Recombinant microorganism with improved glycerol utilization ability and glycerol utilization method using same

Abstract

The present invention relates to a recombinant microorganism with improved glycerol utilization ability and a glycerol utilization method using same and, in particular, to: a recombinant microorganism of the genus Corynebacterium, which has improved glycerol utilization ability by using a gene involved in the use of glycerol derived from a heterologous strain; and a glycerol utilization method using same. The recombinant microorganism of the present invention is a recombinant microorganism with improved glycerol utilization ability, in which the glycerol conversion pathway operates more efficiently, thereby providing a recombinant microorganism which can facilitate the production of products via the glycerol conversion pathway and is useful for mass production.

Description

Recombinant microorganism with improved glycerol utilization ability and glycerol utilization method using the same

[0001] The present invention relates to a recombinant microorganism having improved glycerol utilization ability and a method for utilizing glycerol using the same, and more particularly, to a recombinant microorganism of the genus Corynebacterium having improved glycerol utilization ability using a gene involved in glycerol utilization derived from a heterologous strain, and a method for utilizing glycerol using the same.

[0002] Glycerol can be converted into DHAP, propanol, glyceric acid, propylene glycol, 1,3-propanediol (1,3-PDO), 3-hydroxypropionic acid (3-HP), etc. by chemical / biological methods. Among the compounds that can be produced by oxidizing glycerol, 1,3-PDO, 3-HP, DHAP, etc. are considered high value-added compounds. Many microorganisms used industrially do not have the ability to utilize glycerol and cannot use it as a carbon source. For example, Corynebacterium glutamicum strains cannot naturally metabolize glycerol, so glycerol metabolism is possible only through the introduction of a glycerol utilization pathway. According to a previously reported study, there is a report that glycerol utilization ability was improved by introducing the glpFKD gene derived from Escherichia coli (Korean Patent Publication No. 10-2019-0133940). Furthermore, glycerol is widely used as a carbon source for the industrial production of 1,3-PDO and 3-HP as a C3 carbon source. Therefore, the development of novel microorganisms capable of more efficiently producing high-value-added compounds such as 1,3-PDO and 3-HP through glycerol oxidation has been demanded. Therefore, the present invention provides a recombinant microorganism with improved glycerol utilization ability using a novel glycerol utilization gene derived from a heterologous strain, thereby completing the present invention.

[0003] The purpose of the present invention is to provide a recombinant microorganism having improved glycerol utilization ability, wherein the glycerol conversion pathway operates more efficiently.

[0004] Another object of the present invention is to provide a product produced by the glycerol conversion pathway from the recombinant microorganism and a method for producing the same.

[0005] Another object of the present invention is to provide a use for utilizing glycerol from the recombinant microorganism.

[0006] The objectives of the present invention are not limited to those mentioned above. Other objectives and advantages of the present invention not mentioned above can be understood through the following description and will be more clearly understood through the embodiments of the present invention. Furthermore, it will be readily apparent that the objectives and advantages of the present invention can be realized by the means and combinations thereof set forth in the claims.

[0007] To achieve the above objectives, the present invention provides a novel recombinant microorganism with improved glycerol utilization ability, wherein the glycerol conversion pathway operates more efficiently. Specifically, a recombinant microorganism of the genus Corynebacterium with improved glycerol utilization ability can be provided by utilizing a novel glycerol utilization-related gene derived from a heterologous strain.

[0008] In addition, according to the present invention, a product by a glycerol conversion pathway can be provided from the recombinant microorganism.

[0009] In addition, according to the present invention, a method for producing a product through a glycerol conversion pathway from the novel recombinant microorganism can be provided.

[0010] In addition, according to the present invention, it is possible to provide a use for producing a product by the glycerol conversion pathway of the novel recombinant microorganism.

[0011] The recombinant microorganism of the present invention is a recombinant microorganism with improved glycerol utilization ability, and thus provides a recombinant microorganism in which the glycerol conversion pathway operates more efficiently, thereby providing a recombinant microorganism useful for mass-producing products through the glycerol conversion pathway.

[0012] The recombinant microorganism of the present invention is a recombinant microorganism with improved glycerol utilization ability, and thus provides a recombinant microorganism in which the glycerol conversion pathway operates more efficiently, thereby providing a recombinant microorganism capable of easily producing a product by the glycerol conversion pathway.

[0013] The recombinant microorganism of the present invention enables efficient conversion of glycerol to DHAP, thereby activating glycolysis and facilitating cell growth. The recombinant microorganism of the present invention utilizes glycerol as a carbon source and enables conversion into 1,3-PDO and 3-HP, facilitating the mass production of high-value-added materials.

[0014] In addition to the effects described above, specific effects of the present invention are described below while explaining specific details for carrying out the invention.

[0015] Figure 1 illustrates a glycerol conversion pathway according to one embodiment of the present invention. Specifically, it illustrates the biosynthetic pathways for G-3-P, DHAP, 3-HPA, 1,3-PDO, and 3-HP, which are products of the glycerol conversion pathway from glycerol.

[0016] FIG. 2 is a cleavage map of the pCES208-H36-Bli-glpFKD vector according to one embodiment of the present invention.

[0017] FIG. 3 is a cleavage map of the pEKEX1-yqhD-pduCDEGH vector according to one embodiment of the present invention.

[0018] Figure 4 is a cleavage map of the pEKEX1-ydcW-pduCDEGH vector according to one embodiment of the present invention.

[0019] FIG. 5 is a graph showing the glycerol utilization ability of a recombinant Corynebacterium strain of the present invention as an example of a recombinant microorganism into which a glycerol conversion pathway has been introduced.

[0020] FIG. 6 is a graph showing the glycerol utilization ability and 1,3-PDO production ability of a recombinant Corynebacterium strain of the present invention as an example of a recombinant microorganism into which a glycerol conversion pathway has been introduced.

[0021] Figure 7 is a graph showing the glycerol utilization ability and 3-HP production ability of a recombinant Corynebacterium strain of the present invention as an example of a recombinant microorganism into which a glycerol conversion pathway has been introduced.

[0022] The above-described objects, features, and advantages are described in detail below, so that those skilled in the art can easily practice the technical concepts of the present invention. In describing the present invention, if a detailed description of known technologies related to the present invention is judged to unnecessarily obscure the gist of the present invention, a detailed description thereof will be omitted. The terms described below are terms that were described in consideration of their functions and actions in the present invention, and the meaning of each term should be interpreted based on the contents throughout this specification.

[0023] Hereinafter, a recombinant microorganism having improved glycerol utilization ability according to the present invention, in which a glycerol conversion pathway operates more efficiently, a product synthesized by the glycerol conversion pathway using the recombinant microorganism, and a method for producing the same are described in detail.

[0024] The present invention provides a recombinant microorganism for producing DHAP via a glycerol conversion pathway. The present invention provides a recombinant microorganism for producing 1,3-PDO via a glycerol conversion pathway. The present invention provides a recombinant microorganism for producing 3-HP via a glycerol conversion pathway. The present invention provides a recombinant microorganism for producing G-3-P via a glycerol conversion pathway. The present invention provides a recombinant microorganism for producing 3-HPA via a glycerol conversion pathway.

[0025] The abbreviations used in the present invention are as follows.

[0026] GLY, glycerol;

[0027] G-3-P, glycerol-3-phosphate;

[0028] DHAP, DiHydroxyAcetone Phosphate;

[0029] 3-HPA, 3-hydroxypropionaldehyde

[0030] 1,3-PDO, 1,3-propanediol

[0031] 3-HP, 3-hydroxypropionic acid

[0032] glpF, glycerol uptake facilitator

[0033] glpK, glycerol kinase

[0034] glpD, glycerol-3-phosphate dehydrogenase

[0035] pduCDEGH, glycerol dehydratases and glycerol reactivases

[0036] yqhD, 1,3-propanediol oxidoreductase

[0037] ydcW, gamma-aminobutyraldehyde dehydrogenase

[0038] Figure 1 shows a glycerol conversion pathway according to one embodiment of the present invention. Specifically, it shows a pathway in which a recombinant microorganism produces DHAP, 1,3-PDO, and 3-HP, which are products of the glycerol conversion pathway, from glycerol. More specifically, it shows a reaction pathway in which glycerol present in a medium is taken up into recombinant microorganism cells by glycerol uptake facilitator protein (glpF), converted to glycerol-3-phosphate (G-3-P) by glycerol kinase (glpK), and then produced from G-3-P by glycerol-3-phosphate dehydrogenase (glpD). In addition, glycerol absorbed into the recombinant microbial cell is converted to 3-HPA by glycerol dehydratases and glycerol reactivases (pduCDEGH), then converted to 1,3-PDO by 1,3-propanediol oxidoreductase (yqhD), or converted to 3-HP by gamma-aminobutyraldehyde dehydrogenase (ydcW).

[0039] According to the glycerol conversion pathway provided by the present invention, glycerol is converted into G-3-P, and G-3-P is converted into DHAP. According to the glycerol conversion pathway provided by the present invention, glycerol is converted into 3-HPA, and 3-HPA is converted into 1,3-PDO. According to the glycerol conversion pathway provided by the present invention, glycerol is converted into 3-HPA, and 3-HPA is converted into 3-HP.

[0040] The recombinant microorganism of the present invention may be a recombinant microorganism of the genus Corynebacterium. For example, the recombinant microorganism of the genus Corynebacterium may be, but is not limited to, Corynebacterium glutamicum, Corynebacterium efficiens, Corynebacterium jeikeium, etc.

[0041] Corynebacterium glutamicum, Corynebacterium efficiens, and Corynebacterium jeikeium lack glycerol uptake facilitator proteins involved in glycerol utilization, which prevents the Corynebacterium microorganisms from effectively utilizing glycerol.

[0042] The present invention provides a recombinant microorganism of the genus Corynebacterium transformed with a gene involved in glycerol utilization derived from a heterologous strain.

[0043] The gene involved in the above glycerol utilization is a gene encoding a polypeptide that allows glycerol from outside the microorganism to enter the microorganism, phosphorylate it to convert it into glycerol-3-phosphate, and then convert it into DHAP for metabolism, and any gene that operates in a microorganism of the genus Corynebacterium is included. The gene involved in the above glycerol utilization may be a glpF gene encoding a glycerol uptake facilitator protein. The gene involved in the above glycerol utilization may be a glpK gene encoding glycerol kinase, an enzyme that produces glycerol-3-phosphate by phosphorylation using ATP. The gene involved in the above glycerol utilization may be the glpD gene encoding glycerol-3-phosphodihydrogenase, an enzyme that oxidizes glycerol-3-phosphate to produce DHAP.

[0044] The gene involved in the above glycerol utilization is a gene encoding a polypeptide that allows glycerol from outside the microorganism to enter the microorganism, biosynthesizes 3-HPA therefrom, and then converts it into 1,3-PDO, and any gene that operates in a microorganism of the genus Corynebacterium is included. The gene involved in the above glycerol utilization may be a glpF gene encoding a glycerol uptake facilitator protein. The gene involved in the above glycerol utilization may be a pduCDEGH gene encoding glycerol dehydratases and glycerol reactivases. The gene involved in the above glycerol utilization may be a yqhD gene encoding 1,3-propanediol oxidoreductase.

[0045] The gene involved in the above glycerol utilization is a gene encoding a polypeptide that allows glycerol from outside the microorganism to enter the microorganism, biosynthesizes 3-HPA therefrom, and then converts it into 3-HP, and any gene that operates in a microorganism of the genus Corynebacterium is included. The gene involved in the above glycerol utilization may be a glpF gene encoding a glycerol uptake facilitator protein. The gene involved in the above glycerol utilization may be a pduCDEGH gene encoding glycerol dehydratases and glycerol reactivases. The gene involved in the above glycerol utilization may be a ydcW gene encoding gamma-aminobutyraldehyde dehydrogenase.

[0046] In one embodiment, the heterologous strain may be a Bacillus genus. The present invention provides a recombinant microorganism of the genus Corynebacterium transformed with a gene involved in glycerol utilization derived from the genus Bacillus.

[0047] The gene involved in the utilization of the above glycerol may be derived from a microorganism of the genus Bacillus. The gene involved in the utilization of the above glycerol may be derived from Bacillus licheniformis. The gene involved in the utilization of the above glycerol may be derived from Bacillus licheniformis GSC4071, but is not limited thereto.

[0048] For example, a vector into which a gene involved in utilizing glycerol derived from Bacillus licheniformis is inserted can be transformed into a Corynebacterium strain to produce a recombinant microorganism. Specifically, a vector including a glpF gene encoding a glycerol uptake facilitator protein derived from Bacillus licheniformis can be transformed into a Corynebacterium strain to produce a recombinant microorganism. Specifically, a vector including a glpK gene encoding a glycerol kinase derived from Bacillus licheniformis can be transformed into a Corynebacterium strain to produce a recombinant microorganism. Specifically, a vector containing the glpD gene encoding glycerol-3-phosphate dehydrogenase derived from Bacillus licheniformis can be transformed into a Corynebacterium strain to produce a recombinant microorganism. At least one of the glpF gene, glpK gene, and glpD gene may be produced in the form of an operon.

[0049] The gene involved in utilizing glycerol derived from Bacillus licheniformis may be the glpF gene of SEQ ID NO: 1. The gene involved in utilizing glycerol derived from Bacillus licheniformis may be the glpK gene of SEQ ID NO: 3. The gene involved in utilizing glycerol derived from Bacillus licheniformis may be the glpD gene of SEQ ID NO: 5.

[0050] The above glycerol uptake facilitator protein may be expressed by a nucleic acid sequence of SEQ ID NO: 1, but is not limited thereto. The above glycerol uptake facilitator protein may be expressed by a nucleic acid sequence having a sequence homology of 99% or more, 95% or more, 90% or more, or 80% or more to the nucleic acid sequence of SEQ ID NO: 1.

[0051] The above glycerol kinase may be expressed by a nucleic acid sequence of SEQ ID NO: 3, but is not limited thereto. The above glycerol kinase may be expressed by a nucleic acid sequence having a sequence homology of 99% or more, 95% or more, 90% or more, or 80% or more to the nucleic acid sequence of SEQ ID NO: 3.

[0052] The above glycerol-3-phosphate dehydrogenase may be expressed by a nucleic acid sequence of SEQ ID NO: 5, but is not limited thereto. The above glycerol-3-phosphate dehydrogenase may be expressed by a nucleic acid sequence having a sequence homology of 99% or more, 95% or more, 90% or more, or 80% or more to the nucleic acid sequence of SEQ ID NO: 5.

[0053] In one embodiment, the heterologous strain may be of the genus Klebsiella. The present invention provides a recombinant microorganism of the genus Corynebacterium transformed with a gene involved in glycerol utilization derived from the genus Klebsiella.

[0054] The gene involved in the above glycerol utilization may be derived from a microorganism of the genus Klebsiella. The gene involved in the above glycerol utilization may be derived from Klebsiella pneumoniae. The gene involved in the above glycerol utilization may be derived from Klebsiella pneumoniae GSC123 (KCTC12133BP), but is not limited thereto.

[0055] For example, a vector into which a gene involved in utilizing glycerol derived from Klebsiella pneumoniae has been inserted can be transformed into a Corynebacterium strain to produce a recombinant microorganism. Specifically, a vector containing the pduCDEGH genes encoding glycerol dehydratases and glycerol reactivases derived from Klebsiella pneumoniae can be transformed into a Corynebacterium strain to produce a recombinant microorganism. The pduCDEGH genes may be produced in the form of an operon comprising the pduC gene, the pduD gene, the pduE gene, the pduG gene, and the pduH gene. Specifically, a vector containing the ydcW gene encoding gamma-aminobutyraldehyde dehydrogenase derived from Klebsiella pneumoniae can be transformed into a strain of the genus Corynebacterium to produce a recombinant microorganism.

[0056] The gene involved in utilizing glycerol derived from the above Klebsiella pneumoniae may be the pduCDEGH gene of SEQ ID NO: 7. The gene involved in utilizing glycerol derived from the above Klebsiella pneumoniae may be the ydcW gene of SEQ ID NO: 9.

[0057] The above glycerol dehydratases and glycerol reactivases may be expressed by a nucleic acid sequence of SEQ ID NO: 7, but are not limited thereto. The above glycerol dehydratases and glycerol reactivases may be expressed by a nucleic acid sequence having a sequence homology of 99% or more, 95% or more, 90% or more, or 80% or more to the nucleic acid sequence of SEQ ID NO: 7.

[0058] The gamma-aminobutyraldehyde dehydrogenase may be expressed by a nucleic acid sequence of SEQ ID NO: 9, but is not limited thereto. The gamma-aminobutyraldehyde dehydrogenase may be expressed by a nucleic acid sequence having a sequence homology of 99% or more, 95% or more, 90% or more, or 80% or more to the nucleic acid sequence of SEQ ID NO: 9.

[0059] In one embodiment, the heterologous strain may be Escherichia coli. The present invention provides a recombinant microorganism of the genus Corynebacterium transformed with a gene involved in the utilization of glycerol derived from Escherichia coli.

[0060] The gene involved in the above glycerol utilization may be derived from Escherichia coli. The gene involved in the above glycerol utilization may be derived from Escherichia coli. The gene involved in the above glycerol utilization may be derived from Escherichia coli W3110, but is not limited thereto.

[0061] For example, a vector into which a gene involved in utilizing glycerol derived from Escherichia coli is inserted can be transformed into a Corynebacterium strain to produce a recombinant microorganism. Specifically, a vector containing the yqhD gene encoding 1,3-propanediol oxidoreductase derived from Escherichia coli can be transformed into a Corynebacterium strain to produce a recombinant microorganism.

[0062] The yqhD gene encoding the 1,3-propanediol oxidoreductase derived from Escherichia coli may be the yqhD gene of SEQ ID NO: 8.

[0063] The above glycerol 1,3-propanediol oxidoreductase may be expressed by a nucleic acid sequence of SEQ ID NO: 8, but is not limited thereto. The above 1,3-propanediol oxidoreductase may be expressed by a nucleic acid sequence having a sequence homology of 99% or more, 95% or more, 90% or more, or 80% or more to the nucleic acid sequence of SEQ ID NO: 8.

[0064] The present invention provides a recombinant microorganism obtained by transforming a vector into a strain of the genus Corynebacterium, into which a gene involved in utilizing glycerol of one heterologous strain has been inserted.

[0065] The present invention provides a recombinant microorganism obtained by transforming a vector into a strain of the genus Corynebacterium, into which a gene involved in utilizing glycerol of one or more heterologous strains has been inserted.

[0066] The above heterologous strain may be at least one of Bacillus genus, Klebsiella genus, and Escherichia coli. The above heterologous strain may be at least one of Bacillus licheniformis, Klebsiella pneumoniae, and Escherichia coli.

[0067] For example, a recombinant microorganism belonging to the genus Corynebacterium can be produced, characterized by being transformed with a vector containing a glpF gene encoding a glycerol uptake facilitator protein derived from Bacillus licheniformis, a glpK gene encoding a glycerol kinase, and a glpD gene encoding a glycerol-3-phosphate dehydrogenase.

[0068] For example, a Corynebacterium characterized in that it is transformed with a first vector including a glpF gene encoding a glycerol uptake facilitator protein derived from Bacillus licheniformis, a glpK gene encoding a glycerol kinase, and a glpD gene encoding a glycerol-3-phosphate dehydrogenase, and a second vector including a pduCDEGH gene encoding a glycerol dehydratases and glycerol reactivases derived from Klebsiella pneumoniae, and a ydcW gene encoding a gamma-aminobutyraldehyde dehydrogenase. Recombinant microorganisms can be produced.

[0069] For example, a recombinant microorganism of the genus Corynebacterium can be produced, characterized by being transformed with a vector containing a glpF gene encoding a glycerol uptake facilitator protein from Bacillus licheniformis, a pduCDEGH gene encoding glycerol dehydratases and glycerol reactivases from Klebsiella pneumoniae, and a ydcW gene encoding gamma-aminobutyraldehyde dehydrogenase from Klebsiella pneumoniae.

[0070] For example, a first vector comprising a glpF gene encoding a glycerol uptake facilitator protein derived from Bacillus licheniformis, a glpK gene encoding a glycerol kinase, and a glpD gene encoding a glycerol-3-phosphate dehydrogenase, and a second vector comprising a pduCDEGH gene encoding a glycerol dehydratases and glycerol reactivases derived from Klebsiella pneumoniae, and a yqhD gene encoding a 1,3-propanediol oxidoreductase derived from Escherichia coli, characterized in that the vector is transformed. Recombinant microorganisms of the genus Corynebacterium can be produced.

[0071] For example, a recombinant microorganism of the genus Corynebacterium can be produced, characterized by being transformed with a vector containing the glpF gene encoding a glycerol uptake facilitator protein from Bacillus licheniformis, the pduCDEGH gene encoding glycerol dehydratases and glycerol reactivases from Klebsiella pneumoniae, and the yqhD gene encoding 1,3-propanediol oxidoreductase from Escherichia coli.

[0072] For example, a first vector comprising a glpF gene encoding a glycerol uptake facilitator protein from Bacillus licheniformis, a glpK gene encoding a glycerol kinase, and a glpD gene encoding a glycerol-3-phosphate dehydrogenase, a second vector comprising a pduCDEGH gene encoding a glycerol dehydratases and glycerol reactivases from Klebsiella pneumoniae, and a yqhD gene encoding a 1,3-propanediol oxidoreductase from Escherichia coli, and a Klebsiella A recombinant microorganism of the genus Corynebacterium can be produced, characterized by being transformed with a third vector containing the pduCDEGH gene encoding glycerol dehydratases and glycerol reactivases from Klebsiella pneumoniae and the ydcW gene encoding gamma-aminobutyraldehyde dehydrogenase from Klebsiella pneumoniae.

[0073] For example, a recombinant Corynebacterium spp. characterized by being transformed with a vector containing the glpF gene encoding a glycerol uptake facilitator protein from Bacillus licheniformis, the pduCDEGH gene encoding glycerol dehydratases and glycerol reactivases from Klebsiella pneumoniae, the yqhD gene encoding 1,3-propanediol oxidoreductase from Escherichia coli, and the ydcW gene encoding gamma-aminobutyraldehyde dehydrogenase from Klebsiella pneumoniae. Microorganisms can be manufactured.

[0074] The term "transformation" as used herein refers to introducing a gene into a host cell so that it can be expressed within the host cell. The transformed gene may be either integrated into the chromosome of the host cell or located extrachromosomally, as long as it can be expressed within the host cell. Furthermore, the gene is a polynucleotide capable of encoding a polypeptide, including DNA and RNA. The gene may be introduced in any form, as long as it can be introduced into the host cell and expressed. Furthermore, the gene may be introduced into the host cell on its own or in the form of a polynucleotide construct, and operably linked to a sequence necessary for expression in the host cell.

[0075] According to one aspect disclosed herein, a new recombinant microorganism of the genus Corynebacterium is provided.

[0076] The recombinant microorganism of the genus Corynebacterium may comprise a glpF gene encoding a glycerol uptake facilitator protein. The recombinant microorganism of the genus Corynebacterium may comprise a glpK gene encoding a glycerol kinase. The recombinant microorganism of the genus Corynebacterium may comprise a glpD gene encoding a glycerol-3-phosphate dehydrogenase.

[0077] The recombinant microorganism of the genus Corynebacterium may comprise a pduCDEGH gene encoding glycerol dehydratases and glycerol reactivases. The recombinant microorganism of the genus Corynebacterium may comprise a ydcW gene encoding gamma-aminobutyraldehyde dehydrogenase. The recombinant microorganism of the genus Corynebacterium may comprise a yqhD gene encoding 1,3-propanediol oxidoreductase.

[0078] The glpF gene encoding the glycerol uptake facilitator protein may have a nucleic acid sequence of SEQ ID NO: 1. The glpF gene may be derived from the genus Bacillus. The glpF gene may be derived from Bacillus licheniformis. The glpF gene may be derived from Bacillus licheniformis GSC4071 (KCTC 14485BP).

[0079] The glpK gene encoding the above glycerol kinase may have a nucleic acid sequence of SEQ ID NO: 3. The glpK gene may be derived from the genus Bacillus. The glpK gene may be derived from Bacillus licheniformis. The glpK gene may be derived from Bacillus licheniformis GSC4071 (KCTC 14485BP).

[0080] The glpD gene encoding the above glycerol-3-phosphate dehydrogenase may have a nucleic acid sequence of SEQ ID NO: 5. The glpD gene may be derived from the genus Bacillus. The glpD gene may be derived from Bacillus licheniformis. The glpD gene may be derived from Bacillus licheniformis GSC4071 (KCTC 14485BP). The Bacillus licheniformis strain was deposited at the Microbial Resource Center of the Korea Research Institute of Bioscience and Biotechnology on March 5, 2021 (KCTC14485BP).

[0081] The pduCDEGH gene encoding the above glycerol dehydratases and glycerol reactivases may have a nucleic acid sequence of SEQ ID NO: 7. The pduCDEGH gene may be derived from the genus Klebsiella. The pduCDEGH gene may be derived from Klebsiella pneumoniae. The pduCDEGH gene may be derived from Klebsiella pneumoniae GSC123 (KCTC12133BP).

[0082] The ydcW gene encoding the above-mentioned gamma-aminobutyraldehyde dehydrogenase may have a nucleic acid sequence of SEQ ID NO: 9. The ydcW gene may be derived from the genus Klebsiella. The ydcW gene may be derived from Klebsiella pneumoniae. The ydcW gene may be derived from Klebsiella pneumoniae GSC123 (KCTC12133BP).

[0083] The above Klebsiella pneumoniae strain was deposited at the Korea Research Institute of Bioscience and Biotechnology on February 16, 2012 (deposit number KCTC12133BP).

[0084] The yqhD gene encoding the above 1,3-propanediol oxidoreductase may have a nucleic acid sequence of SEQ ID NO: 8. The yqhD gene may be derived from Escherichia coli. The yqhD gene may be derived from Escherichia coli W3110 (Escherichia coliW3110). Escherichia coliW3110 is a model microorganism widely used for research and industrial purposes.

[0085] The recombinant microorganism may have an improved ability to utilize glycerol. The recombinant microorganism may produce G-3-P from glycerol. The recombinant microorganism may produce DHAP from glycerol. The recombinant microorganism may produce 3-HPA from glycerol. The recombinant microorganism may produce 1,3-PDO from glycerol. The recombinant microorganism may produce 3-HP from glycerol.

[0086] In another aspect disclosed herein, a product of a glycerol conversion pathway produced from a novel recombinant microorganism is provided.

[0087] Here, the product by the glycerol conversion pathway may be G-3-P. The product by the glycerol conversion pathway may be a compound synthesized from G-3-P. The product by the glycerol conversion pathway may be DHAP. The product by the glycerol conversion pathway may be a compound synthesized from DHAP. The product by the glycerol conversion pathway may be 3-HPA. The product by the glycerol conversion pathway may be a compound synthesized from 3-HPA. The product by the glycerol conversion pathway may be 1,3-PDO. The product by the glycerol conversion pathway may be a compound synthesized from 1,3-PDO. The product by the glycerol conversion pathway may be 3-HP. The product by the glycerol conversion pathway may be a compound synthesized from 3-HP.

[0088] DHAP catalyzes the formation of glyceraldehyde-3-phosphate by triose phosphate isomerase. This glyceraldehyde-3-phosphate then passes through the remaining glycolytic pathways and ultimately enters the tricarboxylic acid cycle, where it can accumulate any of the metabolic intermediates, depending on the type of genetic alteration occurring in the glycolytic, fermentative, and tricarboxylic acid cycle pathways.

[0089] According to another aspect disclosed by the present specification, a method for producing a product by a glycerol conversion pathway from a novel recombinant microorganism is provided.

[0090] According to one embodiment, a method for producing a product by a glycerol conversion pathway and a product by a glycerol conversion pathway according to the method may be provided, including a step of inoculating and culturing the recombinant microorganism in a culture medium containing glycerol in part or solely as a carbon source and a step of isolating a product by the glycerol conversion pathway from the culture.

[0091] In one embodiment, the recombinant microorganism may be cultured in a culture medium partially or solely containing glycerol as a carbon source, and G-3-P secreted into the culture solution may be recovered. In one embodiment, the recombinant microorganism may be cultured in a culture medium partially or solely containing glycerol as a carbon source, and DHAP secreted into the culture solution may be recovered. In one embodiment, the recombinant microorganism may be cultured in a culture medium partially or solely containing glycerol as a carbon source, and 3-HPA secreted into the culture solution may be recovered. In one embodiment, the recombinant microorganism may be cultured in a culture medium partially or solely containing glycerol as a carbon source, and 1,3-PDO secreted into the culture solution may be recovered. In one embodiment, the recombinant microorganism may be cultured in a culture medium partially or solely containing glycerol as a carbon source, and 1,3-PDO secreted into the culture solution may be recovered.

[0092] The above culturing may be performed under aerobic conditions. The above culturing may be performed under constant temperature conditions of 30°C to 40°C.

[0093] In another embodiment, the recombinant microorganism may be cultured in a culture medium partially or solely containing glycerol as a carbon source, and G-3-P may be recovered from the cell-free extract. In another embodiment, the recombinant microorganism may be cultured in a culture medium partially or solely containing glycerol as a carbon source, and DHAP may be recovered from the cell-free extract. In another embodiment, the recombinant microorganism may be cultured in a culture medium partially or solely containing glycerol as a carbon source, and 3-HPA may be recovered from the cell-free extract. In another embodiment, the recombinant microorganism may be cultured in a culture medium partially or solely containing glycerol as a carbon source, and 1,3-PDO may be recovered from the cell-free extract. In another embodiment, the recombinant microorganism may be cultured in a culture medium partially or solely containing glycerol as a carbon source, and 3-HP may be recovered from the cell-free extract.

[0094] The above culturing may be performed under aerobic conditions. The above culturing may be performed under constant temperature conditions of 30°C to 40°C.

[0095] According to another aspect disclosed by the present disclosure, there is provided a use of the novel recombinant microorganism for producing a product by a glycerol conversion pathway.

[0096] The present invention provides a recombinant microorganism in which a glycerol conversion pathway operates more efficiently, and thus can provide a recombinant microorganism for mass-producing a product by the glycerol conversion pathway. The present invention provides a recombinant microorganism for providing a product by the glycerol conversion pathway, and thus can provide a recombinant microorganism capable of more easily producing a product by the glycerol conversion pathway. The product by the glycerol conversion pathway may be G-3-P or a compound synthesized using G-3-P as an intermediate. The product by the glycerol conversion pathway may be DHAP or a compound synthesized using DHAP as an intermediate. The product by the glycerol conversion pathway may be 3-HPA or a compound synthesized using 3-HPA as an intermediate. The product by the glycerol conversion pathway may be 1,3-PDO or a compound synthesized using 1,3-PDO as an intermediate. The product by the above glycerol conversion pathway may be 3-HP or a compound synthesized using 3-HP as an intermediate.

[0097] Accordingly, in one embodiment, the present invention can provide a use for producing G-3-P as a new recombinant microorganism. The present invention can provide a use for producing a compound synthesized using G-3-P as an intermediate as a new recombinant microorganism. The present invention can provide a use for producing DHAP as a new recombinant microorganism. The present invention can provide a use for producing a compound synthesized using DHAP as an intermediate as a new recombinant microorganism. The present invention can provide a use for producing 3-HPA as a new recombinant microorganism. The present invention can provide a use for producing a compound synthesized using 3-HPA as an intermediate as a new recombinant microorganism. The present invention can provide a use for producing 1,3-PDO as a new recombinant microorganism. The present invention can provide a use for producing a compound synthesized using 1,3-PDO as an intermediate as a new recombinant microorganism. The present invention can provide a use for producing 3-HP as a new recombinant microorganism. The present invention can provide a use for producing a compound synthesized using 3-HP as an intermediate as a new recombinant microorganism.

[0098] Hereinafter, the present invention will be described in more detail through examples and experimental examples. However, the following examples and experimental examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples and experimental examples.

[0099] <Example 1> Production of a recombinant microorganism containing a glycerol production pathway gene derived from a heterologous strain

[0100] Bacillus licheniformis GSC4071 (KCTC 14485BP), a wild-type strain isolated from a soil sample near Daejeon, was used. Corynebacterium glutamicum ATCC13032 strain was used. Klebsiella strain Klebsiella pneumoniae GSC123 (KCTC12133BP) strain was used. Escherichia coli strain Escherichia coli W3110 strain was used.

[0101] <1-1> Construction of vectors expressing Bacillus licheniformis GSC4071 glpF, glpK, and glpD in Corynebacterium

[0102] For the expression of the glpFKD gene derived from Bacillus licheniformis in Cornebacterium, the universal plasmid pCES208-H36 was utilized. To amplify sequences 1 / 3 / 5 in operon form at once from the B. licheniformis genome, PCR amplification was performed using sequences 10 / 11, and sequences 12 / 13 were used to amplify the pCES208-H36 plasmid. The two amplified fragments were assembled into pCES208-H36-Bli-glpFKD using Gibson assembly (see Fig. 2). Similarly, to amplify sequences 2 / 4 / 6 in operon form at once from the E. coli genome, PCR amplification was performed using sequences 14 / 15, and sequences 16 / 17 were used to amplify the pCES208-H36 plasmid. The two amplified fragments were assembled into pCES208-H36-Ecj-glpFKD by Gibson assembly.

[0103] <1-2> Production of vectors expressing the 1,3-PDO and 3-HP production pathways in Corynebacterium

[0104] For the expression of 1,3-PDO and 3-HP production pathways in Cornebacterium, a universal plasmid, pEKEX1, was utilized. The pduCDEGH operon and ydcW genes were introduced from Klebsiella pneumoniae, and the yqhD gene was introduced from Escherichia coli. First, the construction of a plasmid for 1,3-PDO production was performed. The yqhD gene was amplified from E. coli genomic DNA using SEQ ID NOs: 18 / 19, the pduCDEGH operon was amplified from Klebsiella pneumoniaegenomic DNA using SEQ ID NOs: 20 / 21, and the pEKEX1 plasmid was amplified using SEQ ID NOs: 22 / 23. The three amplified fragments were combined through Gibson assembly, and the pEKEX1-yqhD-pduCDEGH plasmid was finally obtained (see Fig. 3). Similarly, for the construction of a plasmid for 3-HP production, the ydcW gene was amplified from Klebsiella pneumoniaegenomic DNA using SEQ ID NOs: 24 / 25, the pduCDEGH operon was amplified from Klebsiella pneumoniaegenomic DNA using SEQ ID NOs: 26 / 27, and the pEKEX1 plasmid was amplified using SEQ ID NOs: 28 / 29. The three amplified fragments were assembled through Gibson assembly, and the pEKEX1-ydcW-pduCDEGH plasmid was finally obtained (see Fig. 4).

[0105] <1-3> Production of recombinant Corynebacterium with improved glycerol utilization ability

[0106] To construct a Corynebacterium strain with improved glycerol utilization, ATCC13032 strain was used as a model microorganism. Plasmid introduction was performed into the ATCC13032 strain via electroporation under conditions of 25 uF, 200 Ω, and 2.5 kV / cm. Screening was performed on BHI agar medium containing 200 μg / mL Spectinomycin to select strains containing pCES208-based plasmids. Gene expression was performed using a constitutive expression system, and no separate inducer was added. The Corynebacterium ATCC13032 strain was used as Comparative Example 1, the strain in which pCES208-H36-Ecj-glpFKD was introduced into the ATCC13032 strain was used as Production Example 1, and the strain in which pCES208-H36-Bli-glpFKD was introduced into the ATCC13032 strain was used as Production Example 2.

[0107] <1-4> Production of recombinant Corynebacterium with 1,3-PDO and 3-HP production ability

[0108] A recombinant microorganism was created by introducing plasmids for producing 1,3-PDO and 3-HP into a Corynebacterium microorganism with improved glycerol utilization ability. For plasmid introduction, the strain was transferred to the production example 2 strain via electroporation under the conditions of 25 uF, 200 Ω, and 2.5 kV / cm, and screening was performed on BHI agar medium containing 200 μg / mL Spectinomycin and 25 μg / mL Kanamycin to select strains containing the two plasmids. 1 mM IPTG was added as an inducer for pEKEX1 plasmid-based gene expression. The strain in which the pEKEX1-yqhD-pduCDEGH plasmid was introduced into the strain in Manufacturing Example 2 was used as Manufacturing Example 3, and the strain in which the pEKEX1-ydcW-pduCDEGH plasmid was introduced into the strain in Manufacturing Example 2 was used as Manufacturing Example 4.

[0109]

[0110] <Example 2> Verification of glycerol utilization in recombinant Corynebacterium The glycerol utilization ability of wild type Corynebacterium ATCC13032 and Preparation Examples 1 and 2 was evaluated.

[0111] First, flask culture was performed for the above microorganisms as follows.

[0112] Modified CGXII medium was used for cultivation, and 100 g / L glycerol was added as a carbon source. A 250 mL baffled flask was used, and the working volume was 25 mL. Cultivation was performed for 24 hours at an initial pH of 7.0, a temperature of 30°C, and a 200 rpm shaking incubator. The detailed modified CGXII medium composition is as follows. Samples were collected after the completion of cultivation for the wild-type and recombinant Corynebacterium, and the collected samples were centrifuged at 13,000 rpm for 10 minutes, and the glycerol concentration of the supernatant was analyzed by high-performance liquid chromatography (HPLC).

[0113] Modified MR medium composition for culturing wild-type and recombinant CorynebacteriumComponent (g / L)KH2PO41K2HPO41(NH4)2.SO420Urea2MnSO4.4H2O0.014ZnSO4.7H2O0.001CuSO4.5H2O0.0003NiCl2.6H2O0.00002FeSO4.7H2O (HCl 2ml)0.01CaCl2.2H2O0.017MgSO4.7H2O0.5Yeast extract10Citric acid0.03Thiamine-HCl0.009d-Biotin0.0018CaCO310

[0114] As a result, it was confirmed that the wild-type ATCC13032 strain did not utilize glycerol at all. The microorganism introduced with glpFKD of E. coli in Manufacturing Example 1 had 59.8 g / L of residual glycerol after 48 hours of culture at an initial concentration of 114 g / L, and consumed approximately 54.2 g / L of glycerol. On the other hand, the microorganism introduced with glpFKD of Bacillus licheniformis in Manufacturing Example 1 had 8.7 g / L of residual glycerol after 48 hours of culture at an initial concentration of 114 g / L, and consumed approximately 105.3 g / L of glycerol. As is known, when glpFKD derived from Bacillus licheniformis was introduced into a Corynebacterium microorganism that has no glycerol metabolic ability, it was confirmed that a higher glycerol utilization ability could be secured than when the E. coli gene was introduced (see Fig. 5, Corynebacterium ATCC13032 corresponds to GSB1840; Production Example 1 corresponds to GSB1840 / ecj-glpFKD; Production Example 2 corresponds to GSB1840 / bli-glpFKD). The glpF of E. coli is composed of 281 amino acids, and the glpF of Bacillus licheniformis is composed of 271 amino acids. Since the identity is at the level of 35%, it can be confirmed that the glpF genes of E. coli and Bacillus licheniformis are genes with low homology. The glpK of E. coli consists of 502 amino acids, while the glpK of Bacillus licheniformis consists of 496 amino acids. Since the identity is approximately 62%, it can be confirmed that the glpK genes of E. coli and Bacillus licheniformis have low homology. In addition, the glpD of E. coli consists of 501 amino acids, while the glpD of Bacillus lichenformis consists of 555 amino acids.Since the identity is at the 30% level, it is confirmed that the glpD genes of E. coli and Bacillus licheniformis are genes with low homology.

[0115] <Example 3> Production of 1,3-PDO and 3-HP using recombinant Corynebacterium with glycerol utilization ability

[0116] The production capacity of 1,3-PDO and 3-HP was confirmed through Manufacturing Examples 3 and 4.

[0117] First, flask culture was performed for the above microorganisms as follows.

[0118] Modified CGXII medium was used for cultivation, and 100 g / L glycerol was added as a carbon source for 1,3-PDO production and 70 g / L glycerol was added as a carbon source for 3-HP production. A 250 mL baffled flask was used, and the working volume was 25 mL. The culture was performed at an initial pH of 7.0, a temperature of 30°C, and a 200 rpm shaking incubator for 24 hours. The detailed modified CGXII medium composition is as follows. After the completion of the culture, samples were collected for the wild-type and recombinant Corynebacterium, and the collected samples were centrifuged at 13,000 rpm for 10 minutes, and the glycerol concentration in the supernatant was analyzed by high-performance liquid chromatography (HPLC).

[0119] Modified MR medium composition for culturing wild-type and recombinant CorynebacteriumComponent (g / L)KH2PO41K2HPO41(NH4)2.SO420Urea2MnSO4.4H2O0.014ZnSO4.7H2O0.001CuSO4.5H2O0.0003NiCl2.6H2O0.00002FeSO4.7H2O (HCl 2ml)0.01CaCl2.2H2O0.017MgSO4.7H2O0.5Yeast extract10Citric acid0.03Thiamine-HCl0.009d-Biotin0.0018CaCO310

[0120] As a result, the strain of Manufacturing Example 3 for 1,3-PDO production consumed all 100 g / L glycerol through 48 hours of culture and produced 14 g / L of 1,3-PDO as the final product. It was confirmed that efficient production of 1,3-PDO was possible based on high glycerol utilization ability (see Fig. 6). In addition, the strain of Manufacturing Example 4 for 3-HP production consumed all 70 g / L glycerol through 48 hours of culture and produced 14.7 g / L of 3-HP as the final product. Similarly, it was confirmed that efficient production of 3-HP was possible based on high glycerol utilization ability (see Fig. 7). Although the examples of the present specification have been described in more detail above, the present specification is not necessarily limited to these examples, and various modifications may be made within a scope that does not depart from the technical spirit of the present specification. Accordingly, the embodiments disclosed in this specification are intended to illustrate, rather than limit, the technical concepts of the present invention, and the scope of the technical concepts of the present invention is not limited by these embodiments. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of protection of this specification and the present invention should be interpreted by the claims, and all technical concepts within the scope equivalent thereto should be interpreted as being included within the scope of the rights of this specification and the present invention.

[0121] Name of depositor: Korea Research Institute of Bioscience and Biotechnology

[0122] Accession number: KCTC14485BP

[0123] Date of acceptance: 20210305

[0124]

Claims

1. A vector comprising a glpF gene encoding a glycerol uptake facilitator protein derived from Bacillus licheniformis, a glpK gene encoding a glycerol kinase, and a glpD gene encoding a glycerol-3-phosphate dehydrogenase.

2. In paragraph 1, A vector characterized in that the above glpF gene has a nucleic acid sequence of SEQ ID NO:

1.

3. In paragraph 1, A vector characterized in that the above glpK gene has a nucleic acid sequence of sequence number 3.

4. In paragraph 1, A vector characterized in that the above glpD gene has a nucleic acid sequence of sequence number 5.

5. In paragraph 1, The above Bacillus licheniformis is a vector characterized by being Bacillus licheniformisGSC4071 (KCTC 14485BP).

6. A recombinant microorganism of the genus Corynebacterium characterized by being transformed with the vector of paragraph 1.

7. In paragraph 6, The recombinant microorganism is Corynebacterium glutamicum, Corynebacterium efficiens or Corynebacterium jeikeium.

8. In paragraph 6, The above recombinant microorganism is a recombinant microorganism, Corynebacterium glutamicum ATCC13032.

9. In paragraph 6, A recombinant microorganism characterized in that the above recombinant microorganism is transformed to further include a vector including a pduCDEGH gene encoding glycerol dehydratases and glycerol reactivases derived from Klebsiella pneumoniae and a ydcW gene encoding gamma-aminobutyraldehyde dehydrogenase.

10. In paragraph 9, A recombinant microorganism characterized in that the above pduCDEGH gene has a nucleic acid sequence of sequence number 7.

11. In paragraph 9, A recombinant microorganism characterized in that the above ydcW gene has a nucleic acid sequence of sequence number 9.

12. In paragraph 9, The above Klebsiella pneumoniae is a recombinant microorganism characterized in that it is Klebsiella pneumoniae GSC123 (Klebsiella pneumoniaeGSC123) (KCTC12133BP).

13. In paragraph 6, A recombinant microorganism characterized in that the above recombinant microorganism is transformed to further include a vector including a pduCDEGH gene encoding glycerol dehydratases and glycerol reactivases derived from Klebsiella pneumoniae and a yqhD gene encoding 1,3-propanediol oxidoreductase derived from Escherichia coli.

14. In paragraph 13, A recombinant microorganism characterized in that the above pduCDEGH gene has a nucleic acid sequence of sequence number 7.

15. In paragraph 13, A recombinant microorganism characterized in that the above yqhD gene has a nucleic acid sequence of sequence number 8.

16. In paragraph 13, The above Klebsiella pneumoniae is a recombinant microorganism characterized in that it is Klebsiella pneumoniae GSC123 (Klebsiella pneumoniaeGSC123) (KCTC12133BP).

17. In paragraph 13, The above E. coli is a recombinant microorganism characterized by being Escherichia coli W3110.

18. A step of inoculating and culturing a recombinant microorganism of the genus Corynebacterium according to any one of claims 6 to 17 into a culture medium containing glycerol as a carbon source, in part or solely; and A step of separating a product by the glycerol conversion pathway from the above culture A method for producing a product by a glycerol conversion pathway, comprising:

19. In paragraph 18, A method characterized in that the product by the above glycerol conversion pathway is at least one of glycerol-3-phosphate (G-3-P), dihydroxyacetone phosphate (DHAP), 3-hydroxypropionaldehyde (3-HPA), 1,3-propanediol (1,3-PDO), and 3-hydroxypropionic acid (3-HP).

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