Microorganisms simultaneously producing 1, 3-propanediol and 3-hydroxypropionic acid and uses thereof

Abstract

The present specification relates to a microorganism having introduced therein a GPD gene, a GPP gene and dhaB, gdrAB, aldH and / or yqhD genes, which is capable of simultaneously producing 1, 3-PDO and 3-HP, and / or a use thereof.

Description

Technical Field

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority based on Korean Patent Application No. 10-2022-0165039 filed on November 30, 2022 and Korean Patent Application No. 10-2022-0165127 filed on November 30, 2022, and all contents disclosed in the files of the corresponding Korean patent applications are incorporated as a part of this specification.

[0003] The present specification relates to microorganisms and / or their uses that introduce a gene encoding glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate dehydrogenase, GPD) (hereinafter referred to as the GPD gene) and / or a gene encoding glycerol-3-phosphate phosphatase (glycerol-3-phosphate phosphatase, GPP) (hereinafter referred to as the GPP gene), dhaB, gdrAB, aldH and / or yqhD genes. Background Art

[0004] 1,3-propanediol (1,3-PDO), an aliphatic diol, is an organic chemical substance with a relatively simple structure, also known as trimethylene glycol or 1,3-dihydroxypropane, etc., and is used in various applications including polymers, cosmetics, and lubricants. Polytrimethylene terephthalate (PTT), which is produced and commercialized by the polymerization reaction of 1,3-PDO and terephthalic acid, has received great attention due to its superior properties compared to polyester and nylon in terms of elasticity and optical stability.

[0005] 3-Hydroxypropionic Acid (3-HP) is a platform compound that can be converted into various chemicals such as acrylic acid, methyl acrylate, acrylamide, etc., and because it was selected as one of the 12 major value-added biochemicals by the U.S. Department of Energy in 2004, it is actively studied in academia and industry.

[0006] Various microorganisms have been developed to simultaneously biosynthesize 1,3-PDO and 3-HP, which are high-value-added monomers. However, conventional strains have low titers or use a method of mixed culture of two microorganisms, etc., and there is a lack of research on the simultaneous biosynthesis of 1,3-PDO and 3-HP from glucose or glycerol, so there are limitations in applying them to practical processes. Summary of the invention

[0007] Technical issues

[0008] Under the above background, the present inventors have repeatedly studied microorganisms for the simultaneous biosynthesis of 1,3-PDO and 3-HP, and as a result, it has been confirmed that microorganisms introduced with a gene encoding glycerol-3-phosphate dehydrogenase (GPD), a gene encoding glycerol-3-phosphate phosphatase (GPP), dhaB, gdrAB, aldH and yqhD can simultaneously produce 1,3-PDO and 3-HP from glucose and / or glycerol, thereby completing the present invention.

[0009] One embodiment of the present specification provides a microorganism comprising at least one (2 or more, 3 or more, or 4) gene selected from dhaB, gdrAB, aldH, and yqhD (a first recombinant microorganism). The first recombinant microorganism may have the ability to produce 1,3-PDO and 3-HP from glycerol (e.g., the ability to produce both simultaneously).

[0010] The microorganism may further comprise at least one gene selected from 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 have the ability to produce 1,3-PDO and 3-HP from glucose (e.g., the ability to produce both).

[0011] Another embodiment provides a recombinant vector (first recombinant vector) comprising at least one (2 or more, 3 or more, or 4) selected from dhaB, gdrAB, aldH, and yqhD.

[0012] The recombinant vector may further comprise at least one gene selected from the group consisting of a GPD gene and a GPP gene (a second recombinant vector).

[0013] The first recombinant vector and the second recombinant vector may have the purpose of producing 1,3-PDO and / or 3-HP. In one embodiment, the first recombinant vector may be a recombinant vector for producing 1,3-PDO and / or 3-HP from glycerol. In another embodiment, the second recombinant vector may be a recombinant vector for producing 1,3-PDO and / or 3-HP from glucose.

[0014] Another embodiment provides a composition for producing 1,3-PDO and / or 3-HP, comprising the microorganism and / or the recombinant vector. In one embodiment, a composition for producing 1,3-PDO and / or 3-HP from glycerol is provided, comprising a first recombinant microorganism, a first recombinant vector, or both. In another embodiment, a composition for producing 1,3-PDO and / or 3-HP from glucose is provided, comprising a second recombinant microorganism, a second recombinant vector, or both.

[0015] Another embodiment provides a method for producing 1,3-PDO and / or 3-HP comprising culturing the microorganism.

[0016] Another embodiment provides a fermentation composition obtained by fermenting a microorganism having 1,3-PDO and 3-HP producing activity, and comprising 1,3-PDO and 3-HP.

[0017] Technical Solution

[0018] One embodiment of the present specification provides a microorganism comprising at least one (2 or more, 3 or more, or 4) selected from dhaB, gdrAB, aldH, and yqhD.

[0019] The microorganism may further comprise at least one gene selected from a gene encoding glycerol-3-phosphate dehydrogenase (GPD) (GPD gene) and a gene encoding glycerol-3-phosphate phosphatase (GPP) (GPP gene) (second recombinant microorganism).

[0020] The microorganism may contain all of the dhaB, gdrAB, aldH and yqhD genes, but is not limited thereto.

[0021] The microorganism may include all of the GPD gene and the GPP gene, but is not limited thereto.

[0022] The microorganism may produce 1,3-propanediol (1,3-PDO) and / or 3-hydroxypropionic acid (3-HP), and may simultaneously produce 1,3-PDO and 3-HP, but is not limited thereto.

[0023] The microorganism may have an activity of producing 1,3-propanediol (1,3-PDO) and / or 3-hydroxypropionic acid (3-HP), and may have an activity of simultaneously producing 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP), but is not limited thereto.

[0024] The microorganism provided in the present specification can produce 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP) from glucose and / or glycerol, but is not limited thereto.

[0025] Another embodiment provides a composition for producing 1,3-PDO and / or 3-HP, which comprises the microorganism and / or recombinant vector provided in the present specification.

[0026] Another embodiment

[0027] A recombinant vector is provided, which comprises at least one (2 or more, 3 or more, or 4) selected from dhaB, gdrAB, aldH and yqhD.

[0028] The recombinant vector may further comprise at least one gene selected from the group consisting of a GPD gene and a GPP gene (a second recombinant vector).

[0029] The first recombinant vector may include dhaB, gdrAB, aldH and yqhD genes, but is not limited thereto.

[0030] The second recombinant vector may contain a GPD gene and a GPP gene, but is not limited thereto.

[0031] Another embodiment provides a method for producing 1,3-PDO and / or 3-HP, comprising culturing a microorganism provided in the present specification.

[0032] Hereinafter, the present application will be described in more detail.

[0033] In this specification, "microorganism" means a very small organism that cannot be seen with the naked eye. In this specification, the microorganism may have the ability to synthesize organic compounds, for example, it may have the ability to synthesize 1,3-propanediol (1,3-PDO) and / or 3-hydroxypropionic acid (3-HP) (or, 1,3-PDO production ability and / or 3-HP production ability). The microorganism according to one embodiment itself can be a strain that is genetically modified to produce organic compounds, or has the ability to produce organic compounds, or enhances the ability to produce organic compounds by including genes involved in the production of organic compounds. The production of organic compounds includes production within strains, intracellular production and extracellular secretion, or a combination thereof.

[0034] The microorganism of the present specification may have enhanced activity of the introduced gene due to the introduction of at least one gene selected from 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, but is not limited thereto.

[0035] The microorganism of the present specification may also include a feature of attenuated or inactivated activity of at least one gene selected from yqhD, glpK, ldhA, ack-pta, gldA and ptsG, but is not limited thereto.

[0036] The microorganism of the present specification may also include at least one feature selected from the following (a) and (b), but is not limited thereto:

[0037] (a) the activity of at least one gene selected from yqhD, glpK, ldhA, ack-pta, gldA and ptsG is weakened or inactivated; and

[0038] (b) The activity of at least one gene selected from galP and glk is enhanced.

[0039] Features (a) and (b) may be features of the microorganism before at least one gene selected from 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 is introduced into the microorganism of the present specification, and the corresponding features may or may not be maintained after the introduction of the genes, but are not limited thereto.

[0040] The microorganism of the present specification can be a microorganism of the genus Escherichia (for example, Escherichia coli (E. coli), Escherichia Alberti (E. albertii), Escherichia fergusonii (E. fergusonii), Escherichia hermannii (E. hermannii) and Escherichia marmotae (E. marmotae)), a microorganism of the genus Klebsiella (for example, Klebsiella pneumoniae (K. pneumoniae), Klebsiella aerogenes (K. aeroge nes), Klebsiella granulomatis, Klebsiella grimontii, Klebsiella huaxiensis), and / or Lactobacillus microorganisms (e.g., Lactobacillus reuteri, Lactobacillus acetotolerans, Lactobacillus acidophilus, Lactobacillus thermophilus), but are not limited thereto.

[0041] The microorganism provided in the present specification may have the ability to produce 1,3-propanediol (1,3-PDO) and / or the ability to produce 3-HP, but is not limited thereto.

[0042] In the present specification, “comprising a gene” may mean not only comprising a gene contained in an object (eg, a microorganism) but also introducing and comprising a foreign gene into the object.

[0043] In the present specification, "having 1,3-propanediol (1,3-PDO)-producing ability" may mean a cell and / or microorganism that naturally has 1,3-PDO-producing ability, or a microorganism in which a parent strain that does not have 1,3-PDO-producing ability is imparted with 1,3-PDO-producing ability. It may be used to mean a case where a microorganism has 1,3-PDO-producing ability by introducing a mutation according to one embodiment and / or a case where a microorganism has 1,3-PDO-producing ability by introducing a mutation according to one embodiment into a microorganism that does not have 1,3-PDO-producing ability. For example, an Escherichia microorganism having 1,3-PDO-producing ability may mean a natural microorganism itself, or an Escherichia microorganism having improved 1,3-PDO-producing ability by inserting an exogenous gene related to the 1,3-PDO production mechanism or enhancing the activity of an inherent gene or inactivating the activity of an inherent gene.

[0044] In this specification, "having 3-HP production ability" can mean a cell and / or microorganism with natural 3-HP production ability or a parent strain that does not have 3-HP production ability is endowed with 3-HP production ability. It can be used to mean a situation in which a microorganism has 3-HP production ability by introducing a mutation according to an embodiment and / or a situation in which a microorganism has 3-HP production ability by introducing a mutation according to an embodiment in a microorganism that does not have 3-HP production ability. For example, an Escherichia microorganism with 3-HP production ability can mean a natural microorganism itself, or an Escherichia microorganism with improved 3-HP production ability by inserting an exogenous gene related to the 3-HP production mechanism or enhancing the activity of an intrinsic gene or inactivating the activity of an intrinsic gene.

[0045] In one embodiment, the microorganism can be a strain having the ability to produce 1,3-PDO and / or 3-HP, or a strain genetically modified to have the ability to produce 1,3-PDO and / or 3-HP. The production of 1,3-PDO and / or 3-HP includes production within the strain, intracellular production and extracellular secretion, or a combination thereof.

[0046] The microorganism (or recombinant cell) may further comprise a mutation for increasing 1,3-PDO production and / or 3-HP production, and the position of the mutation and / or the type of gene and / or protein subjected to the mutation may be included without limitation, as long as it increases 1,3-PDO production and / or 3-HP production. The microorganism may be used without limitation, as long as it is a cell capable of transformation.

[0047] In the present application, in order to distinguish a microorganism before a mutation according to one embodiment is introduced from a microorganism in which 1,3-PDO production ability and / or 3-HP production ability is improved or 1,3-PDO production ability and / or 3-HP production ability is imparted due to the introduction of a mutation according to one embodiment, the microorganism before the mutation according to one embodiment is introduced may be referred to as a host microorganism (or parent strain).

[0048] In the present specification, "glycerol-3-phosphate dehydrogenase (GPD)" refers to a polypeptide having an enzyme activity that catalyzes the conversion of dihydroxyacetone phosphate (DHAP) into glycerol-3-phosphate (G3P). Glycerol-3-phosphate dehydrogenase may be NADH-, NADPH- or FAD-dependent. Glycerol-3-phosphate dehydrogenase, regardless of its origin, is included in the scope of the present application as long as it is an enzyme having an enzyme activity that catalyzes the conversion of DHAP into G3P, and GPD1 or GPD2 may be exemplified as a gene encoding it.

[0049] In the present specification, "glycerol-3-phosphate phosphatase (GPP)" refers to a polypeptide having an enzyme activity that catalyzes the conversion of glycerol-3-phosphate (G3P) into glycerol. Glycerol-3-phosphate phosphatase, regardless of its origin, is included in the scope of the present application as long as it is an enzyme having an enzyme activity that catalyzes the conversion of G3P into glycerol, and GPP1 or GPP2 can be exemplified as a gene encoding it.

[0050] In the present specification, "glycerol dehydratase" or "diol dehydratase" may mean a polypeptide having an enzymatic activity that catalyzes the conversion of glycerol into 3-hydroxypropionaldehyde (3-HPA). Glycerol dehydratase or diol dehydratase may be coenzyme B12-dependent or independent. The enzyme may be derived from Klebsiella pneumoniae, Citrobacter freundii, Clostridium pasteurianum, Salmonella typhimurium, Klebsiella oxytoca and / or Clostridium butyricum, etc., but is not limited thereto. Regardless of its source, the enzyme may be included in the scope of the present application as long as it is an enzyme having an enzymatic activity that catalyzes the conversion of glycerol into 3-hydroxypropionaldehyde (3-HPA), for example, the gene encoding the enzyme may be dhaB.

[0051] In the present specification, "glycerol dehydratase reactivation enzyme" may mean an enzyme for maintaining catalytic activity by activating a substance that is irreversibly inactivated when the glycerol dehydratase acts. As the glycerol dehydratase reactivation enzyme, any commonly known glycerol dehydratase reactivation enzyme can be used, for example, it can be derived from Klebsiella (e.g., Klebsiella pneumoniae), Citrobacter, Lactobacillus, Salmonella strains, etc., but is not limited thereto. The glycerol dehydratase reactivation enzyme can be DhaFG, GdrAB, DdrAB, etc., for example, the gene encoding it can be dhaFG, gdrAB and / or ddrAB.

[0052] In the present specification, “yqhD” is a gene encoding alcohol dehydrogenase, and encodes an enzyme that converts 3-hydroxypropionaldehyde into 1,3-PDO when glycerol is degraded into 3-hydroxypropionaldehyde and water by glycerol dehydratase.

[0053] In the present specification, "aldehyde dehydrogenase" is an enzyme that catalyzes the oxidation of aldehydes. The enzyme can convert aldehydes (RC(=O)-H) into carboxylic acids (RC(=O)-OH). The enzyme can be encoded by an aldH gene, and the gene can be an aldH (GenBank accession number U00096.3; EaldH) gene derived from Escherichia coli or Escherichia coli K12 MG1655 18-3 cell line, a puuC gene derived from Klebsiella pneumoniae (K.pneumonia), and / or a KGSADH gene derived from Azospirillumbrasilense, but is not limited thereto. The aldehyde hydrogenase protein and the gene encoding it may contain mutations in the gene and / or amino acid sequence within the range of maintaining the activity of producing 3-HP from 3-HPA.

[0054] In the present specification, "enhanced activity" or "activity enhancement" may include not only effects beyond the original function due to the newly introduced or increased activity of the protein itself, but also means the state of the activity of the microorganism after the transformation is improved compared to the activity of the microorganism before the transformation, such as the increase in gene expression, the increase in intrinsic gene activity, the amplification of intrinsic genes from internal or external factors, the deletion of inhibitory regulatory factors of gene expression, the increase in gene copy number, the introduction of genes from the outside, the modification of expression regulatory sequences, and / or promoter replacement or modification, and the improvement of enzyme activity by intragenic mutations, etc.

[0055] In this specification, the activity of enhancing proteins and / or enzymes (e.g., GPD, GPP, dhaB, gdrAB, aldH and / or yqhD) can be achieved by applying various methods well known in the art. For the method for enhancing or improving the activity of proteins and / or enzymes, various methods well known in the art can be applied. For example, it is not limited thereto, but can include a method for inserting a polynucleotide comprising a sequence encoding the corresponding enzyme (or protein) into a chromosome in addition, a method for increasing the copy number of the sequence encoding the enzyme (or protein) by introducing a polynucleotide into a vector system, a method for replacing a promoter capable of expressing polynucleotides with a strong promoter, and a method for introducing mutations into a promoter, and a method for mutating into an enzyme (or protein) with strong activity by gene mutation, etc. The method for inserting a polynucleotide (e.g., GPD gene, GPP gene, dhaB, gdrAB, aldH and / or yqhD) into the host cell genome (chromosome) can be carried out by appropriately selecting a known method by a person skilled in the art, for example, this can be carried out using: (a) an RNA-guided endonuclease (e.g., Cas9 protein, etc.), a gene encoding it, or a vector comprising the gene; and (b) a mixture (a mixture of an RNA-guided endonuclease protein and a guide RNA, etc.) or a complex (e.g., selected from at least one of a ribonucleic acid fusion protein (RNP), a recombinant vector (e.g., a vector simultaneously comprising a gene encoding an RNA-guided endonuclease and a DNA encoding a guide RNA, etc.), but is not limited thereto.

[0056] In the present specification, "reduced activity" may mean a situation where the activity level of a protein and / or enzyme (e.g., GPD, GPP, yqhD, glpK, ldhA, ack-pta, gldA, and / or ptsG) in a cell is reduced compared to a host cell (or parent strain), such as a wild-type strain or a strain before modification, etc. Reduction is a concept that includes the following situations: due to a mutation in a gene encoding an enzyme (or protein), the activity of the enzyme (or protein) itself is reduced compared to the enzyme activity initially possessed by the microorganism; and due to transcriptional inhibition and / or translational inhibition of a gene encoding an enzyme (or protein), the expression level of the enzyme (or protein) is reduced, and thus the overall enzyme (or protein) activity level in the cell is lower than that of a wild-type strain or a strain before modification; and combinations thereof.

[0057] In the present specification, "inactivation" may mean a state in which the expression of a gene encoding a protein and / or enzyme (e.g., GPD gene, GPP gene, yqhD, glpK, ldhA, ack-pta, gldA and / or ptsG) is not performed at all, or it is expressed but has no activity.

[0058] In the present specification, the activity reduction or inactivation of proteins and / or enzymes (e.g., GPD, GPP, yqhD, glpK, ldhA, ack-pta, gldA and / or ptsG) can be achieved by applying various methods known in the art. For example, there are methods of replacing a gene encoding an enzyme with a mutated gene to reduce the activity of the enzyme (or protein) (including the case of completely eliminating the activity of the enzyme (or protein)); methods of introducing mutations into expression regulatory sequences (e.g., promoters) of genes encoding enzymes (or proteins) on chromosomes; methods of replacing expression regulatory sequences of genes encoding enzymes (or proteins) with weakly active or inactive sequences; methods of deleting all or some of the genes encoding enzymes (or proteins) on chromosomes; methods of introducing antisense oligonucleotides (e.g., antisense RNA) that complementarily bind to the transcriptome of the gene on the chromosome to inhibit translation from mRNA to enzyme (or protein); methods of artificially adding a complementary sequence to the SD sequence in front of the SD sequence of the gene encoding the enzyme (or protein) to form a secondary structure so that ribosomes cannot attach; and / or a RTE (Reverse transcription engineering) method of adding a promoter to be reverse transcribed to the 3' end of the ORF (open reading frame) of the corresponding sequence, etc., and it can be achieved by a combination of the above methods, but is not limited thereto.

[0059] In this specification, the reduction of gene expression can mean that the expression level of gene is reduced compared with the expression level of host cell (parent strain) and / or wild type before mutation, and can include the situation that expression is suppressed completely so as not to realize expression.When reducing the expression of gene in microorganism, the initiation codon of gene translation can be replaced to reduce its expression, or it can be genetically modified to reduce the expression level of conventional gene.The method for modifying sequence to regulate expression can be carried out by inducing the mutation on expression regulation sequence by deletion, insertion, non-conservative or conservative replacement or its combination in the nucleotide sequence of expression regulation sequence, or can be carried out by replacing the promoter with the method of weaker promoter etc. Expression regulation sequence includes promoter, operator sequence, sequence encoding ribosome binding site and sequence regulating the termination of transcription and translation, but is not limited thereto.When there is the gene reducing expression in the microorganism to be mutated, the expression of gene can be reduced, for example, by replacing the natural promoter of the manipulation expression of gene with weak promoter or introducing mutation.In addition, the expression of gene can be reduced by making all or some deletions in conventional genes.

[0060] In this specification, "vector" refers to any carrier for cloning and / or transferring bases to a host cell. A vector can be a replicon that can replicate a combined fragment when another DNA fragment is combined. "Replicon" can mean any genetic unit (e.g., plasmid, phage, clay, chromosome, virus), which functions as its own unit of DNA replication in vivo, i.e., can be replicated by 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 construct a recombinant vector can be a plasmid, clay, virus and / or phage in a natural state or a recombinant state. For example, as phage vector or cosmid vector, pWE15, M13, λEMBL3, λEMBL4, λFIXII, λDASHII, λZAPII, λgt10, λgt11, Charon4A and / or Charon21A etc. can be used, and as plasmid vector, pDZ vector, based on pBR, based on pUC, based on pBluescriptII, based on pGEM, based on pTZ, based on pCL and / or based on pET etc. can be used. Available vector is not particularly limited, and known expression vector can be used. In one embodiment, vector can be such that the gene delivered by being inserted into the vector is irreversibly fused into the genome of the host cell, thereby allowing gene expression to continue stably for a long time in the cell. Such vector can include transcription and translation expression regulatory sequences, which allow corresponding genes to be expressed in selected hosts. As expression regulatory sequences, any operon sequence for regulating transcription and / or the sequence for regulating the termination of transcription and translation can be included. In one embodiment, the start codon and the stop codon can be generally considered to be part of the nucleic acid sequence encoding the target protein, and should function in the subject when the gene construct is applied, and can be in frame with the coding sequence. In addition, in the case of a replicable expression vector, it can include a replication origin. In addition, it can appropriately include an enhancer, a non-translated region at the 3' end of the target gene, a selection marker (e.g., an antibiotic resistance marker) and / or a replicable unit, etc. The vector can self-replicate or integrate into the host genomic DNA.

[0061] According to one embodiment, the elements in the vector should be operably linked to each other, and the connection of these element sequences can be carried out by ligation (ligation) at convenient restriction enzyme sites, and when such sites do not exist, it can be carried out using synthetic oligonucleotide adapters or linkers according to common methods.

[0062] In this specification, "transduction" refers to the phenomenon that bacterial DNA moves to another bacterium via a bacteriophage, and is one of the methods of horizontal gene transfer (HGT). Phages that can transduce in this way are called "transducing particles" or "transducing phages," and cells that receive DNA from another bacterium are called "transductants."

[0063] In this specification, "conversion" is to introduce a gene into a host cell to express the gene in the host cell, and may include a converted gene, as long as it can be expressed in the host cell, and is not limited to whether it is inserted into the chromosome of the host cell or is located outside the chromosome. In addition, the gene includes DNA and / or RNA encoding the target protein. As long as the gene can be introduced into the host microorganism and expressed, the form in which it is introduced is not limited. For example, the gene can be introduced into the host microorganism in the form of an expression cassette, and the expression cassette form is a gene structure comprising all elements required for self-expression. The expression cassette may generally include an expression regulatory element operably connected to a gene, such as a promoter, a transcription termination signal, a ribosome binding site and / or a translation termination signal, etc. The expression cassette may be in the form of an expression vector that can self-replicate. In addition, the gene can be introduced into the host cell in its own form and operably connected to the sequence required for expression in the host cell.

[0064] In this specification, "promoter" means a non-translated nucleic acid sequence upstream of the coding region, which contains a polymerase binding site and has transcription initiation activity for the mRNA of the gene downstream of the promoter, that is, a DNA region to which the polymerase binds to initiate gene transcription, and may be located in the 5' region of the mRNA transcription initiation region.

[0065] In one embodiment, the microorganism provided in this specification can produce 1,3-propanediol (1,3-PDO) and / or 3-hydroxypropionic acid (3-HP) by further comprising a constitutive promoter and a GPD gene, a GPP gene, dhaB, gdrAB, aldH and / or yqhD operably linked thereto, but is not limited thereto. A constitutive promoter can induce expression of an operably linked gene in the absence of a separate inducer. For example, the constitutive promoter may be based on J23 (e.g., the promoter is J23100, J23101, J23102, J23103, J23104, J23105, J23106, J23107, J23109, J23111, J23112, J23113, J23114, J23115, J23116, J23117 and / or J23118), but is not limited thereto, and may be selected and used by those skilled in the art without limitation as needed within the scope of the purpose of producing 1,3-PDO and / or 3-HP. At least one (2 or more, 3 or more, or 4) gene selected from GPD gene, GPP gene, dhaB, gdrAB, aldH and yqhD (in which a constitutive promoter is introduced) may be cloned into a vector, respectively, and introduced into a cell (or microorganism). Alternatively, the four genes can be cloned into one vector and introduced into cells (or microorganisms).

[0066] In this specification, “generation rate” may mean a generation amount per hour, but is not limited thereto.

[0067] The 1,3-PDO production rate of the microorganism provided in the present specification can be: 0.1 g / L / hour or more, 0.5 g / L / hour or more, 1 g / L / hour or more, 1.2 g / L / hour or more, 1.3 g / L / hour or more, 1.4 g / L / hour or more, 1.45 g / L / hour or more, 1.6 g / L / hour or more, 1.9 g / L / hour or more, 2 g / L / hour or more, or 2.1 g / L / hour or more, for example, 1.45 g / L / hour or 2.1 g / L / hour, and the upper limit of the 1,3-PDO production rate is not particularly limited, for example, the 1,3-PDO production rate thereof can be 100 g / L / hour or less, or 10 g / L / hour or less, but is not limited thereto.

[0068] The 3-HP production rate of the microorganism provided in this specification can be: 0.1g / L / hour or higher, 0.5g / L / hour or higher, 1g / L / hour or higher, 1.2g / L / hour or higher, 1.3g / L / hour or higher, 1.4g / L / hour or higher, 1.45g / L / hour or higher, 1.6g / L / hour or higher, 1.9g / L / hour or higher, or 2g / L / hour or higher, for example, 1.45g / L / hour or 2g / L / hour, and the upper limit of the 3-HP production rate is not particularly limited, for example, its 3-HP production rate can be 100g / L / hour or lower or 10g / L hour or lower, but is not limited thereto.

[0069] The present specification provides a recombinant vector comprising at least one gene (2 or more, 3 or more, or 4) selected from dhaB, gdrAB, aldH, and yqhD.

[0070] The present specification provides a recombinant vector comprising at least one (2 or more, 3 or more, or 4) selected from dhaB, gdrAB, aldH, and yqhD (a first recombinant vector).

[0071] The recombinant vector may further comprise at least one gene selected from the group consisting of a GPD gene and a GPP gene (a second recombinant vector).

[0072] The microorganism into which the recombinant vector is introduced can produce 1,3-PDO and / or 3-HP, and can produce 1,3-PDO and / or 3-HP simultaneously, but is not limited thereto.

[0073] The microorganism into which the recombinant vector is introduced may have 1,3-PDO and / or 3-HP producing activity, and may have the activity of simultaneously producing 1,3-PDO and / or 3-HP, but is not limited thereto.

[0074] The present specification provides a method for producing 1,3-PDO and / or 3-HP, which comprises culturing the microorganism (specifically, comprising culturing a microorganism comprising at least one (2 or more, 3 or more, or 4) selected from dhaB, gdrAB, aldH, and yqhD and / or at least one gene selected from a gene encoding glycerol-3-phosphate dehydrogenase (GPD) (GPD gene) and a gene encoding glycerol-3-phosphate phosphatase (GPP) (GPP gene)).

[0075] Another embodiment provides a method for producing 1,3-PDO and / or 3-HP from glycerol, comprising culturing a first recombinant microorganism.Another embodiment provides a method for producing 1,3-PDO and / or 3-HP from glucose, comprising culturing a second recombinant microorganism.

[0076] Cultivating the microorganism (specifically, the first recombinant microorganism and / or the second recombinant microorganism) may include reacting the microorganism with a substrate. The substrate may be glycerol and / or glucose, but is not limited thereto.

[0077] The method for producing 1,3-PDO and / or 3-HP may further include recovering 1,3-PDO and / or 3-HP from the culture medium or the microorganism, but is not limited thereto.

[0078] In one embodiment, the culturing may include primary culturing and / or primary culturing and subculturing, but is not limited thereto.

[0079] In one embodiment, culturing may include culturing by inoculating into LB medium;

[0080] Cultivate by inoculating into M9 medium; and / or

[0081] The culture is performed by inoculation into MR medium, but is not limited thereto.

[0082] The culture may be performed using culture by inoculation into LB medium as primary culture and using culture by inoculation into M9 medium and / or MR medium as subculture, but is not limited thereto.

[0083] The 1,3-PDO production rate of the method for producing 1,3-PDO and / or 3-HP may be 0.1 g / L / hour or more, 0.5 g / L / hour or more, 1 g / L / hour or more, 1.2 g / L / hour or more, 1.3 g / L / hour or more, 1.4 g / L / hour or more, 1.45 g / L / hour or more, 1.6 g / L / hour or more, 1.9 g / L / hour or more, 2 g / L / hour or more, or 2.1 g / L / hour or more, for example, 1.45 g / L / hour or 2.1 g / L / hour, and the upper limit of the 1,3-PDO production rate is not particularly limited, for example, it may be 100 g / L / hour or less, or 10 g / L / hour or less, but is not limited thereto.

[0084] The 3-HP production rate of the method for producing 1,3-PDO and / or 3-HP can be 0.1 g / L / hour or more, 0.5 g / L / hour or more, 1 g / L / hour or more, 1.2 g / L / hour or more, 1.3 g / L / hour or more, 1.4 g / L / hour or more, 1.45 g / L / hour or more, 1.6 g / L / hour or more, 1.9 g / L / hour or more, or 2 g / L / hour or more, for example, 1.45 g / L / hour or more or 2 g / L / hour, and the upper limit of the 3-HP production rate is not particularly limited, for example, it can be 100 g / L / hour or less, or 10 g / L / hour or less, but is not limited thereto.

[0085] The culture can be carried out according to appropriate culture media and culture conditions known in the art and should meet the requirements of specific strains, and can be appropriately modified by those skilled in the art.

[0086] The culture method may include, for example, batch culture, continuous culture, fed-batch culture, or a combination thereof, but is not limited thereto.

[0087] According to one embodiment, various carbon sources, nitrogen sources and trace element components may be included in the culture medium, and the recombinant cells may be cultured in a common culture medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc. while adjusting the temperature and / or pH, etc. As available carbon sources, sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and / or cellulose are included; as well as oils and fats such as soybean oil, sunflower oil, castor oil and / or coconut oil, etc.; 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 may be used alone or in a mixture, but are not limited thereto. As available nitrogen sources, peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea may be included; or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate may also be used alone or in a mixture, but are not limited thereto. As available phosphorus supply sources, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or corresponding sodium salts may be included, but are not limited thereto. In addition, the culture medium may contain metal salts required for growth, such as magnesium sulfate or iron sulfate, but is not limited thereto. In addition, essential growth substances such as amino acids and vitamins may be contained. In addition, precursors suitable for the culture medium may be used. During the culture process, the culture medium or individual components may be added to the culture solution in a fed-batch or continuous process by an appropriate method, but is not limited thereto.

[0088] Separating and / or recovering 1,3-PDO and / or 3-HP from cultured microorganisms (or recombinant cells) and / or culture medium can be collecting the desired substance (e.g., 1,3-PDO and / or 3-HP) from the culture medium, culture solution or microorganism using appropriate methods known in the art. For example, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation) and / or chromatography (e.g., ion exchange, affinity, hydrophobic, liquid and size exclusion) and the like can be used, but are not limited thereto. Culture medium can mean a culture medium in which a microorganism (or recombinant cell) is cultured.

[0089] In one embodiment, the method for producing 1,3-PDO and / or 3-HP may further include purifying the 1,3-PDO and / or 3-HP prior to, simultaneously with, or after separation and / or recovery.

[0090] In the present specification, "fermentation composition" means a composition prepared by adding strains and / or microorganisms to specific materials through a culture and / or fermentation process.

[0091] The present specification provides a fermented composition obtained by fermenting a microorganism having 1,3-PDO and 3-HP producing activity and comprising 1,3-PDO and / or 3-HP.

[0092] The fermentation 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, and the upper limit of the 1,3-PDO concentration is not particularly limited, for example, it may contain 1,3-PDO at a concentration of 1000 g / L or less, or 100 g / L or less, but is not limited thereto.

[0093] The fermentation 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, such as 26 g / L or 77 g / L, and the upper limit of the 3-HP concentration is not particularly limited, for example, it may contain 3-HP at a concentration of 1000 g / L or less, or 100 g / L or less, but is not limited thereto.

[0094] The fermentation composition may include both 1,3-PDO and 3-HP, but is not limited thereto.

[0095] The fermented composition can be prepared by the microorganisms provided in this specification, but is not limited thereto.

[0096] The fermented composition may be prepared by the method for producing 1,3-PDO and / or 3-HP provided in the present specification, but is not limited thereto.

[0097] Beneficial Effects

[0098] The present invention relates to a microorganism into which a GPD gene, a GPP gene, a dhaB, a gdrAB, aldH and / or a yqhD gene is introduced and / or uses thereof, and has the effect of producing 1,3-PDO and / or 3-HP. BRIEF DESCRIPTION OF THE DRAWINGS

[0099] Figure 1 It is a graph showing the results of confirming the production of 1,3-PDO and 3-HP after culturing the microorganism into which the dhaB, gdrAB, aldH and / or yqhD gene was introduced.

[0100] Figure 2 It is a graph showing the results of confirming the production of 1,3-PDO and 3-HP after culturing the microorganism into which the GPD gene, GPP gene, dhaB, gdrAB, aldH and / or yqhD gene were introduced. DETAILED DESCRIPTION

[0101] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are only for illustrating the present invention, but the present invention is not limited by the following examples.

[0102] Example 1. Plasmid design for strain recombination

[0103] Example 1-1. Construction of pCDF-dhaB1,2,3-gdrA,B-aldH-yqhD vector

[0104] Construct a vector containing yqhD to overexpress the yqhD gene. Specifically, the yqhD gene (SEQ ID NO: 1) was prepared by amplifying the genome of the Escherichia coli W3110 strain (yqhD, glpK, ldhA, ack-pta, gldA and ptsG deletion; galP and glk overexpression; named DKALGP::PK strain; KCCM40219) using a primer pair consisting of the nucleic acid sequence of SEQ ID NO: 2 and SEQ ID NO: 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, and finally extended at 72°C for 2 minutes, then maintained and stored at 4°C, and the final product size: 1.1 kb), and the information of the yqhD gene and primers is shown in Tables 1 and 2 below.

[0105] [Table 1]

[0106]

[0107] [Table 2]

[0108]

[0109] The prepared yqhD gene was cloned into the pCDF-dhaB1,2,3-gdrA,B-aldH plasmid for producing 3HP.

[0110] 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) is replaced by the J23101 and J23100 promoters, and the dhaB (dhaB1, dhaB2, dhaB3) and gdrAB genes (gdrA, gdrB) of Klebsiella pneumoniae (Klebsiella pneumoniae genomic DNA; KCTC12385) and the aldH gene derived from Escherichia coli K12 MG1655 are cloned. Using the primers in Table 3 below and NEB (New England Biolabs) Q5 DNA polymerase in the Klebsiella pneumoniae chromosome, dhaB (about 2.7 kb; dhaB1, dhaB2, dhaB3) and gdrAB genes (about 2.2 kb; gdrA, gdrB) were amplified using the conditions provided by NEB. In addition, the information of the promoter is shown in Table 4 below.

[0111] [Table 3]

[0112]

[0113] [Table 4]

[0114]

[0115] On the chromosome of Klebsiella pneumoniae, dhaB123 and gdrA genes are located together, so they are amplified together, and gdrB is located in the opposite direction to dhaB123 and gdrA, so only gdrB is amplified alone, and NEBQ5 DNA polymerase is used, and as conditions, conditions provided by NEB are used. Thereafter, under the J23101 promoter, dhaB123 and gdrA genes are cloned using restriction enzymes EcoRI and HindIII, and gdrB gene is cloned using restriction enzymes HindIII and AflII, and pCDF-dhaB1,2,3-gdrA,B plasmid (hereinafter pCDF-dhaB-gdrAB vector) is prepared.

[0116] aldH was amplified from the E. coli K12 MG1655 chromosome using the J23100 promoter and the primers in Table 5 below and NEB Q5 DNA polymerase, using conditions provided by NEB.

[0117] [Table 5]

[0118]

[0119] aldH was cloned by T4 DNA ligase reaction under the J23100 promoter of the pCDF-dhaB-gdrAB vector using restriction enzymes KpniI and NdeI, and pCDF-dhaB1,2,3-gdrA,B-aldH plasmid (hereinafter pCDF-dhaB-gdrAB-aldH vector) was prepared.

[0120] For the CDF-dhaB1,2,3-gdrA,B-aldH plasmid prepared as above, the prepared yqhD gene was cloned by fusion reaction using PacI restriction enzyme, and finally, the recombinant vector pCDF-dhaB1,2,3-gdrA,B-aldH-yqhD (hereinafter pCDF-dhaB-gdrAB-aldH-yqhD vector) was prepared. In addition, the pCDF-dhaB-gdrAB-yqhD vector was prepared, in which yqhD was cloned into the pCDF-dhaB-gdrAB vector instead of aldH by the same method.

[0121] Example 1-2. Construction of pRSF-pLTTR-GPD-GPP vector

[0122] For the GPD1 and GPP2 genes derived from Saccharomyces cerevisiae (BY4741; ATCC 201388), PCR was performed using the primer pairs of SEQ ID Nos: 12 and 13 or SEQ ID Nos: 14 and 15 in Table 6 below, respectively (preliminary 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; holding and storing at 4°C), and each of them was treated with BamHI / SacI (using the primer pair of SEQ ID Nos: 12 and 13) and KpnI / XhoI (using the primer pair of SEQ ID Nos: 14 and 15) and cloned into the pRSFDuet-1 vector (Invitrogen) containing the LTTR promoter, and a vector containing pRSF-pLTTR-GPD-GPP (hereinafter referred to as pRSF-pLTTR-GPD-GPP vector) was constructed. The sequence of the LTTR promoter used is described in Table 7 below.

[0123] [Table 6]

[0124]

[0125] [Table 7]

[0126]

[0127] Example 2. Preparation and cultivation of transformed strains

[0128] Example 2-1. Preparation of strains

[0129] In order to prepare the transformation strain with the vector prepared in Example 1, Escherichia coli W3110 (yqhD, glpK, ldhA, ack-pta, gldA and ptsG deletion; galP and glk overexpression; named DKALGP::PK strain) strain was prepared. The strain was streaked on LB plates (10g tryptone, 5g yeast extract, 10g NaCl, 15g agar per 1L) and cultured overnight at 37°C. The cultured single colony was inoculated into 5mL LB liquid culture medium (the culture medium composition was the same as the LB plate, but agar was not included) and stirred and cultured overnight at 37°C. The next morning, the culture solution was diluted to 1:100 in 5mL NL liquid culture medium and cultured for 3 to 6 hours until OD reached about 0.5. The cells were collected by cooling on ice for 10 to 15 minutes and then centrifuging at 4000rpm for 10 minutes at 4°C. The cells were resuspended in 1 mL of ice-cooled sterile DI water (deionized water) and washed, and then centrifuged again at 4000 rpm for 10 minutes at 4° C. to collect the cells. The collected cells were resuspended in 1 mL of DI water and washed, and centrifuged again at 4000 rpm for 10 minutes at 4° C. to collect the cells. The collected cells were resuspended in 0.08 mL of DI water, and cell aliquots were added to separate pre-cooled microcentrifuge tubes.

[0130] Example 2-2. dhaB, gdrAB, aldH and / or yqhD gene transformation

[0131] pCDFDuet-1 (pCDF) of Example 1 and the plasmid DNA constructed in Example 1 (each of the pCDF-dhaB-gdrAB vector, the pCDF-dhaB-gdrAB-yqhD vector and the pCDF-dhaB-gdrAB-aldH-yqhD vector) were added to the cells collected in Example 2-1 by pipetting 1 μL of them, and then 0.08 ml to 0.09 ml of the mixture was transferred to a pretreated 0.1 cm electroporation cuvette (pre-cooled electroporation cuvette).

[0132] Electroporation was performed at 1.8 kV, and 1 mL of room temperature LB liquid medium was added to the cells immediately after the pulse, and they were cultured at 37°C for 1 hour. The culture solution was spread on an LB plate containing streptomycin, and the excess liquid was dried and absorbed into the plate. The plate was inverted and cultured at 37°C to prepare single colonies.

[0133] Example 2-3. Additional transformation of GPD and GPP genes

[0134] In addition, pCDFDuet-1 (pCDF) of Example 1 and plasmid DNAs constructed in Example 1 (each of the pCDF-dhaB-gdrAB vector, pCDF-dhaB-gdrAB-aldH vector, pCDF-dhaB-gdrAB-yqhD vector and pCDF-dhaB-gdrAB-aldH-yqhD vector) (1 μL) were added to the cells collected in Example 2-1 together with 1 μL of the plasmid DNA pRSF-pLTTR-GPD-GPP vector prepared in Example 1 (hereinafter referred to as pRSF-GG vector) by pipetting them, and then 0.08 ml to 0.09 ml of the mixture was transferred to a pretreated 0.1 cm electroporation cuvette (pre-cooled electroporation cuvette). Electroporation was performed at 1.8 kV, and 1 mL of room temperature LB liquid medium was added to the cells immediately after the pulse, and they were cultured at 37°C for 1 hour. The culture solution was spread on an LB plate containing streptomycin, and the excess liquid was dried and absorbed into the plate. The plate was inverted and cultured at 37°C to prepare single colonies.

[0135] Example 3. Confirmation of the production amount of 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP) in strains transformed with dhaB, gdrAB, aldH and / or yqhD genes

[0136] Example 3-1. Confirmation of 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid production of transformed strains by flask culture Production

[0137] 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 in a 250 mL conical flask. Then, the pH was maintained at 6.0 using Ca(OH)2.

[0138] 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, and the absorbance of the strain culture solution was measured at OD600 using a UV spectrometer under the analytical conditions of Table 8 below using HPLC, and the production of 3-PDO and 3-HP by the transformed strain of Example 2-2 was confirmed, and the results are shown in Table 9 below.

[0139] [Table 8]

[0140] HPLC Model Agilent Technologies 1200 Series column Bio-Rad Aminex HPX-87H ion exclusion column 300mm×7.8mm Detector Refractive Index (RI) / Ultraviolet (UV) Detector Mobile phase <![CDATA[0.5mM H2SO4]]> flow 0.4mL / min Run time 35 minutes Column temperature 35℃ Detector temperature 35℃ Injection volume 10μL

[0141] [Table 9]

[0142] Vector type in transformed strain 1,3-PDO(g / L) 3-HP(g / L) pCDF 0 0 pCDF-dhaB-gdrAB 0 0 pCDF-dhaB-gdrAB-yqhD 1.42 0 pCDF-dhaB-gdrAB-aldH-yqhD 2.40 1.31

[0143] As a result, when only the empty vector (pCDF) or dhaB / gdrAB was expressed, both 1,3-PDO and 3-HP were not produced. However, it was confirmed that when dhaB / gdrAB was expressed together with yqhD, 1,3-PDO was produced at 1.42 g / L, and when aldH and yqhD were expressed simultaneously, 1,3-PDO and 3HP were simultaneously produced. It was confirmed that the total target production amount was increased, such as 1,3-PDO production was increased by about 70% compared to single production, and 3HP was additionally produced during simultaneous fermentation.

[0144] Example 3-2. Confirmation of 1,3-propanediol (1,3-PDO) and 3-hydroxypropanediol (3-hydroxypropanediol) of the transformed strain by high-concentration cell culture Propionic acid (3-HP) production

[0145] A single colony of Example 2-2 was inoculated into an LB liquid medium having the same composition as that of the LB liquid medium of Example 2-2, and pre-cultured (seed culture) at 37°C for 18 hours.

[0146] Then, for high-concentration cell culture, in a 5L fermentor, the preculture solution (seed culture) in the 150mL flask was inoculated into 2L of a modified MR medium (containing MgSO4·7H2O 0.8g / L, (NH4)2HPO4 4g / L, KH2PO4 6.67g / L, and citrate 0.8g / L) containing 20g / L glucose, 25mg / L streptomycin, and 50mg / L kanamycin, and cultured at 35°C, 500rpm, and pH 6.95 (adjusted with NH4(OH)). After the glucose contained in the medium was depleted, a feed solution (containing glucose 700g / L, MgSO4 15g / L, trace metals 10mL / 1L, 10×MR salts 100mL / 1L, and 25mg / L streptomycin) was added at a rate of 40mL / hour, and cultured for 24 hours.

[0147] After high-density cell culture, for 3-HP production, at an initial OD of 25 in the culture medium, the culture solution was inoculated into 2 L of new MR medium (containing (NH4)2HPO4 4 g / L, KH2PO4 6.67 g / L, and citrate 0.8 g / L), and 1 μM vitamin B was added. 12 To produce 3-HP, a feed solution of 200 g / L glucose and 500 g / L glycerol was added at a rate of 35 mL / hour, and Mg(OH)2 was added to maintain the pH condition of 6.95.

[0148] Thereafter, the production amounts of 1,3-PDO and 3-HP of the strain transformed with pCDF-dhaB-gdrAB-aldH-yqhD were confirmed using HPLC by substantially the same method as in Example 3-1 above, and the results were Figure 1 and are shown in Table 10. The yield was calculated by the following Equation 1.

[0149] [Equation 1]

[0150] Yield (%) = (production of 1,3-PDO and 3-HP) / (amount of glucose and glycerol used) * 100

[0151] [Table 10]

[0152] type Measurements 1,3-PDO(g / L) 81 3-HP(g / L) 77 1,3-PDO production rate (g / L / hour) 2.1 3-HP production rate (g / L / hour) 2.0 Yield (%, g / g) 63

[0153] (In the above Table 10, the production rate means the production amount per hour)

[0154] As a result, high production amounts and production rates of 1,3-PDO and 3-HP were confirmed, and it was confirmed that the yield was also high.

[0155] Example 4. Confirmation of the production of 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid (3-HP) by strains transformed with GPD and GPP genes

[0156] Example 4-1. Confirmation of 1,3-propanediol (1,3-PDO) and 3-hydroxypropionic acid of transformed strains by flask culture (3-HP) production

[0157] 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 in a 250 mL conical flask. Then, the pH was maintained at 6.0 using Ca(OH)2.

[0158] 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 strain culture solution was measured at OD600 using a UV spectrometer under the analytical conditions of Table 11 below, and the production of 1,3-PDO and 3-HP by the transformed strain of Example 2-3 was confirmed. The results are shown in Table 12 below.

[0159] [Table 11]

[0160] HPLC Model Agilent Technologies 1200 Series column Bio-Rad Aminex HPX-87H ion exclusion column 300mm×7.8mm Detector Refractive Index (RI) / Ultraviolet (UV) Detector Mobile phase <![CDATA[0.5mM H2SO4]]> flow 0.4mL / min Run time 35 minutes Column temperature 35℃ Detector temperature 35℃ Injection volume 10μL

[0161] [Table 12]

[0162] Vector type in transformed strain 1,3-PDO(g / L) 3-HP(g / L) pRSF-GG, pCDF 0 0 pRSF-GG, pCDF-dhaB-gdrAB 0 0 pRSF-GG, pCDF-dhaB-gdrAB-aldH 0 3.43 pRSF-GG, pCDF-dhaB-gdrAB-yqhD 1.42 0 pRSF-GG, pCDF-dhaB-gdrAB-aldH-yqhD 5.21 2.76

[0163] As a result, when only the empty vector (pCDF) or dhaB / gdrAB was expressed, both 1,3-PDO and 3HP were not produced. However, it was confirmed that when dhaB / gdrAB and GPD-GPP were expressed together with aldH or yqhD, 3HP or 1,3-PDO was produced at 3.43 g / L, 1.42 g / L, respectively, and when aldH and yqhD were expressed simultaneously, 1,3-PDO and 3HP were simultaneously produced. Since the production of 1,3-PDO was increased by about 3.7 times compared to single production, and 3HP was additionally produced during simultaneous fermentation, the total target production was confirmed to be increased.

[0164] Example 4-2. Confirmation of 1,3-propanediol (1,3-PDO) and 3-hydroxypropanediol (3-hydroxypropanediol) of the transformed strain by high-concentration cell culture Propionic acid (3-HP) production

[0165] A single colony of Example 2-3 was inoculated into an LB liquid medium having the same composition as that of the LB liquid medium of Example 2-3, and pre-cultured (seed culture) at 37° C. for 18 hours.

[0166] Then, for high-concentration cell culture, in a 5-L fermentor, the preculture solution in the 150-mL flask was inoculated into 2 L of a modified MR medium (containing MgSO4·7H2O 0.8 g / L, (NH4)2HPO4 4 g / L, KH2PO4 6.67 g / L, and citrate 0.8 g / L) containing 20 g / L glucose, 25 mg / L streptomycin, and 50 mg / L kanamycin, and cultured at 35°C, 500 rpm, and pH 6.95 (adjusted with NH4(OH)). After the glucose contained in the medium was depleted, a feed solution (containing glucose 700 g / L, MgSO4 15 g / L, trace metals 10 mL / 1 L, 10×MR salts 100 mL / 1 L, 25 mg / L streptomycin, and 50 mg / L kanamycin) was added at a rate of 40 mL / hour and cultured for 24 hours.

[0167] After high-density cell culture, for 3-HP production, at an initial OD of 25 in the culture medium, the culture solution was inoculated into 2 L of new MR medium (containing (NH4)2HPO4 4 g / L, KH2PO4 6.67 g / L, and citrate 0.8 g / L), and 1 μM vitamin B was added. 12 To produce 3-HP, a feed solution of 600 g / L of glucose was added at a rate of 35 mL / hour, and Mg(OH)2 was added to maintain the pH condition of 6.95.

[0168] Afterwards, the production amounts of 1,3-PDO and 3-HP were confirmed using HPLC by substantially the same method as in Example 4-1 above, and the results were Figure 2 and are shown in Table 13. The yield was calculated by the following Equation 2.

[0169] [Equation 2]

[0170] Yield (%) = (production of 1,3-PDO and 3-HP) / (amount of glucose used) * 100

[0171] [Table 13]

[0172] type Measurements 1,3-PDO(g / L) 26 3-HP(g / L) 26 1,3-PDO production rate (g / L / hour) 1.45 3-HP production rate (g / L / hour) 1.45 Yield (%, g / g) 30

[0173] (In Table 13 above, the production rate refers to the total production per hour)

[0174] As a result, high production amounts and production rates of 1,3-PDO and 3-HP were confirmed, and it was confirmed that the yield was also high.

Claims

1. A microorganism comprising at least one gene selected from the group consisting of dhaB, gdrAB, aldH and yqhD.

2. The microorganism according to claim 1, further comprising at least one gene 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 according to claim 1, comprising all of the dhaB, gdrAB, aldH and yqhD genes. The microorganism according to claim 2 , comprising both the GPD gene and the GPP gene.

5. The microorganism according to claim 1, wherein the microorganism further comprises a feature of attenuated or inactivated activity of at least one gene selected from yqhD, glpK, ldhA, ack-pta, gldA and ptsG.

6. The microorganism of claim 2, wherein the microorganism further comprises at least one feature selected from the group consisting of (a) and (b): (a) the activity of at least one gene selected from yqhD, glpK, ldhA, ack-pta, gldA and ptsG is weakened or inactivated; and (b) The activity of at least one gene selected from 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, a microorganism of the genus Klebsiella, or a microorganism of the genus Lactobacillus.

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

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

12. A recombinant vector comprising at least one gene selected from the group consisting of dhaB, gdrAB, aldH and yqhD. The recombinant vector according to claim 12 , further comprising at least one gene 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 claim 13.

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

16. The method for producing 1,3-PDO and 3-HP according to claim 15, wherein the 1,3-PDO production rate of the method for producing 1,3-PDO and 3-HP is 0.5 g / L / hour or more, or The method for producing 1,3-PDO and 3-HP has a 3-HP production rate of 0.5 g / L / hour or more.

17. A fermentation composition obtained by fermenting a microorganism having 1,3-PDO and 3-HP producing activities, and comprising 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 composition according to claim 17, which is prepared by the microorganism according to any one of claims 1 to claim 6.

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

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