# Patent

## Title
Transformant comprising gene coding for ws/dgat and method of producing fatty acid ethyl esters using the same

## Abstract

The present invention relates to a transformant comprising a gene encoding ws/dgat (wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase) and a method of producing fatty acid ethyl esters using the same. Specifically, the transformant for producing fatty acid ethyl esters is constructed such that glycerol is used as a fermentation substrate, and comprises an atfA gene encoding ws/dgat (wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase) from Acinetobacter sp., so that the atfA gene is expressed in the transformant.

## Background
TECHNICAL FIELD
The present invention relates to a transformant, which uses glycerol as a fermentation substrate and can produce fatty acid ethyl esters by expressing a ws/dgat-encoding gene therein, and to a method of producing fatty acid ethyl esters using the transformant.
BACKGROUND ART
Since the United Nations Conference on Environment and Development (UNCED) in Rio de Janeiro in 1992 and the Kyoto Convention in 1997, the emission of air pollutants has been regulated, and biofuel has received a great deal of attention as a renewable energy source for reducing the emission of carbon dioxide. Currently, there is an urgent need to develop biodiesel that can be used as a clean alternative fuel for diesel vehicles, which account for 30% or more of transportation vehicles in the world and generate 50% or more air pollution. Biodiesel is made from organic materials by esterifying animal and vegetable oils with alcohols such as methanol or ethanol and removing glycerol from the esterified oils, thus obtaining fatty acid methyl esters or fatty acid ethyl esters. Biodiesel has properties similar to petroleum-derived light oil and causes little air pollution upon combustion, and thus when it is mixed with petroleum-based light oil, it can be used as a clean alternative fuel that can significantly reduce vehicle air pollution, which is the main cause of air pollution. Thus, the demand and need for biodiesel is increasing. In addition to an alternative fuel for light oil, biodiesel can be used as raw materials and additives for lubricant oils and can also be used in various applications, including pollution-free solvents and agricultural biochemicals. In addition, biodiesel can also be used as a catalyst to promote bioremediation for cleaning the seashore when contaminated with crude oils. So far, biodiesel has been produced from a variety of animal and vegetable oils by chemical catalytic methods using a strong acid or a strong base. However, in the chemical catalytic methods, a multi-step reaction process that consumes large amounts of energy is required, it is difficult to recover catalysts and byproducts, and a large amount of wastewater is generated, thus causing secondary environmental contamination. Due to these problems, a low-energy-consuming, environmentally friendly new biological process that can satisfy the strong demand for biodiesel is required. In 2006, Rainer et al. reported the production of fatty acid ethyl esters in E. coli (Rainer Kalscheuer et al, Microbiology. 152, 2529-2536, 2006), suggesting that fatty acid ethyl esters can be processed by a biological method. However, the production of alcohols such as ethanol in E. coli is insignificant, and E. coli has low resistance to alcohols. In recent years, studies on the increase in the production of fatty acid ethyl esters in E. coli have been conducted by Eric et al. (Eric J. Steen et al., Nature. 463. 559-556, 2010), but even in an E. coli strain transformed such that ethanol can be produced therein, the production of ethanol was significantly low. This led to a study that reported an increase in the production of fatty acid ethyl esters when ethanol was added to a medium. The present inventors have developed a more efficient system for producing fatty acid ethyl esters as a result of selecting yeast having ethanol resistance and high ethanol productivity as a host for producing fatty acid ethyl esters.
Also, glycerol (C3H8O3), which is used as a substrate in the present invention, is chemically more reduced than glucose (C6H12O6), and thus provides a higher reducing power for the metabolism of microorganisms. Since a lot of materials produced during fermentation are generally required to have reducing power in their metabolism, the use of glycerol as a substrate can lead to a significant improvement in the yield and productivity of desired fermentation products. Currently, as the production of biodiesel increases, the production of glycerol also increases, and thus the price thereof is decreasing rapidly. As described above, because a rapid increase in the production of biodiesel leads to an increase in the production of the byproduct glycerol, the effective treatment of byproducts including glycerol will be issued. Thus, if glycerol can be effectively used to produce useful fermentation products, it can provide a lot of additional effects.
DISCLOSURE
Technical Problem
It is an object of the present invention to provide a transformant for producing fatty acid ethyl esters that comprises a gene encoding ws/dgat (wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase), a glycerol dehydrogenase gene, a dihydroxyacetone kinase gene and a glycerol uptake protein gene, and a method for preparing the transformant.
Another object of the present invention is to provide a method for producing fatty acid ethyl esters using said transformant.
Technical Solution
To achieve the above objects, the present invention provides a transformant for producing fatty acid ethyl esters, which comprises a gene encoding ws/dgat (wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase), a glycerol dehydrogenase gene, a dihydroxyacetone kinase gene and a glycerol uptake protein gene.
The present invention also provides a method for preparing a transformant for preparing fatty acid ethyl esters, the method comprising the steps of: constructing a recombinant vector comprising a gene encoding ws/dgat (wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase) and a glycerol uptake protein gene; constructing a recombinant vector comprising a glycerol dehydrogenase gene and a dihydroxyacetone kinase gene; and transforming the recombinant vectors.
The present invention also provides a method for producing fatty acid ethyl esters, the method comprising a step of culturing said transformant using glycerol as a substrate.
Advantageous Effects
The transformant according to the present invention is constructed so that it uses glycerol as a carbon source and contains genes causing the synthesis of fatty acid ethyl esters. Accordingly, the transformant of the present invention can be used to produce large amounts of fatty acid ethyl esters using glycerol, which is a byproduct of biodiesel production, and thus is highly useful.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows the results of performing agarose gel electrophoresis of the PCR product of the ws/dgat-encoding gene in the present invention. In FIG. 1, lane 1: 1-kb DNA marker; and lane 2: Ws/dgat PCR product.
FIG. 2 shows the results of performing agarose gel electrophoresis of PCR products, wherein FIG. 2(A) shows the PCR product of the glycerol dehydrogenase gene (lane 1: 1-kb DNA marker; and lane 2, Gcy PCR product), FIG. 2(B) shows the PCR product of the dihydroxyacetone kinase gene (lane 1: 1-kb DNA marker; and lane 2: Dak PCR product), and FIG. 2(C) shows the PCR product of the glycerol uptake protein gene (lane 1: 1-kb DNA marker; and lane 2: Gup PCR product).
FIG. 3a is a schematic diagram showing the process of constructing the recombinant vector pGup1a ws.dgat cas containing a ws/dgat-encoding gene according to the present invention.
FIG. 3b is a schematic diagram showing the process of constructing the recombinant vector pGcyaDak containing glycerol dehydrogenase gene and a dihydroxyacetone kinase gene in both sense and antisense directions according to the present invention.
FIG. 3c is a schematic diagram showing the process of constructing the recombinant vector pGupCas containing a glycerol uptake protein gene according to the present invention.
FIG. 4 shows the results of GC-MS analysis before (a) and after (b) the addition of oleate.
FIG. 5a shows the results of measuring the synthesis of ethanol (left) and fatty acid ethyl esters (right) when the transformant containing the ws/dgat-encoding gene according to the present invention was cultured in a medium not containing sodium oleate.
FIG. 5b shows the results of measuring the synthesis of ethanol (left) and fatty acid ethyl esters (right) when the transformant containing the ws/dgat-encoding gene according to the present invention was cultured in a medium containing sodium oleate. In FIGS. 5a and 5b, lane 1: YPH499 (pESC-TRP); lane 2: YPH499 (pGcyaDak, pGup1aWS.DGATcas); ♦: YPH499 (pESC-TRP); ▪: YPH499 (pGcyaDak,pGupCas); and ▴: YPH499 (pGcyaDak, pGup1a ws.dgat cas).



MODE FOR INVENTION
The present invention relates to a transformant for producing fatty acid ethyl esters, which comprises a gene encoding ws/dgat (wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase), a glycerol dehydrogenase gene, a dihydroxyacetone kinase gene and a glycerol uptake protein gene.
In the present invention, the ws/dgat-encoding gene is preferably a gene from Acinetobacter sp., but is not limited thereto.
The present invention, the ws/dgat preferably has the amino acid sequence set forth in SEQ ID NO: 1, but may also be a mutant protein having ws/dgat activity, which contains at least one mutation (such as a substitution, deletion or addition) in the amino acid sequence of SEQ ID NO: 1.
In the present invention, the gene encoding ws/dgat preferably has the nucleotide sequence set forth in SEQ ID NO: 2, but may also be a gene having a homology of 80% or greater, preferably 85% or greater, even more preferably 90% or greater, and most preferably 95% or greater, with the nucleotide sequence of SEQ ID NO: 2, in view of the degeneracy of the genetic code.
In one embodiment of the present invention, the glycerol dehydrogenase preferably has the amino acid sequence set forth in SEQ ID NO: 3, but may also be a mutant protein having glycerol dehydrogenase activity, which contains at least one mutation (such as a substitution, deletion or addition) in the amino acid sequence of SEQ ID NO: 3.
In one embodiment of the present invention, the glycerol dehydrogenase gene preferably has the nucleotide sequence set forth in SEQ ID NO: 4, but may also be a gene having a homology of 80% or greater, preferably 85% or greater, even more preferably 90% or greater, and most preferably 95% or greater, with the nucleotide sequence of SEQ ID NO: 4, in view of the degeneracy of the genetic code.
In another embodiment of the present invention, the dihydroxyacetone kinase preferably has the amino acid sequence set forth in SEQ ID NO: 5, but may also be a mutant protein having dihydroxyacetone kinase activity, which contains at least one mutation (such as a substitution, deletion or addition) in the amino acid sequence of SEQ ID NO: 5.
In still another embodiment of the present invention, the dihydroxyacetone kinase gene preferably has the nucleotide sequence set, forth in SEQ ID NO: 6, but may also be a gene having a homology of 80% or greater, preferably 85% or greater, even more preferably 90% or greater, and most preferably 95% or greater, with the nucleotide sequence of SEQ ID NO: 6, in view of the degeneracy of the genetic code.
In still another embodiment of the present invention, the glycerol uptake protein preferably has the amino acid sequence set forth in SEQ ID NO: 7, but may also be a mutant protein having glycerol uptake activity, which contains at least one mutation (such as a substitution, deletion or addition) in the amino acid sequence of SEQ ID NO: 7.
In still another embodiment of the present invention, the glycerol uptake protein gene preferably has the nucleotide sequence set forth in SEQ ID NO: 8, but may also be a gene having a homology of 80% or greater, preferably 85% or greater, even more preferably 90% or greater, and most preferably 95% or greater, with the nucleotide sequence of SEQ ID NO: 8, in view of the degeneracy of the genetic code.
The transformant of the present invention is yeast, and preferably Saccharomyces cerevisiae, but the scope of the present invention is not limited thereto.
The transformant of the present invention is preferably, but not limited to, the yeast Saccharomyces cerevisiae YPH499 (pGcyaDak, pGupla ws.dgat cas) deposited under accession number KCCM11094P. Saccharomyces cerevisiae YPH499 (pGcyaDak, pGupla ws.dgat cas) has been deposited at the Korean Culture Center of Microorganisms, at 361-221, Yurim B/D, Hongje-1-dong, Seodaemun-gu, Seoul, 120-091, Republic of Korea, on Aug. 18, 2010 and given the deposit reference KCCM11094P. This deposit was made under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms, and all restrictions imposed by the depositor on the availability to the public of the deposited material will be irrevocably removed upon the grant of the patent.
Also, the present invention relates to a method for preparing a transformant for preparing fatty acid ethyl esters, the method comprising the steps of: constructing a recombinant vector comprising a gene encoding ws/dgat (wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase) and a glycerol uptake protein gene; constructing a recombinant vector comprising a glycerol dehydrogenase gene and a dihydroxyacetone kinase gene; and transforming the recombinant vectors.
As used herein, the term “recombinant vector” describes a vector capable of expressing a target protein in a suitable host cell, and refers to a genetic construct that comprises essential regulatory elements to which a gene insert is operably linked in such a manner as to be expressed in a host cell. Examples of the vector include, but are not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector and a viral vector. Suitable expression vectors may include a signal sequence or a leader sequence for membrane targeting or secretion, as well as regulatory sequences such as a promoter, an operator, an initiation codon, a termination codon, a polyadenylation signal, an enhancer and the like, and can be prepared in various ways depending on the desired purpose. The promoter of the vector may be constitutive or inducible. Further, the expression vector may include one or more selective markers for selecting a host cell containing the expression vector, and, in the case of a replicable expression vector, may include a nucleotide sequence of replication origin.
Also, the recombinant vector may further have a fragment for regulating expression, which has a variety of functions for suppression, amplification or triggering of expression, as well as a marker for the selection of a transformant, a antibiotic resistance gene, or a gene encoding a signal for extracellular secretion.
The recombinant vector comprising the gene encoding ws/dgat (wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase) and the glycerol uptake protein gene may be, for example, pGup1a ws.dgat cas, but is not limited thereto.
The recombinant vector comprising the glycerol dehydrogenase gene and the dihydroxyacetone kinase gene may be, for example, pGcyaDak, but is not limited thereto.
When yeast is used as a host in the present invention, the expression vector may be, for example, a YEpl3, YCp50, pRS or pYEX vector, and the promoter may be, for example, a GAL promoter or an ADD promoter.
Transformation in the present invention may be carried out according to any method for introducing a nucleic acid molecule into an organism, a cell, a tissue or an organ, and methods that may be used to introduce recombinant DNA into yeast include electroporation (Method Enzymol., 194, 182-187 (1990)), the spheroplast method (Proc. Natl. Acad. Sci. USA, 84, 1929-1933 (1978)), and the lithium acetate method (J. Bacteriol., 153, 163-168 (1983)).
The present invention also relates to a method for producing fatty acid ethyl esters, which comprise a step of culturing the transformant of the present invention using glycerol as a substrate.
In the present invention, glycerol is preferably glycerol that is a byproduct of glycerol production, but is not limited thereto.
In the process of producing fatty acid ethyl esters according to the present invention, a fatty acid salt having 12 to 20 carbon atoms is preferably added to a medium, but the scope of the present invention is not limited thereto.
The transformant is preferably yeast, more preferably yeast Saccharomyces cerevisiae, and more preferably Saccharomyces cerevisiae YPH499 (pGcyaDak, pGup1a ws.dgat cas) [accession number: KCCM11094P].
As used herein, the term “composition for producing fatty acid ethyl esters” means, for example, a composition capable of producing fatty acid ethyl esters by culturing the transformant using glycerol as a substrate. The composition may include a polypeptide, a fermentation broth, a cell lysate, a purified or non-purified yeast extract or the like, that is suitable for the production of fatty acid ethyl esters.
The transformant of the present invention may be cultured using any conventional method that is used for the culture of hosts.
The culture of the transformant may be carried out using any conventional method for microbial culture, including a batch culture method, a fed-batch culture method, a continuous culture method, and a reactor-type culture method. Examples of a medium for culturing the transformant using E. coli or yeast as a host include complete media or synthetic media, for example, LB medium, NB medium and the like. Also, the transformant is cultured at a temperature ranging from 25 to 30° C., so as to facilitate the accumulation of ws/dgat in microbial cells.
Examples of the carbon source that may be used for the growth of microorganisms include sugars, such as glucose, fructose, sucrose, maltose, galactose or starch; lower alcohols, such as ethanol, propanol or butanol; polyhydric alcohols such as glycerol; organic acids, such as acetic acid, citric acid, succinic acid, tartaric acid, lactic acid or gluconic acid; and fatty acids, such as propionic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, undecanoic acid or dodecanoic acid.
Examples of the nitrogen source include ammonium salts, such as ammonia, ammonium chloride, ammonium sulfate or ammonium phosphate, and materials of natural origin, such as peptone, meat juice, yeast extracts, malt extracts, casein hydrolysates, or corn steep liquor. Also, examples of minerals include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride and the like. The medium may be supplemented with antibiotics, such as kanamycin, ampicillin, tetracycline, chloramphenicol and streptomycin.
Also, when microorganisms transformed with an expression vector having an inducible promoter are cultured, an inducer suitable for the kind of promoter may be added to the medium. Examples of the inducer include isopropyl-β-D-thiogalactopyranoside (IPTG), tetracycline, indole acrylic acid (IAA), and the like.
The fatty acid ethyl ester synthase ws/dgat can be obtained by centrifuging the culture medium of the transformant, collecting a cell or a supernatant from the centrifuged medium, and subjecting the collected cell or supernatant to one or a combination of two or more selected from cell lysis, extraction, affinity chromatography, cation or anion exchange chromatography, and gel filtration.
The determination of whether the resulting purified substance is the desired yeast can be performed using a conventional method, for example, SDS-polyacrylamide gel electrophoresis or Western blotting.
In one embodiment of the present invention, the production of fatty acid ethyl esters in Saccharomyces cerevisiae is based on short chain fatty acids such as oleic acid, in which oleic acid is converted into Oleoyl-CoA by acyl CoA synthase, and the Oleoyl-CoA reacts with ethanol to produce ethyl oleate ester, the reaction being catalyzed by wax ester synthase.
Accordingly, in the present invention, the ws/dgat-encoding gene from Acinetobacter sp. is inserted into a strain constructed such that glycerol is used as the carbon source, whereby the synthesis of fatty acid ethyl esters becomes possible.
Hereinafter, the present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited to these examples.
EXAMPLES
Example 1
Amplification of Ws/Dgat-Encoding Gene from Acinetobacter 
In order to insert an atfA gene from Acinetobacter, which encodes ws/dgat (wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase), a PCR reaction was performed using the genomic DNA of Acinetobacter sp. as a template and the following pair of primers, each containing a site for the restriction enzyme spel: forward primer (5′-actagtcccgccgccaccaaggagatgcgcccattacatccgat-3′, SEQ ID NO: 9); and reverse primer (5′-actagtttaattggctgttttaatatcttc-3′, SEQ ID NO: 10). The PCR reaction was performed under the following conditions: step 1 at 95° C. for 10 min; step 2 at 95° C. for 30 sec; step 3 at 50° C. for 30 sec; step 4 at 72° C. for 2 min; step 5 at 95° C. for 10 min; the process from step 2 to step 4 being repeated for 30 cycles [1.2 kb PCR product].
Example 2
Cloning and Transformation of Ws/Dgat-Encoding Gene from Acinetobacter 
The amplification product obtained in Example 1 was electrophoresed on 0.8% agarose gel, and the DNA fragment on the agarose gel was collected using a Biospin gel extraction kit (Bioflux).
Then, the fragment was digested with the restriction enzyme Spe1, after which it was ligated with pGup1 containing glycerol uptake protein and was transformed into E. coli DH5a (Invitrogen). Then, the ligated recombinant plasmid DNA was separated from the transformant. The recombinant vector was named “pGup1/Ws-Dgat” and is shown in FIG. 3a. Also, in order to insert pGup1/Ws-Dgat, a sense primer (5′-ggatccatgtcagcattttaggtaaattccgtg-3′; SEQ ID NO: 11) and an antisense primer (5′-ggatccataatgtcgctgatcagcatcctgtct-3′; SEQ ID NO: 12) were constructed so as to include a BamH1 recognition sequence, and were cloned into the yeast integration vector YIP-5 [ATCC], thereby constructing pGup1a ws.dgat cas. This was inserted into the genomic DNA of Saccharomyces cerevisiae (FIG. 3a).
Example 3
Transformation of Glycerol Dehydrogenase Gene, Dihydroxyacetone Kinase Gene and Glycerol Uptake Protein Gene
1. Amplification of Glycerol Dehydrogenase Gene, a Dihydroxyacetone Kinase Gene and a Glycerol Uptake Protein Gene
In order to efficiently convert glycerol into the intermediate of glycolysis DHAP (dihydroxyacetone phosphate), cloning of the glycerol dehydrogenase gene (Gcy), the dihydroxyacetone kinase gene (Dak) and the glycerol uptake protein gene (Gup1) was performed with reference to the nucleotide sequences of peptide regions from the genomic DNA (BY4741) of Saccharomyces cerevisiae. 
For cloning of Gcy, Dak and Gup1, primers including the following recognition sequences were synthesized: for Gcy, BamH (5-ggatccatgcctgctactttacatgattct-3; SEQ ID NO: 13), Sal (5-gtcgacatacttgaatacttcgaaaggag-3); for Dak, Spe (5-actagtatgtccgctaaatcgtttgaagtc-3, SEQ ID NO: 14), and Cla (5-atcgatatacaaggcgctttgaaccccctt-3, SEQ ID NO: 15); and for Gup1, EcoR (5-gaattcatgtcgctgatcagcatcctg-3, SEQ ID NO: 16), and Spe (5-actagtccagcattttaggtaaattccgtg-3, SEQ ID NO: 17). Using the synthesized primers, PCR was performed for each gene under the following conditions: step 1 at 95° C. for 10 min; step 2 at 95° C. for 30 sec; step 3 at 50° C. for 30 sec; step 4 at 72° C. for 2 min; and step 5 at 95° C. for 10 min; the process from step 2 to step 4 being repeated for 30 cycles. As a result, PCR bands having lengths of 936 bp, 1755 bp and 1683 bp, respectively, could be confirmed (FIG. 2).
2. Cloning of Glycerol Dehydrogenase Gene, a Dihydroxyacetone Kinase Gene and a Glycerol Uptake Protein Gene
Each of the Gcy, Dak and Gup amplification products obtained in Example 3-1 was electrophoresed on 0.8% agarose gel, and the DNA fragments on the agarose gels were collected using a Biospin gel extraction kit (Bioflux).
Then, Gcy, Dak and Gup1 were digested with BamH and Sal, Spe and Cla, and EcoR and Spe, respectively, after which each of the digested genes was ligated with the yeast-E. coli shuttle vector pESC-trp (Clontech) and transformed into E. coli DH5a. Then, the ligated recombinant plasmid DNAs were separated from the transformants. The recombinant vectors were named “pGcy”, “pDak” and “pGup1”, respectively.
Next, Dak was cloned with the pGcy vector and transformed into E. coli DH5a (Invitrogen). From the transformant, the ligated recombinant plasmid DNA was separated. The recombinant vector was named “pGcyaDak”, and is shown in FIG. 3b. 
Also, in order to insert pGup1 into the genomic DNA of Saccharomyces cerevisiae, a sense primer (5′-ggatccatgt cagcattttaggtaaattccgtg-3′, SEQ ID NO: 18) and an antisense primer (5′-ggatccataatgtcgctgatcagcatcctgtct-3′, SEQ ID NO: 19) were constructed so as to include a BamH1 recognition sequence, and were cloned into the yeast integration vector YIP-5, which was then inserted into the genomic DNA of Saccharomyces cerevisiae. The resulting recombinant vector was named “pGupCas”, and is shown in FIG. 3c. 
Example 4
Construction of Transformant Saccharomyces cerevisiae YPH499 (pGcyaDak, pGup1a Ws.Dgat Cas)
Yeast host cells were transformed with the above recombinant vectors (pGcyaDak, and pGup1a ws.dgat cas) according to the experimental method provided in the YEASTMAKER yeast transformation kit2 (Clontech). The resulting transformant was named “Saccharomyces cerevisiae YPH499 (pGup1a ws.dgat cas)”.
The yeast host cells used were Saccharomyces cerevisiae YPH499 (S. cerevisiae YPH499, ura3-52lys2-801amberade2-101ochretrpl-63 his3-200 leu2-1). Then, the transformant was selected using a tryptophan-deficient SD medium (0.67% yeast nitrogen base, 2% glucose, 0.067% yeast nitrogen base w/o trp, 2% agar), and also the strain comprising Gup1 inserted into Saccharomyces cerevisiae YPH499 was selected in an SD medium containing G418 (100 ug/ml). The selected strain was deposited at the Korean Culture Center of Microorganisms (KCCM) on 18 Aug., 2010 and was assigned accession number KCCM11094P.
Example 5
Examination of Conditions for Culture of Transformant Saccharomyces cerevisiae YPH499 (pGcyaDak, pGup1a Ws.Dgat Cas)
In order to examine the synthesis of fatty acid ethyl esters caused by the addition of sodium oleate (short chain fatty acid) during the culture of the transformant Saccharomyces cerevisiae YPH499 (pGcyaDak, pGup1a ws.dgat cas), the synthesis of ethanol and fatty acid ester in each of media (0.67% yeast nitrogen base, 2% glycerol, 0.067% yeast nitrogen base w/o trp, 0.2% galactose, 2% agar) containing or not containing sodium oleate was measured. The constructed transformant was cultured for 96 hours in each of a medium containing 0.1% sodium oleate and a medium not containing sodium oleate, and was then analyzed by GC-MS.
The analysis results are shown in FIG. 4, which shows the results of analysis of the components of the produced fatty acid ethyl esters by GC-MS. The results in FIG. 4 indicate that the most major component of the fatty acid ethyl esters was ethyl oleate and that fatty acid ethyl esters were successfully produced in the transformant constructed according to the present invention.
Example 6
Production of Fatty Acid Ethyl Esters in Transformant Saccharomyces cerevisiae YPH499 (pGcyaDak, pGup1a Ws.Dgat Cas)
Each of YPH499 (pESC-TRP), YPH499 (pGcyaDak, pGupCas) and YPH499 (pGcyaDak, pGup1a ws.dgat cas) was precultured for 24 hours in an SG medium (0.67% yeast nitrogen base, 2% glucose, 0.067% yeast nitrogen base w/o trp, 0.2% galactose) inducing expression by galactose, and was then shake-cultured for 48 hours in a fermentation medium (0.67% yeast nitrogen base, 2% glycerol, 0.067% yeast nitrogen base w/o trp, 0.2% galactose) containing 2% glycerol as a substrate, until the absorbance of the medium at a wavelength of 600 nm reached 1. The fermentation broth was cultured in each of media containing sodium oleate and not containing sodium oleate, at 30° C. and 100 rpm, while the culture broth was sampled at various time points and subjected to gas chromatography, thereby measuring the amount of ethanol produced. Also, the production of fatty acid ethyl esters (oleic acid ethyl ester) was measured by TLC (FIG. 5 a). It was found that, when the strains were cultured so as to produce fatty acid ethyl esters, the synthesis of fatty acid ethyl esters was possible in the medium containing 0.1% sodium oleate. The measurement results are shown in FIG. 5b. FIG. 5 shows the dependence of the transformant of the present invention on oleate. Specifically, FIG. 5a shows that, when no oleate was added during culture, fatty acid ethyl esters were not produced, and FIG. 5b shows that, when oleate was added, fatty acid ethyl esters were successfully produced by efficiently using ethanol produced in the transformant of the present invention.

## Claims

1. A transformant that is Saccharomyces cerevisiae YPH499 (pGcyaDak, pGup1a ws.dgat cas) (accession number: KCCM11094P).

2. A method for preparing a transformant for producing fatty acid ethyl esters, the method comprising the steps of:
constructing a recombinant vector comprising a gene encoding ws/dgat (wax ester synthase/acyl-coenzyme A: diacylglycerol acyltransferase) and a glycerol uptake protein gene;
constructing a recombinant vector comprising a glycerol dehydrogenase gene and a dihydroxyacetone kinase gene; and
transforming Saccharomyces cerevisiae YPH499 with the recombinant vectors, wherein the transformant is Saccharomyces cerevisiae YPH499(pGcyaDak, pGup1 a ws.dgat cas) (accession number: KCCM11094P).

3. A method for producing fatty acid ethyl esters, comprising a step of culturing the transformant of claim 1 using glycerol as a substrate.

4. The method of claim 3, wherein the glycerol is a byproduct of biodiesel production.

5. The method of claim 3 wherein a fatty acid salt having 12 to 20 carbon atoms is added during the culturing step.

6. The method of claim 3, wherein the culturing step is carried out at a temperature ranging from 25° C. to 30° C.