Microorganism capable of producing 1,4-butanediol and method for producing 1,4-butanediol using the same

Abstract

The present invention relates to a microorganism that produces 1,4-butanediol and a method for producing 1,4-butanediol using the same. Unlike naturally occurring microorganisms, the microorganism according to the present invention can efficiently produce 1,4-butanediol using ornithine as a carbon source.

Description

[Technical field]

[0001] The present invention relates to a microorganism capable of producing 1,4-butanediol and a method for producing 1,4-butanediol using the same. [Background technology]

[0002] 1,4-Butanediol is used throughout the chemical industry as a solvent, polymer intermediate, fine chemical intermediate, etc. 1,4-Butanediol is mainly produced by reacting acetylene with formaldehyde and then adding hydrogen, but it can also be produced from maleic anhydride and propylene oxide, but there are problems such as greenhouse gases and waste generation due to the use of fossil raw materials, disruption of raw material supply and demand due to unstable international crude oil prices, and increased production costs.

[0003] To complement these chemical production processes, a low-cost and environmentally friendly process for biologically producing 1,4-butanediol using biomass as a raw material has been developed recently. In general, 1,4-butanediol is produced via α-ketoglutarate or succinyl-CoA in the metabolic process of microorganisms. However, the 1,4-butanediol production process using microorganisms has limitations, such as the relatively low production yield due to the production of unnecessary by-products as well as 1,4-butanediol in the metabolic process of the microorganisms. Therefore, in order to overcome these limitations, efforts are being made to develop microorganisms that can produce 1,4-butanediol at high yields using genetic engineering techniques. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] European Patent No. 3050970 [Patent Document 2] European Patent No. 2782893 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a microorganism that produces 1,4-butanediol.

[0006] Another object of the present invention is to provide a method for producing 1,4-butanediol using the microorganism. [Means for solving the problem]

[0007] In conventional microorganisms, the production pathway for 1,4-butanediol involves the oxidation of pyruvate produced from glucose during glycolysis to acetyl-CoA, which is then synthesized into α-ketoglutarate and succinyl-CoA in the TCA cycle. Here, 4-hydroxybutyric acid is produced via the decarboxylation of α-ketoglutarate or the reduction of succinyl-CoA, from which 1,4-butanediol is ultimately produced.

[0008] However, the present inventors have constructed a novel 1,4-butanediol production pathway that uses ornithine instead of α-ketoglutarate or succinyl-CoA as a starting material, and have confirmed that a microorganism equipped with such a production pathway can efficiently produce 1,4-butanediol due to its high enzyme activity, thereby completing the present invention.

[0009] One aspect of the present invention provides a microorganism that produces 1,4-butanediol from ornithine.

[0010] As used herein, "microorganism" refers to any prokaryotic or eukaryotic organism, including bacteria, yeast, fungi, and the like.

[0011] The term "microorganisms producing 1,4-butanediol" used in the present invention means microorganisms containing enzymes involved in the production pathway of 1,4-butanediol or genes encoding the enzymes. The pathway for producing 1,4-butanediol from ornithine in the present invention is a production pathway that has not been confirmed in any known microorganisms. Therefore, the microorganisms producing 1,4-butanediol in the present invention (hereinafter referred to as "1,4-butanediol-producing microorganisms") refer to microorganisms modified by modifying the endogenous gene sequence (such as deletion, addition, or replacement of nucleic acids), inserting a foreign gene into the gene, and / or introducing a plasmid (vector) containing a foreign gene, for the gene encoding the enzyme involved in the production pathway of ornithine-1,4-butanediol. In the present invention, the term "modified microorganism" can be used interchangeably with the terms "mutated microorganism", "mutated strain", "recombinant microorganism", "recombinant strain", "genetically modified microorganism" or "genetically modified strain". The opposite concept of "pre-transformed microorganism," "pre-transformed strain," "non-mutated microorganism," "non-mutated strain," or "mother strain" refers to a microorganism before its characteristics are changed through genetic mutation due to natural or artificial factors, for example, a microorganism that does not / cannot produce 1,4-butanediol because it lacks an enzyme that acts on the ornithine-1,4-butanediol production pathway.

[0012] According to one embodiment of the present invention, the 1,4-butanediol-producing microorganism may have an enzymatic reaction for converting ornithine to 4-aminobutaneamide, an enzymatic reaction for converting 4-aminobutyric acid to 4-aminobutyric acid, an enzymatic reaction for converting 4-aminobutyric acid to succinate semialdehyde, an enzymatic reaction for converting succinate semialdehyde to 4-hydroxybutyric acid, and an enzymatic reaction for converting 4-hydroxybutyric acid to 1,4-butanediol.

[0013] According to one embodiment of the present invention, the enzyme that catalyzes the enzymatic reaction of converting ornithine to 4-aminobutanamide may be lysine 2-monooxygenase encoded by the davB gene.

[0014] According to one embodiment of the present invention, the enzyme that catalyzes the enzymatic reaction of converting 4-aminobutanamide to 4-aminobutyric acid may be 5-aminovaleramidase encoded by the davA gene.

[0015] According to one embodiment of the present invention, the enzyme that catalyzes the enzymatic reaction of converting 4-aminobutyric acid to succinate semialdehyde may be gamma-aminobutyrate aminotransferase encoded by the gabT gene.

[0016] According to one embodiment of the present invention, the enzyme that catalyzes the enzymatic reaction of converting succinate semialdehyde to 4-hydroxybutyric acid may be alcohol dehydrogenase encoded by the yahK gene.

[0017] According to one embodiment of the present invention, the enzyme catalyzing the enzymatic reaction of converting 4-hydroxybutyric acid to 1,4-butanediol may be alcohol dehydrogenase encoded by the yahK gene or carboxylic acid reductase encoded by the car gene.

[0018] The carboxylic acid reductase can be activated by the 4'-phosphopantetheinyl transferase encoded by the sfp gene.

[0019] The lysine 2-monooxygenase, 5-aminovaleramidase, gamma-aminobutyrate aminotransferase, alcohol dehydrogenase and carboxylic acid reductase contained in such a 1,4-butanediol producing microorganism are enzymes that are not present in the microorganism before transformation. In addition, the 4'-phosphopantetheinyl transferase may or may not be present in the microorganism before transformation.

[0020] According to one embodiment of the invention, the enzyme may be the result of the introduction of an exogenous gene.

[0021] More specifically, the enzyme can be expressed and catalyze the enzymatic reaction for producing 1,4-butanediol from ornithine by artificially introducing a gene selected from the group consisting of davB, davA, gabT, yahK, car, and sfp present in a normal microorganism, or a sequence having substantial identity thereto, into a 1,4-butanediol-producing microorganism.

[0022] As used herein, "substantial identity" means that when the respective gene sequences, i.e., base sequences or nucleotide sequences, are aligned and analyzed with any other nucleotide sequence for maximum correspondence, the any other nucleotide sequence has a sequence homology of 70% or more, 80% or more, 90% or more, or 98% or more with the respective nucleotide sequence.

[0023] According to one embodiment of the present invention, the genes encoding the enzymes, davB, davA, gabT, yahK, car and sfp, may be derived from microorganisms known in the art, and the type of microorganism is not particularly limited.

[0024] More specifically, the genes of the enzymes are expressed in Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Mycobacterium abscessus, Bacillus subtilis, Acinetobacter baumannii, Azotobacter vinelandii, Chromohalobacter salexigens, Citrobacter koseri, Citrobacter youngae, Enterobacter cloacae, and the like. cloacae, Marinobacter aquaeolei, Marinomonas mediterranea, Pantoea ananatis, Pseudoalteromonas haloplanktis, Ralstonia eutropha, Shewanella putrefaciens, and Thiobacillus denitrificans.

[0025] For example, the davB gene may be represented by the nucleotide sequence of SEQ ID NO: 1 or encoded by the amino acid sequence of SEQ ID NO: 2, but is not limited thereto.

[0026] The davA gene may be represented by the nucleotide sequence of SEQ ID NO: 3 or may be encoded by the amino acid sequence of SEQ ID NO: 4, but is not limited thereto.

[0027] The gabT gene may be represented by the nucleotide sequence of SEQ ID NO:5 or encoded by the amino acid sequence of SEQ ID NO:6, but is not limited thereto.

[0028] The yahK gene may be represented by the nucleotide sequence of SEQ ID NO: 7 or encoded by the amino acid sequence of SEQ ID NO: 8, but is not limited thereto.

[0029] The car gene may be represented by the nucleotide sequence of SEQ ID NO: 9 or encoded by the amino acid sequence of SEQ ID NO: 10, but is not limited thereto.

[0030] The sfp gene may be represented by the nucleotide sequence of SEQ ID NO: 11 or encoded by the amino acid sequence of SEQ ID NO: 12, but is not limited thereto.

[0031] The 1,4-butanediol producing microorganism according to the present invention may be produced by introducing or transforming a host cell via one or more vectors containing all or individually of the davB, davA, gabT, yahK, car and sfp genes.

[0032] The term "vector" as used herein refers to any type of nucleic acid sequence delivery structure used as a means for transferring and expressing a gene of interest in a host cell. Unless otherwise specified, the vector may refer to a vector in which a nucleic acid sequence carried therein is inserted into a host cell genome to be expressed and / or expressed independently. Such a vector includes essential regulatory elements operably linked to express a gene insert, and "operably linked" means that a gene of interest and its regulatory sequence are functionally linked to each other to enable gene expression, and "regulatory elements" include a promoter for transcription, an optional operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, and a sequence for regulating the termination of transcription and translation.

[0033] The vector used in the present invention is not particularly limited as long as it can be replicated in a host cell, and any vector known in the art can be used. Examples of the vector include plasmids, cosmids, viruses, and bacteriophages in a natural or recombinant state. For example, phage or cosmid vectors include pWE15, M13, λMBL3, λMBL4, λIXII, λASHII, λAPII, λt10, λt11, Charon4A, and Charon21A, and plasmid vectors include, but are not limited to, pBR, pUC, pBluescriptII, pGEM, pTZ, pCL, and pET.

[0034] The vector can typically be constructed as a vector for cloning or a vector for expression. The vector for expression can be a vector commonly used in the art for expressing foreign genes or proteins in plants, animals, or microorganisms, and can be constructed by various methods known in the art.

[0035] Recombinant vectors can be constructed using prokaryotic or eukaryotic cells as hosts. For example, when the vector used is an expression vector and a prokaryotic cell is used as a host, it generally contains a strong promoter (e.g., pLλ promoter, CMV promoter, trp promoter, lac promoter, tac promoter, T7 promoter) that can cause transcription to proceed, a ribosome binding site for the initiation of decoding, and a transcription / decoding termination sequence. When a eukaryotic cell is used as a host, the replication origin contained in the vector that operates in a eukaryotic cell includes, but is not limited to, the f1 replication origin, SV40 replication origin, pMB1 replication origin, adeno replication origin, AAV replication origin, and BBV replication origin. In addition, promoters derived from the genome of mammalian cells (e.g., metallothionine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, HSV tk promoter) can be used, and generally have a polyadenylation sequence as a transcription termination sequence.

[0036] The recombinant vector may contain a selection marker, which is used to select a transformant (host cell) transformed with the vector, and since only cells expressing the selection marker can survive in a medium treated with the selection marker, the transformed cells can be selected. Representative examples of the selection marker include, but are not limited to, kanamycin, streptomycin, and chloramphenicol.

[0037] A transformant can be produced by inserting a recombinant vector into a host cell, and the transformant may be obtained by introducing a recombinant vector into a suitable host cell. The host cell may be any host cell known in the art as a cell capable of stably and continuously cloning or expressing the expression vector.

[0038] When transforming prokaryotic cells to prepare recombinant microorganisms, host cells may be used, but are not limited to, Escherichia coli strains such as E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, and E. coli XL1-Blue, Bacillus strains such as Bacillus subtilis and Bacillus thuringiensis, Corynebacterium strains, Salmonella typhimurium, Serratia marcescens, and various enterobacteria and strains such as Pseudomonas species.

[0039] When transforming eukaryotic cells to produce recombinant microorganisms, host cells can be used, but are not limited to, yeast (e.g., Saccharomyces cerevisiae), insect cells, plant cells and animal cells, such as Sp2 / 0, CHO K1, CHO DG44, PER.C6, W138, BHK, COS7, 293, HepG2, Huh7, 3T3, RIN, and MDCK cell lines.

[0040] As used herein, "transformation" refers to the phenomenon of artificially causing genetic changes by introducing exogenous DNA into a host cell, and "transformant" refers to a host cell into which exogenous DNA has been introduced and which stably maintains the expression of a target gene.

[0041] The transformation can be carried out by selecting a suitable vector introduction technique depending on the host cell, and expressing the target gene or a recombinant vector containing the same in the host cell. For example, the introduction of the vector can be carried out by, but is not limited to, electroporation, heat shock, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method, lithium acetate-DMSO method, or a combination thereof. The transformed gene can be inserted into the chromosome of the host cell or located extrachromosomally, without limitation, as long as it can be expressed in the host cell.

[0042] The transformant includes cells that have been transfected, transformed, or infected in vivo or in vitro with a recombinant vector according to the invention, and may be used interchangeably as recombinant host cell, recombinant cell, or recombinant microorganism.

[0043] According to one embodiment of the present invention, the transformant is a 1,4-butanediol producing microorganism, and is not particularly limited as long as it is a microorganism known in the art.

[0044] More specifically, the 1,4-butanediol producing microorganism may be a strain of the genus Escherichia or Corynebacterium.

[0045] Examples of the Escherichia genus strain include, but are not limited to, Escherichia coli, Escherichia albertii, Escherichia blattae, Escherichia fergusonii, Escherichia hermannii, and Escherichia vulneris.

[0046] The Corynebacterium genus strains include Corynebacterium glutamicum, Corynebacterium crudilactis, Corynebacterium deserti, Corynebacterium callunae, Corynebacterium suranareeae, Corynebacterium lubricantis, Corynebacterium doosanense, Corynebacterium efficiens, Corynebacterium uterequii, and Corynebacterium spp. Corynebacterium uterequi, Corynebacterium stationis, Corynebacterium pacaense, Corynebacterium singulare, Corynebacterium humireducens, Corynebacterium marinum, Corynebacterium halotolerans, Corynebacterium spheniscorum, Corynebacterium freiburgense, Corynebacterium striatum striatum, Corynebacterium canis, Corynebacterium ammoniagenes, Corynebacterium renale, Corynebacterium portisoriiThe bacteria may be, but is not limited to, Corynebacterium pollutisoli, Corynebacterium imitans, Corynebacterium caspium, Corynebacterium testudinoris, Corynebacterium pseudopelargi, Corynebacterium flavescens, and the like.

[0047] Another aspect of the present invention provides a method for producing 1,4-butanediol, comprising culturing a microorganism as described above in a medium and recovering 1,4-butanediol from the microorganism or the medium in which the microorganism is cultured.

[0048] The culture may be performed using a suitable medium and culture conditions known in the art, and a person skilled in the art can easily adjust the medium and culture conditions. Specifically, the medium may be a liquid medium, but is not limited thereto. The culture method may include, but is not limited to, for example, batch culture, continuous culture, fed-batch culture, or a combination thereof.

[0049] According to one embodiment of the present invention, the medium should meet the requirements of a specific strain in an appropriate manner, and can be modified by a person skilled in the art. Culture media for Escherichia and Corynebacterium can be found in the literature (Manual of Methods for General Bacteriology, American Society for Bacteriology, Washington DC, USA, 1981), but are not limited thereto.

[0050] According to one embodiment of the present invention, the medium may contain a variety of carbon sources, nitrogen sources and trace element components. Usable carbon sources include sugars and carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, cellulose, oils and fats such as soybean oil, sunflower oil, castor oil, coconut oil, etc., fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid. These substances may be used individually or as a mixture, but are not limited to these. Usable nitrogen sources may include peptone, yeast extract, meat juice, malt extract, corn steep liquor, soybean flour and iodine or inorganic compounds such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Nitrogen sources may also be used individually or as a mixture, but are not limited to these. Usable phosphorus sources may include, but are not limited to, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. The culture medium may also contain metal salts necessary for growth, such as magnesium sulfate or iron sulfate, but is not limited thereto. Other essential growth substances, such as amino acids and vitamins, may also be included. Furthermore, suitable precursors may be used for the culture medium. The medium or individual components may be added to the culture solution during the culture process in a suitable manner, such as batch or continuous, but is not limited thereto.

[0051] According to one embodiment of the present invention, the pH of the culture solution can be adjusted by adding compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid to the microorganism culture solution in an appropriate manner during the culture. In addition, the generation of bubbles can be suppressed using an antifoaming agent such as fatty acid polyglycol ester during the culture. Additionally, oxygen or oxygen-containing gas (e.g., air) can be injected into the culture solution to maintain the aerobic state of the culture solution. The temperature of the culture solution may usually be 20°C to 45°C, for example, 25°C to 40°C. The culture period can be continued until a desired amount of useful substances is produced, and may be, for example, 10 to 160 hours.

[0052] According to one embodiment of the present invention, the step of recovering 1,4-butanediol from the cultured mutant and the medium in which the mutant is cultured can be performed by collecting or recovering the 1,4-butanediol produced from the medium using a suitable method known in the art depending on the culture method, such as, but not limited to, centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), and chromatography (e.g., ion exchange, affinity, hydrophobic, and size exclusion).

[0053] According to one embodiment of the present invention, the step of recovering 1,4-butanediol can be performed by centrifuging the culture medium at low speed to remove the biomass and separating the resulting supernatant by ion exchange chromatography.

[0054] According to one embodiment of the present invention, the step of recovering 1,4-butanediol may include a step of purifying 1,4-butanediol. Effect of the Invention

[0055] The microorganism according to the present invention is different from naturally occurring microorganisms and can efficiently produce 1,4-butanediol using ornithine as a carbon source. [Brief description of the drawings]

[0056] [Figure 1] 1 shows the results of evaluating the conversion ability of ornithine to 4-aminobutyric acid (GABA) in three types of Escherichia coli expressing davB and davA according to one embodiment of the present invention. [Diagram 2] 1 shows the results of evaluating the conversion ability of ornithine to 4-aminobutyric acid (GABA) in three types of E. coli expressing LAAO / MOG according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] The present invention will be described in more detail below. However, such description is merely presented as an example for understanding the present invention, and the scope of the present invention is not limited by such exemplary description.

[0058] Example 1. Substrate activity evaluation in E. coli expressing davB and davA genes 1-1. Construction of davB and davA expression vectors Using the chromosomal DNA of Pseudomonas putida as a template, davB and davA were amplified by PCR using primers and then cloned into the plasmid pKM212-MCS. In the case of the davB gene, a 6x His-tag was inserted at the N-terminal. This plasmid was named pKM212-DavBHisA.

[0059] Here, the Wizard Genomic DNA Purification Kit (Promega, USA) was used to extract DNA from each strain. The nucleotide sequences of the amplified genes davB and davA are shown in Table 1 below, and the primers and plasmid information used are shown in Tables 2 and 3 below, respectively.

[0060] PCR was performed using a Thermocycler (TP600, TAKARA BIO Inc., Japan) by adding 1 pM of oligonucleotide and 10 ng of template DNA to a reaction solution containing 100 μM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP), and performing 25 to 30 cycles under the following conditions: 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute in the presence of 1 unit of PrimeSTAR Max DNA Polymerase (Takara, Japan).

[0061] The pKM212-DavBHisA vector was transformed into E. coli DH5a (HIT Competent cells TM The vector was transformed into Escherichia coli (Cat. No. RH618), spread onto an LB-agar plate containing 50 μg / ml kanamycin, and cultured at 37° C. for 24 hours. The final colonies formed were isolated to confirm that the insert was correctly present in the vector, and the vector was then isolated and used to produce E. coli expressing davB and davA.

[0062] [Table 1]

[0063] [Table 2]

[0064] [Table 3]

[0065] 1-2. Construction of E. coli expressing davB and davA Competent cells produced by E. coli XL1-Blue (HIT Competent cells TM, Cat No. RH118), WL3110 (Park et al., Proc Natl Acad Sci USA. 2007 104(19):7797-802) and DH5a (HIT Competent cells TM The pKM212-DavBHisA vector was introduced into E. coli XL1-Blue harboring pKM212DavBHisA, E. coli WL3110 harboring pKM212DavBHisA, and E. coli DH5a harboring pKM212DavBHisA, respectively (Cat. No. RH618) by heat shock at 42°C for 1 minute and 30 seconds, and then the vector was spread on an LB-agar plate containing 50 μg / ml kanamycin and cultured at 37°C for 24 hours. The selected colonies were finally confirmed by PCR and sequence analysis to confirm that the davB and davA genes were introduced as intended. These were named E. coli XL1-Blue harboring pKM212DavBHisA, E. coli WL3110 harboring pKM212DavBHisA, and E. coli DH5a harboring pKM212DavBHisA, respectively.

[0066] 1-3. Evaluation of activity towards ornithine in E. coli expressing davB and davA The conversion of ornithine to 4-aminobutyric acid (GABA) was evaluated in E. coli XL1-Blue harboring pKM212DavBHisA, E. coli WL3110 harboring pKM212DavBHisA, and E. coli DH5a harboring pKM212DavBHisA, which contained the davB and davA genes.

[0067] Each strain was inoculated into a 50mL flask containing 5mL of MR medium (containing 200g / L ornithine) and cultured at 37℃ for 96 hours. After the culture was completed, the culture was centrifuged to separate the culture medium from the settled cells, and the culture was treated with DEEMM (Kim et al., J. Mol. Catal. B: Enzym. 2015 115:151-154) and filtered through a 0.22μm filter. The ornithine and GABA contents in the culture were analyzed using a high performance liquid chromatography (HPLC) (Agilent, 1260 infinity II) equipped with a column (C18 column, RStech) and a VWD (Variable Wavelength Detector). The results are shown in Figure 1.

[0068] As shown in FIG. 1, the amount of GABA produced varied depending on the type of host strain, and in particular, E. coli WL3110 expressing davB and davA was confirmed to have the highest ornithine bioconversion rate.

[0069] Example 2. Evaluation of substrate activity in E. coli expressing LAAO / MOG genes Pseudomonas-derived L-amino acid oxidase / monooxygenase (LAAO / MOG) is known to exhibit not only oxidase activity but also lysine 2-monooxygenase activity that converts ornithine to 4-aminobutyric acid (GABA). It has been reported that when the 254th cysteine ​​in the amino acid that constitutes LAAO / MOG is replaced with isoleucine or leucine, the substrate activity for ornithine increases (FEBS Open Bio, 2014 4:220-228).

[0070] Therefore, in order to produce GABA from ornithine, we constructed E. coli expressing LAAO / MOG and evaluated its substrate activity for ornithine.

[0071] 2-1. Construction of LAAO / MOG expression vector The LAAO / MOG of Pseudomonas sp. AIU 813 strain was synthesized (Bioneer) using Escherichia coli-based codon optimization and then cloned into the plasmid pKM212-MCS using primers, which was named pKM212-LAAO / MOG.

[0072] To induce mutations in the synthesized LAAO / MOG gene, the 254th amino acid, cysteine, was replaced with isoleucine and leucine, respectively, to synthesize the LAAO / MOG(C254I) and LAAO / MOG(C254L) genes, which were then cloned into the plasmid pKM212-MCS using primers. These were named pKM212-LAAO / MOG(C254I) and pKM212-LAAO / MOG(C254L), respectively.

[0073] Here, the Wizard Genomic DNA Purification Kit (Promega, USA) was used to extract DNA from each strain. The base sequence of the amplified gene LAAO / MOG is shown in Table 4 below, and the primers and plasmid information used are shown in Tables 5 and 6 below, respectively.

[0074] PCR was performed using a Thermocycler (TP600, TAKARA BIO Inc., Japan) by adding 1 pM of oligonucleotide and 10 ng of template DNA to a reaction solution containing 100 μM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP), and performing 25 to 30 cycles under the following conditions: 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute in the presence of 1 unit of PrimeSTAR Max DNA Polymerase (Takara, Japan).

[0075] The pKM212-LAAO / MOG, pKM212-LAAO / MOG(C254I) and pKM212-LAAO / MOG(C254L) vectors were transformed into E. coli WL3110 (Park et al., Proc Natl Acad Sci USA. 2007 104(19):7797-802), spread onto LB-agar plates containing 50 μg / ml kanamycin and cultured at 37° C. for 24 hours. The final colonies formed were isolated to confirm that the insert was correctly present in the vector, and the vector was then isolated and used to produce LAAO / MOG-expressing E. coli.

[0076] [Table 4-1] [Table 4-2]

[0077] [Table 5]

[0078] [Table 6]

[0079] 2-2. Preparation of LAAO / MOG-expressing E. coli pKM212-LAAO / MOG, pKM212-LAAO / MOG(C254I) and pKM212-LAAO / MOG(C254L) vectors were introduced into E. coli WL3110 cells made from competent cells by heat shock at 42°C for 1 minute and 30 seconds, and then the cells were spread on LB-agar plates containing 50 μg / ml kanamycin and cultured at 37°C for 24 hours. The selected colonies were finally confirmed by PCR and sequence analysis to see whether the LAAO / MOG, LAAO / MOG(C254I) or LAAO / MOG(C254L) gene was introduced as intended. Finally, E. coli strains carrying the LAAO / MOG genes were constructed and named E. coli WL3110 harboring pKM212LAAO / MOG, E. coli WL3110 harboring pKM212LAAO / MOG(C254I), and E. coli WL3110 harboring pKM212LAAO / MOG(C254L), respectively.

[0080] 2-3. Evaluation of activity of LAAO / MOG-expressing E. coli towards ornithine The conversion of ornithine to 4-aminobutyric acid (GABA) was evaluated in E. coli WL3110 harboring pKM212LAAO / MOG, E. coli WL3110 harboring pKM212LAAO / MOG(C254I), and E. coli WL3110 harboring pKM212LAAO / MOG(C254L) into which the LAAO / MOG gene had been introduced.

[0081] Each strain was inoculated into 1.5 mL e-tubes containing 1 mL of medium (containing Tris-HCl (pH 7.6) and 500 mM ornithine) and cultured at 37°C for 96 hours. After the culture was completed, the culture was centrifuged to separate the culture medium from the settled cells, and the culture was treated with DEEMM (Kim et al., J Mol Catal B Enzym. 2015 115:151-154) and filtered through a 0.22 μm filter. The ornithine and GABA contents in the culture were analyzed using a high performance liquid chromatography (HPLC) (Agilent, 1260 infinity II) equipped with a column (C18 column, RStech) and a VWD (Variable Wavelength Detector). The results are shown in Figure 2.

[0082] As shown in Figure 2, it was confirmed that the expression of the mutated LAAO / MOG resulted in a higher ornithine bioconversion rate and a higher GABA yield than the expression of the wild-type LAAO / MOG.

[0083] Example 3. Evaluation of 1,4-butanediol productivity in Corynebacterium glutamicum with the ornithine-1,4-butanediol pathway 3-1. Construction of davB, davA, gabT, yahK and car expression vectors The chromosomal DNA of Corynebacterium glutamicum ATCC13032 strain was used as a template to amplify the DNA fragments by PCR using primers 1 and 2, primers 3 and 4, primers 7 and 8, and primers 11 and 12, respectively. The davB and davA expression vector pKM212-DavBHisA of Example 1 was used as a template to amplify the DNA fragments by PCR using primers 5 and 6, and primers 9 and 10, respectively. The PCR products obtained by this were amplified by crossover PCR and then inserted into the restriction enzyme sites HindIII (NEB, R3104S) and XbaI (NEB, R0145L) of the pK19mobSacB vector (Jaeger et al., Journal of Bacteriology. 1992 174:5462-65). This vector was named pK19ms / davBA.

[0084] The chromosomal DNA of Corynebacterium glutamicum ATCC13032 strain was used as a template for PCR amplification using primers 13 and 14, primers 15 and 16, primers 19 and 20, primers 21 and 22, and primers 23 and 24, respectively. The chromosomal DNA of Escherichia coli K-12 MG1655 strain containing GabT (from Corynebacterium glutamicum) and YahK (from Escherichia coli) was used as a template for PCR amplification using primers 17 and 18. The PCR product obtained was amplified by crossover PCR and inserted into the HindIII and XbaI sites of the pK19mobSacB vector. This vector was named pK19ms / yahK-gabT.

[0085] To insert the sfp gene, which is necessary for activating the enzyme activity of the car gene, the chromosomal DNA of Corynebacterium glutamicum ATCC13032 strain was used as a template and amplified by PCR using primers 25 and 26, primers 27 and 28, and primers 31 and 32, respectively. The E. coli expression vector pKE112CAR3pptase (Polymers (Basel). 2019 11(7):1184) containing sfp (derived from Bacillus subtilis) was used as a template and amplified by PCR using primers 29 and 30. The PCR product obtained by this was amplified by crossover PCR and then inserted into the restriction enzyme sites of pK19mobSacB vector at the HindIII and XbaI sites. This vector was named pK19ms / sfp.

[0086] The chromosomal DNA of Corynebacterium glutamicum ATCC13032 was used as a template for PCR amplification using primers 33 and 34, primers 35 and 36, and primers 39 and 40, respectively. The E. coli expression vector pKE112CAR3pptase (Polymers (Basel). 2019 11(7):1184) containing car (derived from Mycobacterium abscessus) was used as a template for PCR amplification using primers 37 and 38. The PCR product obtained was amplified by crossover PCR and then inserted into the restriction enzyme sites of pK19mobSacB vector at the HindIII and XbaI sites. This vector was named pK19ms / car.

[0087] Here, the Wizard Genomic DNA Purification Kit (Promega, USA) was used to extract DNA from each strain. The base sequences of the amplified genes gabT, yahK, and car are shown in Table 7 below, and the primers in Table 8 below were used for PCR.

[0088] PCR was performed using a Thermocycler (TP600, TAKARA BIO Inc., Japan) by adding 1 pM of oligonucleotide and 10 ng of template DNA to a reaction solution containing 100 μM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP), and performing 25 to 30 cycles under the following conditions: 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute in the presence of 1 unit of PrimeSTAR Max DNA Polymerase (Takara, Japan).

[0089] pK19ms / davBA, pK19ms / yahK-gabT, pK19ms / sfp and pK19ms / car vectors were transformed into E. coli DH5a (HIT Competent cells) TM The vector was transformed into Corynebacterium glutamicum (Cat. No. RH618), spread on an LB-agar plate containing 50μg / ml kanamycin, and cultured at 37℃ for 24 hours. The final colonies were isolated to confirm that the insert was correctly present in the vector, and the vector was then isolated and used to construct Corynebacterium glutamicum carrying an ornithine-1,4-butanediol pathway.

[0090] [Table 7-1] [Table 7-2] [Table 7-3]

[0091] [Table 8-1] [Table 8-2]

[0092] 3-2. Construction of Corynebacterium glutamicum expressing davB, davA, gabT, yahK and car Using an electrophorator (BIO-RAD, USA), the pK19ms / davBA vector prepared in 3-1 above was introduced by electroporation into Corynebacterium glutamicum ATCC13032 strain prepared in competent cells, and then the vector was spread on 2YT KM AGAR medium (containing 2YT AGAR and kanamycin 15 mg / L) and cultured in an incubator at 30°C for 2 days to obtain colonies. Colonies in which primary homologous recombination was induced and confirmed by PCR were cultured in 2YT liquid medium (containing tryptophan 16 g / L, yeast extract 10 g / L, and NaCl 5 g / L) for 12 hours, and then spread on 2YT Sucrose AGAR medium (containing 2YT AGAR and sucrose 100 g / L) to remove the antibiotic marker by secondary homologous recombination. The selected colonies were subjected to PCR and sequence analysis to confirm whether the davB and davA genes had been introduced as intended.

[0093] Then, the above process was carried out sequentially using the pk19ms / yahK-gabT, pk19ms / car and pk19ms / sfp vectors to construct a Corynebacterium glutamicum strain in which davB, davA, gabT, yahK and car were introduced to construct a pathway for producing 1,4-butanediol from ornithine, and the strain was named WB01-P001.

[0094] 3-3. Construction of LAAO / MOG, gabT, yahK and car expression vectors The chromosomal DNA of Corynebacterium glutamicum ATCC13032 strain was used as a template for PCR amplification using primers 1 and 2, primers 3 and 41, primers 7 and 8, and primers 44 and 12, respectively. The LAAO / MOG expression vectors pKM212-LAAO / MOG, pKM212-LAAO / MOG(C254I), and pKM212-LAAO / MOG(C254L) of Example 2 were used as templates for PCR amplification using primers 42 and 43, respectively. The PCR products thus obtained were amplified by crossover PCR and then inserted into the restriction enzyme sites HindIII and XbaI of the pK19mobSacB vector. The vectors were named pK19ms / LAAO / MOG, pK19ms / LAAO / MOG(C254I), and pK19ms / LAAO / MOG(C254L), respectively.

[0095] Here, the Wizard Genomic DNA Purification Kit (Promega, USA) was used to extract DNA from each strain, and the primers in Table 8 above and Table 9 below were used for PCR.

[0096] PCR was performed using a Thermocycler (TP600, TAKARA BIO Inc., Japan) by adding 1 pM of oligonucleotide and 10 ng of template DNA to a reaction solution containing 100 μM of each deoxynucleotide triphosphate (dATP, dCTP, dGTP, dTTP), and performing 25 to 30 cycles under the following conditions: 94°C for 30 seconds, 58°C for 30 seconds, and 72°C for 1 minute in the presence of 1 unit of PrimeSTAR Max DNA Polymerase (Takara, Japan).

[0097] The pK19ms / LAAO / MOG, pK19ms / LAAO / MOG(C254I), and pK19ms / LAAO / MOG(C254L) vectors were transformed into E. coli DH5a (HIT Competent cells TMThe vector was transformed into Corynebacterium glutamicum (Cat. No. RH618), spread on an LB-agar plate containing 50 μg / ml kanamycin, and cultured at 37°C for 24 hours. The final colonies were isolated to confirm that the insert was correctly present in the vector, and the vector was then isolated and used to construct Corynebacterium glutamicum carrying an ornithine-1,4-butanediol pathway.

[0098] [Table 9]

[0099] 3-4. Construction of Corynebacterium glutamicum expressing LAAO / MOG, gabT, yahK and car Using an electrophorator (BIO-RAD, USA), the pK19ms / LAAO / MOG, pK19ms / LAAO / MOG(C254I), or pK19ms / LAAO / MOG(C254L) vector prepared in 3-3 was introduced by electroporation into the Corynebacterium glutamicum ATCC13032 strain prepared in competent cells, and then the cells were smeared on 2YT KM AGAR medium (containing 2YT AGAR and 15 mg / L kanamycin) and cultured in an incubator at 30°C for 2 days to obtain colonies. Colonies in which primary homologous recombination was induced and confirmed by PCR were cultured in 2YT liquid medium (containing 16 g / L tryptophan, 10 g / L yeast extract, and 5 g / L NaCl) for 12 hours, and then smeared on 2YT Sucrose AGAR medium (containing 2YT AGAR and 100 g / L sucrose) to remove the antibiotic marker by secondary homologous recombination. The selected colonies were finally confirmed by PCR and sequence analysis to determine whether the LAAO / MOG, LAAO / MOG(C254I), or LAAO / MOG(C254L) gene had been introduced as intended.

[0100] Then, the above process was carried out sequentially using pK19ms / LAAO / MOG and the pk19ms / yahK-gabT, pk19ms / car and pk19ms / sfp vectors prepared in 3-1 above, to construct a Corynebacterium glutamicum strain in which LAAO / MOG, gabT, yahK and car were introduced to construct a pathway for producing 1,4-butanediol from ornithine, and this strain was named WBP-0002.Then, Corynebacterium glutamicum strains in which LAAO / MOG(C254I) and LAAO / MOG(C254L) mutations were introduced using pK19ms / LAAO / MOG(C254I) and pK19ms / LAAO / MOG(C254L), respectively, were named WBP-0003 and WBP-0004, respectively.

[0101] 3-5.1,4-Butanediol Productivity Evaluation The productivity of 1,4-butanediol in Corynebacterium glutamicum WB01-P001, WBP-0002, WBP-0003, and WBP-0004 introduced with the ornithine-1,4-butanediol pathway prepared in 3-2 and 3-4 was evaluated.

[0102] Each strain was inoculated into flask medium (containing 5% glucose, 0.1% MgSO4, 0.5% yeast extract, 0.2% KH2PO4, 1% (NH4)2SO4, 20 ppm FeSO4, 20 ppm MnSO4, 100 ug / L biotin, 20 ppm thiamine, 20 ppm nicotinamide, and 20 ppm CPN) and cultured at 30°C for 24 hours, with the glucose concentration at 0 g / L. Corynebacterium glutamicum ATCC13032 was used as a control. After the culture was completed, the culture was filtered through a 0.45 μm filter, and the content of 1,4-butanediol in the culture was analyzed using a high performance liquid chromatography (HPLC) (Agilent, 1260 infinity II) equipped with a column (Avantor HPLC Column Apollo C18). The analysis was performed using a 0.1 M phosphate buffer as the mobile phase, at a temperature of 40° C. and a flow rate of 0.8 mL / min for 15 minutes using an RI detector. The results are shown in Table 10 below.

[0103] [Table 10]

[0104] As shown in Table 10, it was confirmed that the strain WB01-P001 expressing davB and davA had significantly improved 1,4-butanediol productivity compared to the strains WBP-0002 to WBP-0004 expressing LAAO / MOG or its mutants.

[0105] The present invention has been described above with a focus on its preferred embodiments. Those skilled in the art will understand that the present invention can be realized in modified forms without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments should be considered from an explanatory perspective, not a restrictive one. The scope of the present invention is defined by the claims, not the above description, and all differences within the scope of the equivalents should be interpreted as being included in the present invention.

Claims

1. A microorganism that produces 1,4-butanediol from ornithine.

2. The microorganism is capable of carrying out an enzymatic reaction to convert ornithine into 4-aminobutanamide. An enzymatic reaction that converts 4-aminobutanamide to 4-aminobutyric acid, An enzymatic reaction that converts 4-aminobutyric acid into succinate semialdehyde, an enzymatic reaction that converts succinate semialdehyde to 4-hydroxybutyric acid; and The microorganism according to claim 1, having an enzymatic reaction for converting 4-hydroxybutyric acid into 1,4-butanediol.

3. The microorganism according to claim 2, wherein the enzyme that catalyzes the enzymatic reaction of converting ornithine to 4-aminobutanamide is lysine 2-monooxygenase.

4. 3. The microorganism according to claim 2, wherein the enzyme that catalyzes the enzymatic reaction of converting 4-aminobutanamide to 4-aminobutyric acid is 5-aminovaleramidase.

5. 3. The microorganism according to claim 2, wherein the enzyme that catalyzes the enzymatic reaction of converting 4-aminobutyric acid to succinate semialdehyde is gamma-aminobutyrate aminotransferase.

6. The microorganism according to claim 2, wherein the enzyme that catalyzes the enzymatic reaction of converting succinate semialdehyde to 4-hydroxybutyric acid is alcohol dehydrogenase.

7. The microorganism according to claim 2, wherein the enzyme that catalyzes the enzymatic reaction of converting 4-hydroxybutyric acid to 1,4-butanediol is alcohol dehydrogenase or carboxylic acid reductase.

8. The microorganism according to any one of claims 3 to 7, wherein the enzyme is produced by introduction of an exogenous gene.

9. The genes of the enzymes are expressed in Escherichia coli, Pseudomonas putida, Corynebacterium glutamicum, Mycobacterium abscessus, Bacillus subtilis, Acinetobacter baumannii, Azotobacter vinelandii, Chromohalobacter salexigens, and the like. salexigens, Citrobacter koseri, Citrobacter youngae, Enterobacter cloacae, Marinobacter aquaeoleyi, Marinomonas mediterranea, Pantoea ananatis, Pseudoalteromonas haloplanktis, Ralstonia eutropha The microorganism according to any one of claims 3 to 7, which is derived from one or more microorganisms selected from the group consisting of Bacillus subtilis, Bacillus eutropha, Shewanella putrefaciens, and Thiobacillus denitrificans.

10. The microorganism according to claim 1 , wherein the microorganism is a strain of the genus Escherichia or Corynebacterium.

11. Cultivating the microorganism according to claim 1 in a medium; and recovering 1,4-butanediol from the microorganism or the medium in which the microorganism is cultured.

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