-aldH-yqhD vector). Additionally, yqhD was cloned into the pCDF-dhaB-gdrAB vector in place of aldH in the same manner as above to prepare the pCDF-dhaB-gdrAB-yqhD vector.

[0102] Example 1-2. Construction of pRSF-pLTTR-GPD-GPP vector The GPD1 and GPP2 genes from Saccharomyces cerevisiae (BY4741; ATCC201388) were prepared by PCR using the primer pair (SEQ ID NOs: 12 and 13) or (SEQ ID NOs: 14 and 15) in Table 6, respectively (pre-denaturation at 95°C for 2 minutes, denaturation at 95°C for 1 minute, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes, repeated 28 times; final extension at 72°C for 5 minutes; storage at 4°C). The PCR products were digested with BamHI / SacI (when the primer pair (SEQ ID NOs: 12 and 13)) or KpnI / XhoI (when the primer pair (SEQ ID NOs: 14 and 15)) and cloned into the pRSFDuet-1 vector (Invitrogen) containing the LTTR promoter to generate a vector containing pRSF-pLTTR-GPD-GPP (hereafter referred to as pRSF-pLTTR-GPD-GPP vector). The sequences of the LTTR promoters used are listed in Table 7 below.

[0103] [Table 6] [Table 7]

[0104] Example 2. Preparation and cultivation of transformed strains Example 2-1. Preparation of strains To prepare a transformant strain using the vector prepared in Example 1, Escherichia coli W3110 (deleted yqhD, glpK, ldhA, ack-pta, gldA, and ptsG; overexpressing galP and glk; designated DKALGP::PK strain) was prepared. The strain was plated on an LB plate (containing 10 g tryptone, 5 g yeast extract, 10 g NaCl, and 15 g agar per liter) and cultured overnight at 37°C. A single colony from the culture was inoculated into 5 mL of LB liquid medium (same composition as the LB plate but without agar) and cultured overnight at 37°C with stirring. The following morning, the culture was diluted 1:100 with 5 mL of LB liquid medium and cultured for 3-6 hours until the OD reached approximately 0.5. After cooling on ice for 10-15 minutes, the cells were collected by centrifugation at 4000 rpm for 10 minutes at 4°C. The cells were washed by resuspending in 1 mL of ice-cold sterile deionized water and then collected by centrifugation at 4000 rpm for 10 minutes at 4°C. The collected cells were washed by resuspending in 1 mL of deionized water and then collected by centrifugation at 4000 rpm for 10 minutes at 4°C. The collected cells were resuspended in 0.08 mL of deionized water and aliquots were placed in individual pre-chilled microfuge tubes.

[0105] Example 2-2. Transformation of dhaB, gdrAB, aldH and / or yqhD genes To the cells collected in Example 2-1, 1 μL each of pCDFDuet-1 (pCDF) from Example 1 and the plasmid DNAs pCDF-dhaB-gdrAB vector, pCDF-dhaB-gdrAB-yqhD vector, and pCDF-dhaB-gdrAB-aldH-yqhD vector prepared in Example 1 was added by pipetting, and then 0.08 to 0.09 ml of the mixture was transferred to a pre-chilled 0.1 cm electroporation cuvette.

[0106] Immediately after electroporation at 1.8 kV, 1 mL of room temperature LB liquid medium was added to the cells and cultured at 37°C for 1 hour. The culture medium was then spread onto an LB plate containing streptomycin, and excess liquid was allowed to dry and absorb into the plate. The plate was then inverted and cultured at 37°C to prepare single colonies.

[0107] Example 2-3. Additional transformation of GPD and GPP genes Additionally, to the cells collected in Example 2-1, 1 μL of the plasmid DNA pRSF-pLTTR-GPD-GPP vector (hereinafter referred to as pRSF-GG vector) prepared in Example 1 was added by pipetting, along with 1 μL of pCDFDuet-1 (pCDF) prepared in Example 1 and the plasmid DNAs pCDF-dhaB-gdrAB vector, pCDF-dhaB-gdrAB-aldH vector, pCDF-dhaB-gdrAB-yqhD vector, and pCDF-dhaB-gdrAB-aldH-yqhD vector prepared in Example 1. Then, 0.08–0.09 mL of the mixture was transferred to a pre-chilled 0.1 cm electroporation cuvette. Immediately after electroporation at 1.8 kV, 1 mL of room-temperature LB liquid medium was added to the cells and they were cultured at 37°C for 1 hour. The culture was spread onto LB plates containing streptomycin, and excess liquid was allowed to dry and absorb into the plate. The plate was then incubated upside down at 37°C to prepare single colonies.

[0108] Example 3: Confirmation of 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP) production amounts of strains transformed with dhaB, gdrAB, aldH, and / or yqhD genes Example 3-1. Confirmation of the production amount of 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP) by the transformed strain in flask culture In a 250 mL Erlenmeyer flask, 100 mL of modified M9 medium (containing 0.6 g / L MgSO₄·7H₂O, 1.5 g / L NaCl, 12.8 g / L Na₂HPO₄·7H₂O, 17.4 g / L K₂HPO₄, 2 g / L NH₄Cl, 0.5 g / L yeast extract, 3 g / L KH₂PO₄, and 0.11 g / L CaCl₂) containing 20 g / L glycerol, 5 g / L glucose, and 25 mg / L streptomycin / kanamycin was prepared. The pH was maintained at 6.0 with Ca(OH)₂.

[0109] 1 mL of a single colony prepared in Example 2-2 was inoculated into the prepared M9 medium flask and cultured in a shaking incubator at 33°C and 250 rpm. The absorbance of the culture medium was measured at OD600 using a UV spectrometer under the analytical conditions in Table 8 below using HPLC to determine the production amounts of 1,3-PDO and 3-HP of the transformed strain of Example 2-2. The results are shown in Table 9 below.

[0110] [Table 8] [Table 9]

[0111] As a result, neither 1,3-PDO nor 3HP was produced when the empty vector (pCDF) or dhaB / gdrAB alone was expressed. However, when dhaB / gdrAB were expressed together with yqhD, 1.42 g / L of 1,3-PDO was produced, and when aldH and yqhD were co-expressed, 1,3-PDO and 3HP were simultaneously produced. Simultaneous fermentation increased 1,3-PDO production by approximately 70% compared to monoculture, and additional 3HP was also produced, confirming an increase in the total target production.

[0112] Example 3-2. Confirmation of 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP) production by transformed strains in high-density cell culture The single colony of Example 2-2 was inoculated into an LB liquid medium having the same composition as the LB liquid medium of Example 2-2, and pre-cultured (seed culture) at 37° C. for 18 hours.

[0113] For high-density cell culture, the seed culture was inoculated into a 150 mL flask and cultured in a 5 L fermentor using 2 L of modified MR medium (containing 0.8 g / L MgSO 7H O, 4 g / L (NH ) HPO, 6.67 g / L KH PO, and 0.8 g / L citrate) containing 20 g / L glucose, 25 mg / L streptomycin, and 50 mg / L kanamycin at 35°C, 500 rpm, and pH 6.95 (adjusted with NH (OH)). After the glucose in the medium was depleted, feeding solution (containing 700 g / L glucose, 15 g / L MgSO , 10 mL / L trace metal, 100 mL / L 10x MR salt, and 25 mg / L streptomycin) was added at a rate of 40 mL / hr, and the culture was continued for 24 hours.

[0114] After high-density cell culture, the culture was inoculated into 2 L of fresh MR medium (containing (NH4)2HPO4 4 g / L, KH2PO4 6.67 g / L, and citrate 0.8 g / L) to the initial OD25 for 3-HP production. 12 (Vitamin B 12 1 μM of 3-HP was added to the culture medium. A feeding solution of 200 g / L glucose and 500 g / L glycerol was added at a rate of 35 mL / hr, and Mg(OH) was added to maintain a pH of 6.95.

[0115] Thereafter, the production amounts of 1,3-PDO and 3-HP in the strain transformed with pCDF-dhaB-gdrAB-aldH-yqhD were determined using HPLC in substantially the same manner as in Example 3-1, and the results are shown in Figure 1 and Table 10. The yields were calculated using the following Equation 1.

number

[0116] [Table 10]

[0117] As a result, high production and productivity of 1,3-PDO and 3-HP were confirmed, and the yield was also high.

[0118] Example 4. Confirmation of the production amount of 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP) in a strain transformed with additional GPD and GPP genes Example 4-1. Confirmation of the production amount of 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP) by the transformed strain in flask culture In a 250 mL Erlenmeyer flask, 100 mL of modified M9 medium (containing 0.6 g / L MgSO₄·7H₂O, 1.5 g / L NaCl, 12.8 g / L Na₂HPO₄·7H₂O, 17.4 g / L K₂HPO₄, 2 g / L NH₄Cl, 0.5 g / L yeast extract, 3 g / L KH₂PO₄, and 0.11 g / L CaCl₂) containing 20 g / L glycerol, 5 g / L glucose, and 25 mg / L streptomycin / kanamycin was prepared. The pH was maintained at 6.0 with Ca(OH)₂.

[0119] 1 mL of a single colony prepared in Example 2-3 was inoculated into the prepared M9 medium flask and cultured in a shaking incubator at 33°C and 250 rpm. The absorbance of the culture medium was measured at OD600 using a UV spectrometer under the analytical conditions shown in Table 11 below using HPLC to determine the production amounts of 1,3-PDO and 3-HP of the transformed strain of Example 2-3. The results are shown in Table 12 below.

[0120] [Table 11] [Table 12]

[0121] As a result, neither 1,3-PDO nor 3HP was produced when the empty vector (pCDF) or dhaB / gdrAB alone was expressed. However, when dhaB / gdrAB and GPD-GPP were expressed together with aldH or yqhD, 3HP and 1,3-PDO were produced at 3.43 g / L and 1.42 g / L, respectively. Simultaneous expression of aldH and yqhD confirmed simultaneous production of 1,3-PDO and 3HP. Simultaneous fermentation increased 1,3-PDO production by approximately 3.7-fold compared to monoculture, and additional 3HP was also produced, confirming an increase in total target production.

[0122] Example 4-2. Confirmation of the production amount of 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP) of the transformed strain by high-density cell culture The single colony of Example 2-3 was inoculated into an LB liquid medium having the same composition as the LB liquid medium of Example 2-3, and pre-cultured (seed culture) at 37° C. for 18 hours.

[0123] Then, for high-density cell culture, the seed culture was inoculated into a 150 mL flask and cultured in a 5 L fermentor at 35°C, 500 rpm, and pH 6.95 (adjusted with NH(OH)) at 35°C, 500 rpm, and 2 L of modified MR medium (containing 0.8 g / L MgSO 7H O, 4 g / L (NH ) HPO, 6.67 g / L KH PO, and 0.8 g / L citrate) containing 20 g / L glucose, 25 mg / L streptomycin, and 50 mg / L kanamycin. After the glucose in the medium was used up, Feeding Solution (containing 700 g / L glucose, 15 g / L MgSO4, 10 mL / L trace metal, 100 mL / L 10x MR salt, 25 mg / L streptomycin, and 50 mg / L kanamycin) was added at a rate of 40 mL / hr, and the medium was cultured for 24 hours.

[0124] After high-density cell culture, the culture was inoculated into 2 L of fresh MR medium (containing (NH4)2HPO4 4 g / L, KH2PO4 6.67 g / L, and citrate 0.8 g / L) to the initial OD25 for 3-HP production. 12 (Vitamin B 12 1 μM of 3-HP was added to the culture medium. 600 g / L glucose feeding solution was added at a rate of 35 mL / hr, and Mg(OH) was added to maintain a pH of 6.95.

[0125] Thereafter, the amounts of 1,3-PDO and 3-HP produced were confirmed using HPLC in substantially the same manner as in Example 4-1, and the results are shown in Figure 2 and Table 13. The yield was calculated using the following mathematical formula 2.

number

[0126] [Table 13]

[0127] As a result, high production and productivity of 1,3-PDO and 3-HP were confirmed, and the yield was also high.

Claims

1. A microorganism comprising one or more genes selected from the group consisting of dhaB, gdrAB, aldH, and yqhD.

2. The microorganism according to claim 1, further comprising one or more genes selected from the group consisting of a gene encoding glycerol-3-phosphate dehydrogenase (GPD) (GPD gene) and a gene encoding glycerol-3-phosphate phosphatase (GPP) (GPP gene).

3. The microorganism of claim 1 , which contains all of the dhaB, gdrAB, aldH, and yqhD genes.

4. The microorganism according to claim 2, which contains both the GPD gene and the GPP gene.

5. The microorganism according to claim 1, further comprising a feature in which the activity of one or more genes selected from the group consisting of yqhD, glpK, ldhA, ack-pta, gldA, and ptsG is weakened or inactivated.

6. The microorganism of claim 2, wherein the microorganism additionally comprises one or more characteristics selected from the group consisting of (a) and (b) below: (a) attenuated or inactivated activity of one or more genes selected from the group consisting of yqhD, glpK, ldhA, ack-pta, gldA, and ptsG; and (b) the activity of one or more genes selected from the group consisting of galP and glk is enhanced.

7. The microorganism according to any one of claims 1 to 6, which simultaneously produces 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP).

8. The microorganism according to any one of claims 1 to 6, wherein the microorganism is a microorganism of the genus Escherichia, Klebsiella, or Lactobacillus.

9. The microorganism according to any one of claims 1 to 5, which produces 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP) from glycerol.

10. The microorganism according to any one of claims 2, 4 and 6, which produces 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP) from glucose.

11. a 1,3-PDO productivity of 0.5 g / L / hr or more; or The microorganism according to any one of claims 1 to 6, having a 3-HP productivity of 0.5 g / L / hr or more.

12. A recombinant vector comprising one or more genes selected from the group consisting of dhaB, gdrAB, aldH and yqhD.

13. The recombinant vector according to claim 12, further comprising one or more genes selected from the group consisting of a GPD gene and a GPP gene.

14. A composition for producing 1,3-PDO and 3-HP, comprising the microorganism according to any one of claims 1 to 6, or the recombinant vector according to claim 12 or 13.

15. A method for producing 1,3-PDO and 3-HP, comprising culturing the microorganism of any one of claims 1 to 6.

16. The 1,3-PDO productivity of the process for producing 1,3-PDO and 3-HP is 0.5 g / L / hr or more; or 16. The method for producing 1,3-PDO and 3-HP according to claim 15, wherein the 3-HP productivity of the method for producing 1,3-PDO and 3-HP is 0.5 g / L / hr or more.

17. A fermentation broth composition obtained by fermenting a microorganism having 1,3-PDO and 3-HP producing activity, and containing 1,3-PDO at a concentration of 10 g / L or more and 3-HP at a concentration of 10 g / L or more.

18. The fermentation liquid composition according to claim 17, which is produced by the microorganism according to any one of claims 1 to 6.

19. The fermentation broth composition according to claim 17, produced by the method for producing 1,3-PDO and 3-HP according to claim 15.

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