KR101914180B1 - Biosynthesis Method of Mevalonates Using a Biodiesel By-Product - Google Patents

Abstract

The present invention discloses a mevalonate biosynthesis process using glycerol-containing biodiesel production by-products as a carbon source. The method of the present invention has a much higher yield than the case of using glucose as a carbon source and has a higher yield than the case of using common glycerol (pure glycerol sold as a product) as a carbon source.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a biosynthesis biosynthetic biosynthetic by-

The present invention relates to a method for biosynthesis of mevalonate using glycerol-containing biodiesel production by-products.

There is no problem of depletion of energy resources, the use of waste resources such as waste cooking oil, the environmental aspect of reducing the generation of various pollutants such as dust, soot, carbon monoxide, and the exhaustion of fossil fuels, The biodiesel industry, which is manufactured from vegetable oils such as Uji, soybean oil, and waste cooking oil, is growing rapidly (Korea Economic Daily, Apr 08, 2014). Article).

Biodiesel is a fatty acid methyl esters or fatty acid ethyl esters obtained by transesterification of glycerol from the triglycerides of animal and vegetable fats in the form of fatty acid and glycerol combined, ). Biodiesel is produced by a chemical method in which triglyceride is produced by reacting triglyceride with a lower alcohol such as methanol or ethanol under an acid or base catalyst, a biological method using an enzyme such as lipase which catalyzes an ester exchange reaction, a method using supercritical methanol or ethanol (Bioresource Technology, 70: 1-15, 1999; Fuel, 80 (2): 225-231, 2001; Fuel, 80 (5): 693-698, 2001) Renewable Energy, 3: 13-16, 2003).

Since biodiesel is produced by separating glycerol from triglyceride, glycerol is essentially produced as a by-product in the biodiesel production process, and glycerol produced at this time is reported to be equivalent to 10% of biodiesel (Green Chem., 9 : 868, 2007). In addition to glycerol, biodiesel production by-products contain unreacted substances such as trichliceride, methanol, and other unreacted biodiesel, acid or base catalysts, and the purity of glycerol in biodiesel production by-products is in the range of 50 to 80% .

(Green Chem., 9: 868, 2007). However, since the price of pure glycerol is not high and the separation and purification process is complicated, the cost is high Therefore, glycerol separated and purified from biodiesel is not competitive in price. Therefore, most of the byproducts of waste glycerol-containing biodiesel are increasingly being discarded due to the increase of biodiesel production.

Mebaronic acid is an intermediate metabolite used in biosynthesis of coenzyme Q10, squalene, cholesterol, isoprenoid, etc. in living organisms and has a structure having alcohol functional groups and carboxyl functional groups at both ends It is soluble in water and soluble in polar solvents. Also, it can be easily converted into mevalonolactone through dehydrogenation reaction by acid treatment. Mebaronic acid is the asymmetric carbon atom in the third carbon and only (R +) is produced and used biologically.

As shown in FIG. 1, the biosynthesis process of mevalonic acid was performed by using two molecules of acetyl-CoA as precursors and using acetoacetyl-coA and hydroxymethylglutaryl coenzyme A (HMGCoA; hydroxymethylglutaryl CoA).

Acetoacetyl CoA synthase, acetyl-CoA acetyltransferase or acetoacetyl-CoA thiolase (hereinafter referred to as " AtoB "), acetoacetyl coenzyme A from two molecules of acetyl coenzyme A, hydroxymethyl For the production of glutaryl coenzyme A, hydroxymethylglutaryl coenzyme A synthetase (HMG-CoA synthase, hereinafter referred to as "HMGS") and hydroxymethylglutaryl coenzyme A are used for generation of mevalonate, (HMG-CoA reductase, hereinafter referred to as " HMGR ") are involved, respectively (Journal of Biotechnology 140: 218.226, 2009; NATURE BIOTECHNOLOGY 21: 796-802, 2003), Metabolic Engineering 11: 13-19 , 2009; Clinical Biochemistry 40: 575-584, 2007; Arch Biochem Biophys. 505 (2): 131-143, 2011)

Mebalonate and mevalonolactone are currently being used in a wide range of industrial applications, and are used in the production of cosmetic additives, biodegradable polymers, and chiral compounds. In particular, when used as cosmetic raw materials, they are absorbed into skin cells, , Moisturizing, regenerating skin barrier, and improving wrinkles. Actual mevalonic acid has been reported to promote skin barrier restoration from damage, which is a membrane that increases skin resistance to damage and protects skin from bacteria and external stimuli and helps maintain skin moisture.

The present invention has been completed by confirming the possibility of using glycerol-containing biodiesel production by-products as a carbon source in the biosynthesis of mevalonate.

The present invention provides a method for biosynthesizing mevalonate using glycerol-containing biodiesel production by-products.

Other and further objects of the present invention will be described below.

As shown in the following examples, the inventors of the present invention found that three genes involved in mevalonate biosynthesis, namely, the atoB gene encoding AtoB , the mvaS gene encoding HMGS, the recombinant Escherichia coli transformed with mvaE encoding HMGR (Yield of mevalonate production relative to the supply amount of carbon source) of 44.85% (500 ml (yield)) was obtained when glycerol-containing biodiesel production by-product was supplied as a carbon source when biosynthesis of mevalonate was carried out through a 500 ml flask and a 2 L fermenter ), 26.43% (2 L of fermenter culture), which was significantly higher than the yield of 9.92% when glucose was supplied as a carbon source. In addition, when the common glycerol (pure glycerol sold as a product) was supplied as a carbon source, The yield was 41.1% (500 mL flask culture) and higher than 26.1% (2L fermenter culture). These results are very meaningful in that the production cost of mevalonate can be reduced since the recycling is not economical as described above, which is obtained by using the by-product of glycerol-containing biodiesel, which is discarded.

(A) preparing a host microorganism transformed with an expression vector capable of expressing AtoB, HMGS and HMGR, (b) preparing a medium for culturing the transformed host microorganism, ; (C) inoculating and culturing the transformed host microorganism in the medium; and (d) obtaining mevalonate from the culture, wherein the medium contains glycerol-containing biodiesel as a carbon source Characterized in that the byproduct is supplied during the medium preparation step of step (b) and / or during the culturing step of the host microorganism of step (c).

In the present specification, mevalonate is used equivalently with mevalonic acid.

As used herein, the term " glycerol-containing biodiesel production by-product " in the present specification means that glycerol is separated from glycerol-linked fatty acid and triglyceride of vegetable fat through transesterification, Byproducts produced when producing biodiesel, fatty acid methyl esters or fatty acid ethyl esters, are meant to include glycerol at levels of 50-80%. More specifically 50 to 80% glycerol, and other unreacted substances such as trichliceride, methanol and the like, unseparated biodiesel, acid or base catalyst, and the like.

In the method of the present invention, the production of an expression vector capable of expressing AtoB, HMGS and HMGR comprises a gene encoding AtoB, HMGS and HMGR, which genes are widely available in the literature . Gene coding for the AtoB is known in the art phaA, mvaE, atoB, etc., a gene coding for HMGS is known as HMGS, mvaS the like in per eopge, genes encoding HMGR is known HMGR, mvaA, mvaE etc. Information on the nucleotide sequence of the corresponding genes can be found in, for example, Korean Patent Publication No. 2003-005950, entitled " Recombinant microorganism having mevalonate or mevalonolactone-producing ability, and method for producing mevalonate or mevalonolactone using the same, 10-2015-0134512, entitled " Microorganisms Containing Genes Encoding Enzymes Involved in the Production of Mevalonic Acid and Methods for Producing Mevalonic Acids Utilizing the Same ", Korean Patent Publication No. 10-2015-0006097, (Metabolic Engineering 11: 13-19, 2009), Clinical Biochemistry 40: 575-584, 2 (2003) 007], Arch Biochem Biophys. 505 (2): 131-143, 2011], or from corresponding genes listed in the NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/).

In the method of the present invention, the expression vector capable of expressing AtoB, HMGS and HMGR is composed of one expression vector capable of expressing both AtoB, HMGS and HMGR, or expresses two of AtoB, HMGS and HMGR and expresses the other Or two expression vectors capable of expressing AtoB, HMGS and HMGR, respectively. In particular, the product of mvaE gene has the function of AtoB and HMGR at the same time. Therefore, when mvaE gene is used, one or two expression vectors may be constructed together with genes such as HMGS and mvaS encoding HMGS .

The expression vector is a DNA construct capable of expressing a gene of interest (a gene coding for AtoB, HMGS and / or HMGR) in a host microorganism, and is replicated independently of the genome of the host microorganism when it is transformed into a host microorganism Function (expression of the target gene) or integrated into the genome of the host microorganism.

In the present invention, the expression vector may be a nucleic acid in the form of a plasmid, a cosmid, a phagemid, a phage or the like. Depending on the host microorganism, a suitable vector may be purchased from a commercialized vector, or used or modified. For example, when E. coli is used as the host microorganism, pUC19, pSTV28, pBBR1MCS, pBluscriptII, pBAD, pTrc99A, pET, pACYC184, pBR322, pJE101, pJE102, pJE103 and the like can be used.

A substantial amount of literature has been accumulated in the art for expression vector constructs involving recombinant DNA technology and is described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, (2001) FM Ausubel et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994), Marston, F (1987) DNA Cloning Techniques, and the like.

The expression vector includes, in addition to the target gene cis-transferase gene or UDP pyrophosphate synthase gene, a regulatory sequence which is operatively linked to the target gene, thereby affecting transcription and translation of the target gene.

Such regulatory sequences typically include promoter sequences, polyadenylation signals, and the like. The term " operably linked " herein means that the transcription and / or translation of a gene is influenced. For example, if a promoter affects the transcription of a gene linked thereto, the promoter and the gene are operably linked.

As used herein, the term " promoter " refers to the conventional meaning as known in the art. Specifically, the term " promoter "Quot; means a nucleic acid sequence having a function of controlling transcription of one or more genes, including transcription initiation point, transcription factor binding site, and the like. The promoter is derived from a prokaryotic organism, such as a Prib-now box (position near the transfer start point (+1) -10) above the transfer start point, a hogness box (position near the transfer start point (+1) (Present at the transcription start point (+1) -20 to -30 position), CAAT box (usually about -75 in comparison with the transcription initiation site) when it is derived from a eukaryote, Position), an enhancer, a transcription repressor, and the like.

The promoter may be a constitutive promoter (a promoter that induces expression constantly in a specific organism) or an inducible promoter (a promoter that induces expression of a target gene in response to a specific external stimulus), if the promoter is capable of expressing a target gene linked thereto And preferably a promoter suitable for a particular host microorganism is used. For example, when E. coli is used as a host microorganism, promoters such as T7A1, T7A2, T7A3, lambda pL, lambda pR, lac, lacUV5, trp, tac, trc, phoA, rrnB, 1PL, and GAL1, GAL10, ADH1, It may be preferable to use promoters such as TDH3 and PGK.

The expression vector comprises a terminator sequence which is a transcription termination sequence in addition to the promoter. The terminator sequence is a sequence acting as a poly (A) addition signal (polyadenylation signal) to increase the completeness and efficiency of transcription. Terminator sequences suitable for the host microorganism are known in the art, for example, tac terminator sequence, rrnB terminator sequence and the like can be used when the host microorganism is E. coli, and ADH1 terminator sequence can be used when the host microorganism is yeast.

The expression vector may further comprise a selectable marker gene. The selectable marker gene is a gene encoding a gene that enables selection of a host microorganism containing such a marker gene, and is generally an antibiotic resistance gene. Examples of antibiotic resistance genes that can be used include puromycin resistant genes (e.g., puromycin N-acetyltransferase genes from Streptomyces alboniger ), neomycin resistant genes (e.g., Streptomyces aminoglycoside phosphotransferase gene derived from fradiae ), hygromycin resistance gene ( Streptomyces Hygromycin phosphotransferase gene from hygroscopicus ), a bleomycin resistance gene (a bleomycin binding protein derived from Streptomyces verticillus), a blasticidin resistance gene (e.g., Streptomyces Blind stitch when the Dean S- acetyltransferase gene), hygromycin resistance gene (e.g. the amino cycle erythritol phospho transferase gene derived from Escherichia coli derived from verticillum), such as ampicillin resistance gene (β- lactamase gene) .

The selectable marker may be a thymidylate synthase gene, a thymidine kinase gene, or a dihydrofolate reductase gene. These genes enable the selection of host microorganisms with the gene in the medium in which the specific component is present (Kaufman RJ. Methods Enzymol 185, 537-56 (1990)).

In the method of the present invention, the expression vector may also contain a restriction enzyme recognition site for easy cloning of the target gene.

In the method of the present invention, after the expression vector of step (a) is prepared, step (b) of transforming it into a host microorganism is performed.

Transformation refers to a modification of the genotype of the host microorganism by introduction of the causal gene, which may be present independently of the genome of the host microorganism or integrated into the genome of the host microorganism. Herein, the homologous gene and the heterologous gene are included in the homologous gene. The term " homologous gene " means the endogenous gene of the host microorganism or the same species as the homologous gene. The " heterologous gene " . For example, when an mvaS gene derived from Enterococcus kingsejongensis Gf1, which is disclosed in SEQ ID NO: 1 of Korean Patent Publication No. H-1, which encodes HMGS, is introduced into Escherichia coli, this gene becomes a heterologous gene, When introduced into Sejong-nessis Gf1, it is a homologous gene.

Methods for transforming a host microorganism with an exogenous gene are well known in the art and any of known methods can be selected and used. For example, when the host microorganism is a prokaryotic cell such as Escherichia coli, transformation can be carried out by a CaCl ₂ method, A calcium phosphate precipitation method, an electroporation method, a liposome-mediated transfection method, a DEAE-mediated transfection method, and the like can be used in the case where the host microorganism is an eukaryotic cell such as yeast. A dextran treatment method, a gene bombardment, and the like. Specific details concerning the transformation method are described in Cohen, SN et al., Proc. Natl. Acac. Sci. USA, 9: 2110-2114 (1973), Hanahan, Acid Res., 16: 6127-6145 (1988)) (Capecchi, MR, Cell, 22: 479 (1980)), 166: 557-580 (1983)), Dower, WJ et al., Nucleic Acids Res. Et al., EMBO J., 1: 841 (1982)), Wong, TK et al. , Gene, 10:87 (1980)), Gopal, Mol. Cell Biol., 5: 1188-1190 (1985), Yang et al., Proc. Natl. Acad Sci., 87: 9568 -9572 (1990)) (Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Har

(1982), Hitzeman et al., J. Biol. Chem., 255, 12073-12080 (1980), Luchansky et al Mol. Microbiol. 2, 637-646 Can be referenced.

The host microorganism that may be used for transformation in the methods of the present invention may be prokaryotic or eukaryotic. As prokaryotic cells, both Gram-positive bacteria and Gram-negative bacteria can be used. Specifically, bacteria belonging to the genus Escherichia, bacteria belonging to the genus Salmonella, bacteria belonging to the genus Shigella, bacteria belonging to the genus Enterobacter, bacteria belonging to the genus Serratia, genus Erwinia A bacterium belonging to the genus Pseudomonas, a bacterium belonging to the genus Caulobacter, a bacterium belonging to the genus Synechocystis (for example, a bacterium belonging to the genus Pseudomonas species PCC 6803 or a bacterium belonging to the genus Pseudomonas species PCC 6301) , Bacteria of the genus Synechococcus, bacteria of the genus Bacillus (for example, Bacillus brevis , Bacillus subtilis , Bacillus thuringiensis Etc.), bacteria of Lactococcus (for example, Lactococcus lactis ), Streptomyces spp. (e.g., Streptomyces spp. lividans , Streptomyces ambofaciens , Streptomyces and the like fradiae, Streptomyces griseofuscus), also cocks course (Rhodococcus) in bacteria (e.g., Rhodococcus erythropolis), Corynebacterium (Corynebacterium) in bacteria (e.g., Corynebacterium gluamicum), Mycobacterium (Mycobacterium) in bacteria (e.g. Mycobacterium smegmatis) Can be used.

Examples of eukaryotic cells include yeast cells such as Pichia spp., Such as Pichia pastoris ), Saccharomyces spp. (e.g., Saccharomyces cerevisiae ), Hansenulla sp. yeast (e.g., Hansenula polymorpha , Kluyveromyces sp. yeast such as Kluyveromyces lactis , Schizosaccharomyces sp. pombe ) can be used.

Preferably, Escherichia coli ) is used. Expression in E. coli may provide several advantages in terms of cost and yield compared to other expression host microorganisms. DH5I ', DH5IM', DH5B, DHIOB / p3, DH1IS, C600, HB101, JM101 (SEQ ID NO: 1), which are suitable for the expression of a high yield of the target gene, as E. coli W3110, E. coli BL21, BL21 , JM105, JM109, JM110, K38, RR1, Y1088, Y1089, CSH18, ER1451, ER1647, NovaBlue, DH5 ?, K12 RV308, K12 C600, K-12, MG1655 and HB101. More details can be found in Brown, Molecular Biology Labfax, Academic Press (1991), which is likewise regarded as part of this specification.

It may be preferable to use Escherichia coli as a host microorganism in which the protease activity is impaired in order to exhibit and maintain the desired protein function in E. coli. Specific details can be found in Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California, 119-128 (1990).

In order to express a high yield of the target gene in E. coli, the sequence of the target gene may be optimized to the preferred codon in E. coli. See Wada et al., Nucleic Acids Res. 20: 2111-2118 (1992) for a discussion of this.

In the method of the present invention, the medium of step (b) is generally prepared containing a carbon source, a nitrogen source, and a trace element.

In general, the host microorganism is cultured in a medium containing a carbon source, a nitrogen source, and a trace element.

The medium of the present invention is a carbon source. It may contain glycerol-containing biodiesel production by-products in the medium preparation step. It may be added during the medium preparation step and may be further supplied during the culture. In the medium preparation step, .

The medium of the present invention is a carbon source. Any carbon source known in the art besides containing or supplying glycerol-containing biodiesel production by-products during its preparation and / or culturing step is further added during the preparation and / .

Such carbon sources are preferably sugars such as monosaccharides, disaccharides or polysaccharides. For example, glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch, cellulose and the like can be used. Complex compounds such as molasses or by-products of the sugar purification process may also be used. In some cases, it may be advantageous to use a variety of sugar components in combination. Fat such as soybean oil, sunflower oil, peanut oil, coconut oil, palmitic acid, stearic acid and linolenic acid, alcohols such as methanol, ethanol and polyalcohol, organic acids such as acetic acid and lactic acid may be advantageous as a carbon source depending on the host microorganism .

Such a carbon source may be contained in the medium preparation step at a content of 1% (w / v) to 15% (w / v) whether the glycerol-containing biodiesel production by-product is used alone or in combination with other carbon sources, Can be supplied during the incubation step to maintain the carbon source concentration in the upper range.

The nitrogen source may typically be an inorganic nitrogen compound, an organic nitrogen compound or a complex compound containing these compounds. Examples of the inorganic nitrogen compound include ammonia, ammonium salts (e.g., ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, ammonium nitrate and the like) and nitrate. Examples of the organic nitrogen compound include urea, And examples of the complex compound include soybean meal, soybean protein, yeast extract, and broth. Such a nitrogen source may range from 0.01% (w / v) to 5% (w / v) in the medium.

Examples of the trace elements include calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper, iron, phosphorus and sulfur. These trace elements may be added to the medium in the form of a salt compound, A chelating agent such as catechol, citric acid or the like may be added together to increase the solubility and maintain the solution state. And in the range of 0.001% (w / v) to 3% (w / v) for trace elements.

The medium for culturing the host microorganism may optionally contain trace amounts of growth promoters such as biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate, pyridoxine and the like.

For optimization of the medium composition, reference can be made to Applied Microbiol. Physiology, A Practical Approach (Editors PM Rhodes, PF Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3) Are considered part of this disclosure.

The medium is preferably sterilized to prevent the growth of unnecessary microorganisms prior to inoculation of the host microorganisms. Methods for sterilization of the medium are well known in the art, and a suitable one among known sterilization methods can be used. For example, high-pressure steam sterilization or ultraviolet sterilization.

The incubation temperature is generally 15 ° C to 45 ° C, preferably 25 ° C to 40 ° C, more preferably 25 to 37 ° C. The pH of the medium is from 5 to 8.5, preferably from about 6.0 to 7.0. The pH of the medium can be adjusted by adding basic compounds such as sodium hydroxide, potassium hydroxide and aqueous ammonia or acidic compounds such as phosphoric acid and sulfuric acid during the course of the culture. Defoamers such as fatty acid polyglycol esters may be added to remove the foam formed during the culture. In order to stably propagate only the transformed host microorganism, a suitable substance having a screening effect, such as the antibiotic mentioned above, may be added to the medium.

It may be advantageous to produce aerobic or anaerobic conditions depending on the propagation characteristics of the host microorganism during the culture.

The culture can be batchwise, semi-batchwise, continuous, fed-batch culture.

The culture is preferably continued until the formation of the desired product reaches its maximum. The incubation time varies depending on the host microorganism, and the optimal incubation time for the host microorganism can be theoretically and experimentally determined within the ordinary skill of those skilled in the art.

Suitable culture methods according to host microorganisms are described in Bioproze < (R) > in Biotechnology [Bioprocess technology 1. Introduction to Bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991), Bioreaktoren und periphere Einrichtungen Peripheral Equipment (Vieweg Verlag, Brunswick / Wiesbaden, 1994), the American Society for Bacteriology (Washington DC, USA, 1981) .

The method of the present invention proceeds with the step of obtaining mevalonate from the culture after the host microorganism has been cultured.

The step of obtaining mevalonate can be carried out by any method known in the art such as centrifugation, filtration, crystallization, ion exchange chromatography, high performance liquid chromatography (HPLC), etc. have. More specifically, after separating the cells and the supernatant by centrifugation, the supernatant containing mevalonate is extracted with a solvent such as acetone and purified by HPLC or crystallization in order to obtain a product of high purity. Can proceed. In connection with other separation methods, the name of the invention as recited above is " a recombinant microorganism having mevalonate or mevalonolactone-producing ability, and a method for producing mevalonate or mevalonolactone using the same " 10-2015-0134512, entitled " Microorganisms Containing Genes Encoding Enzymes Involved in the Production of Mevalonic Acid and Methods for Producing Mevalonic Acids Utilizing the Same ", Korean Patent Publication No. 10-2015-0006097, (Metabolic Engineering 11: 13-19, 2009), Clinical Biochemistry 40: 575-584, 2007), and the like, as described in Journal of Biotechnology 140: 218-226, 2009, NATURE BIOTECHNOLOGY 21: 796-802, , Arch Biochem Biophys. 505 (2): 131-143, 2011].

As described above, the present invention can provide a biosynthesis method of mevalonate using glycerol-containing biodiesel production by-product as a carbon source. The method of the present invention using glycerol-containing biodiesel production by-products as a carbon source has a much higher yield than the case where glucose is used as a carbon source and has a higher yield than the case of using common glycerol (pure glycerol sold as a product) I have.

Figure 1 schematically illustrates the process of mevalonate biosynthesis.
2 is a schematic diagram of pSTVM_mvaS_atoB used for mevalonate biosynthesis.
Figs. 3 to 5 are the results of HPLC analysis of mevalonate standard solution, glycerol standard solution and fermentation broth, respectively.
FIGS. 6 and 7 are the results showing the amount of cell mass and the amount of mevalonate produced when pure glucose and glycerol-containing biodiesel production by-products were used as carbon sources, respectively, at the flask level.
FIGS. 8 to 10 show the results of showing the amount of cell mass and the amount of mevalonate produced when glucose, pure glucose and glycerol-containing biodiesel production by-products were used as carbon sources at the fermenter level, respectively.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

< Example > As a carbon source  Using glycerol Mevalonate  production

One. Materials and Experimental Methods

1.1 Mevalonate Productive  The transformed host microorganism

The E. coli MG1655 strain transformed with two plasmids (pUCM_mvaE and pSTVM_mvaS_atoB) containing the mvaE , mvaS and atoB genes involved in mevalonate biosynthesis and containing a gene resistant to ampicillin and Glucamphenicol , respectively, was used in the experiment. mvaE , mvaS The gene is a recombinant microorganism having the ability to produce mevalonate or mevalonolactone, and a method for producing mevalonate or mevalonolactone using the same, in Korean Patent No. 10-1625898, ( MvaE ) amplified with the primer sequence of SEQ ID NO: 5 and 6 ( mvaS ), and the atoB gene is the gene of KEGG EC: 2.3.1.9 (the gene of Escherichia coli MG1655 strain , http://www.genome.jp/dbget-bin/www_bget?eco:b2224+eco:b2844), and the schematic diagram of pUCM_mvaE is shown in FIG. 3 of Korean Patent No. 10-1625898, and the schematic diagram of pSTVM_mvaS_atoB Is shown in Fig.

1.2 Culture medium composition and culture conditions

The medium used in this experiment was a medium containing 35 g / L Tryprone, 20 g / L yeast extract and 5 g / L sodium chloride as a super broth medium, (Hereinafter referred to as " waste glycerol ") containing glycerol or glycerol at a certain concentration. The medium was prepared by mixing each medium and sterilized, and the waste glycerol was separately sterilized and used as a medium.

1.3 Culture at flask level

A single clone of the transformed E. coli was transformed into LB medium (10 g / L of tryprone, 5 g / L of yeast extract, sodium chloride (pH 7.0), and the like) containing ampicillin of 100 ug / ml and chloramphenicol of 50 ug / ) 5 g / L) and incubated at 30 ° C for 12 hours. After incubation in a cap tube, 70 ml of SB medium (35 g / L of tryptone, 20 g / L of yeast extract, 5 g / L of sodium chloride, pure glycerol or 20 g / L of waste glycerol) containing 50 ug / ml of ampicillin and 50 ug / , And then cultured with shaking at 30 DEG C for 60 hours.

1.3 Culture at the fermenter level

A single clone of each of the transformed Escherichia coli was cultivated in an LB medium (10 g / L of Tryprone, 5 g / L of yeast extract, 5 g / L of sodium chloride, and 10 g / L of yeast extract) containing 100 ug / ml of ampicillin and 50 ug / chloride) 5 g / L) and incubated at 30 ° C for 12 h. cap tube and inoculated into a 500 ml flask containing 100 ug / ml of ampicillin and 100 ml of SB medium (35 g / L of tryptone, 20 g / L of yeast extract and 5 g / L of sodium chloride) containing 50 ug / ml of chloramphenicol And then shake-cultured at 30 ° C for 4 hours. 100 ml of the resulting solution was added to 100 ml of an ampicillin of 100 ug / ml and 50 ml of an SB medium (35 g / L of tryptone, 20 g / L of yeast extract, 5 g / L of sodium chloride, 20 g of glucose or pure glycerol, / L), and then incubated at 30 ° C in a 2 L fermentor at 900 rpm and 50% DO for a certain period of time. The pH was maintained at 6.8 using 30% ammonia water. The concentration of glucose in the medium was 25 g / L, and the concentration of pure glycerol and waste glycerol in the medium was 20 g / L. The pH Stat method (BS Kim et al 2004 bioprocess and biosystems engineering vol.2 issue 3 pp147 -150) and fed with oil. The waste glycerol was quantitatively measured for the glycerol content of the glycerol-containing biodiesel production by-products to be fed at a glycerol concentration of 20 g / L.

1.4 Amount of bacterium  Measure

1 ml of the culture obtained through the flask and the fermenter was taken and diluted. The absorbance was measured at a wavelength of 600 nm using a spectrophotometer, and then the amount of the cells was determined by multiplying by the dilution factor.

When lung glycerol was used, 1 ml was taken and washed three times with PBS buffer (phosphate buffered saline). Absorbance was measured at a wavelength of 600 nm using a spectrophotometer, and the amount of the cells was determined by multiplying by the dilution factor.

1.5 HPLC Through Mevalonate  Quantitative analysis

The mevalonate quantitative analysis was carried out using HPLC. Sample preparation was carried out according to the method of Xiong M. et al. Vol. 111, no. 23 pp8357 ~ 8362). The mevalonate used for the analysis was Sigma Aldrich (Sigma Aldrich) was purchased and used. The mevalonate standard solution was prepared by dissolving 20 g / L of water in water and diluting 6 standard solutions at a concentration of 0.312 to 10 g / L. The calibration curves were prepared by analysis. Pure glycerol was purchased from Daeshwanggae (Korea, Kyunggi province) and waste glycerol was purchased from JC Chemical (Ulsan, Korea). The glycerol standard solution was dissolved in water to prepare 30 g / L, and then diluted with 7 standard solutions at a concentration of 0.468 to 30 g / L. The calibration curves were prepared by analyzing. The fermentation broth was used for the analysis to determine glycerol consumption and mevalonate biosynthesis through fermentation of the transformed host microorganism. After taking 1 ml of the fermentation broth, the cells and supernatant were separated using a centrifugal separator, and the supernatant was collected and filtered through a 0.45 uL filter and analyzed by HPLC.

The HPLC analysis conditions are shown in the table below.

2. Experiment result

2.1 With mevalonate  Glycerol standard curve

HPLC analysis of standard solutions showed that mevalonate was detected at 16.8 min and glycerol was detected at 11.6 min. The same results were obtained when the fermentation broth was analyzed.

The results of HPLC analysis of the mevalonate standard solution, the glycerol standard solution and the fermentation broth are shown in FIG. 3 to FIG.

2.2 Flask Culture

6 and 7 show results obtained by culturing in a 500 ml culture flask for 60 hours using 70 ml of SB medium containing 20 g / L of pure glycerol or waste glycerol.

In FIG. 6, the left graph shows the cell growth curve, the right graph shows the concentration of mevalonate by the incubation time, and FIG. 7 shows the cell growth curve.

When pure glycerol was used, the cells grew up to 14.05 OD and the cells grew up to 16 when the waste glycerol was used.

The yield of mevalonate increased with incubation time up to 8.22 g / L when pure glycerol was used. The yield of mevalonate was 41.1% compared to the amount of carbon source. When the waste glycerol was used, the maximum yield was 8.99 g / L and the yield was 44.95%. When the waste glycerol was used, 36 hr culture and 48 hr culture showed high yield.

2.3 Fermenter culture

8 to 10 show the results of culturing in a 2 L fermenter using SB medium containing 25 g / L of glucose, 20 g / L of pure glycerol or 20 g / L of waste glycerol (Fig. 8: using glucose, Fig. 9: Using pure glycerol, Fig. 10: using waste glycerol).

When the cells were cultured for 80 hours using glucose, the OD value reached up to 56.1. When the cells were cultured for 79 hours with pure glycerol, the cell growth was up to 76 OD, and 67 When incubated for a period of time, cell growth was up to 49.7.

The yield of mevalonate was 9.92%, the yield was 62.17 g / L when pure glycerol was used, the yield was 26.1% at that time, . When the waste glycerol was used, the yield was 36.62 g / L and the yield was 26.43%.

Claims (17)

(a) a host microorganism transformed with an expression vector capable of expressing HMGS, which is an acetoacetyl coenzyme A synthase, AtoB, a hydroxymethylglutaryl coenzyme A synthase, HMGS, and a hydroxymethylglutaryl coenzyme A reductase, , &Lt; / RTI &gt;
(b) preparing a medium for culturing the host microorganism,
(c) inoculating and culturing the host microorganism in the medium, and
(d) obtaining mevalonate from the culture,
The host microorganism is Escherichia coli MG1655 strain
Wherein the medium is fed to at least one of the step of culturing the host microorganism of step (b) and the step of culturing the host microorganism of step (c), wherein glycerol-containing biodiesel production by- Method of mevalonate biosynthesis using diesel - formed by - products.
The method according to claim 1,
The expression vector capable of expressing AtoB, HMGS and HMGR may be composed of one expression vector capable of expressing both AtoB, HMGS and HMGR, or may express two of AtoB, HMGS and HMGR and express the other one Characterized in that it consists of two expression vectors or three expression vectors capable of expressing each of AtoB, HMGS and HMGR.
The method according to claim 1,
The expression vector capable of expressing AtoB, HMGS, and HMGR comprises an expression vector comprising a mvaE gene and a gene encoding the HMGS, which comprises a mvaE gene and a gene encoding the HMGS, &Lt; / RTI &gt; wherein the expression vector comprises each of the expression vectors.
The method according to claim 1,
Wherein the expression vector is one of a plasmid, a cosmid, a phagemid, a phage, and a virus.
The method according to claim 1,
Wherein the expression vector is a plasmid.
The method according to claim 1,
Wherein said expression vector is operably linked to a gene whose coding sequence is encoding said AtoB, HMGS and HMGR.
The method according to claim 1,
The expression vector is characterized in that the coding for AtoB, HMGS and HMGR is regulated by a promoter selected from the group consisting of T7A1, T7A2, T7A3, lambda pL, lac, lacUV5, trp, tac, trc, phoA, rrnB and 1PL .
The method according to claim 1,
Wherein the expression vector further comprises a selectable marker gene.
delete delete delete delete The method according to claim 1,
The host microorganism is Escherichia coli,
Wherein said E. coli is strain MG1655.
The method according to claim 1,
Wherein the medium further comprises other carbon sources selected from monosaccharides, disaccharides and polysaccharides, as well as glycerol-containing biodiesel production by-products as a carbon source, and further comprising a nitrogen source and trace elements.
The method according to claim 1,
Characterized in that the medium comprises a carbon source in a concentration of 1% (w / v) to 15% (w / v).
The method according to claim 1,
Characterized in that said culture consists of batch, semi-batch, continuous or infusion.
The method according to claim 1,
Wherein the culture continues until the amount of mevalonate produced reaches a maximum.

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