KR101492402B1 - Method for increasing total fatty acid content and useful fatty acid ratio in transgenic microalgae overexpressing FAB2 gene - Google Patents

Abstract

The present invention provides a method of overexpressing a gene encoding a microarray-derived stearoyl-acyl carrier protein desaturase (FAB2) protein in microalgae to increase total fatty acid content and useful fatty acid ratio in microalgae and a method for producing biodiesel .

Description

A method for increasing total fatty acid content and useful fatty acid ratio in transgenic microalgae overexpressing FAB2 gene (method for increasing total fatty acid content and useful fatty acid ratio in transgenic microalgae overexpressing FAB2 gene)

The present invention relates to a method for increasing total fatty acid content and useful fatty acid ratio in transgenic microalgae overexpressing FAB2 gene, and more particularly, to a method for increasing the total fatty acid content and useful fatty acid ratio of a gene encoding a microarray-derived stearoyl-acyl carrier protein desaturase (FAB2) To a method for increasing total fatty acid content and useful fatty acid ratio in microalgae, and a method for producing biodiesel.

As renewable energy, biodiesel is currently produced using animal fats, waste cooking oil and vegetable oil. However, this method can not meet the demand of biodiesel. Microalgae is an attractive biodiesel production raw material because it is highly productive compared to oil production using crops. However, the weakness of oil produced by microalgae has the disadvantage that it contains polyunsaturated fatty acids, which makes it difficult to store because of its poor oxidative stability (Chisti et al., 2007 Biotechnol Adv. , 25 (3), 294-306). In order to increase the oxidation stability of oil produced from microalgae, it is necessary to increase the ratio of oleic acid and linoleic acid, which are unsaturated fatty acids and relatively high oxidation stability among total fatty acids.

Fatty acid desaturase plays a role in producing double bonds in fatty acids by removing two hydrogen atoms of fatty acid. This process occurs in pigment and endoplasmic reticulum in higher plants. Several different types of fatty acid unsaturation enzymes are known to be present in higher plant genomes and the higher plant FAB2 (stearoyl-acyl carrier protein desaturase; otherwise known as SSI2) contains saturated stearic acid (18: 0) (18: 1), which is an unsaturated oleic acid. In Arabidopsis, the accumulation of oleic acid was decreased and the accumulation of stearic acid, a precursor of oleic acid, was increased in transformants inhibiting FAB2 expression in Arabidopsis. These results demonstrate that FAB2 is the main enzyme for the synthesis of oleic acid. However, changes in fatty acid composition could not be observed in plants overexpressing FAB2 (Kachroo et al., 2007 Plant Mol Biol , 63 (2), 257-271).

Chlamydomonas reinhardtii, a microalgae used in the present invention, has been utilized as a research model for fatty acid synthesis pathways and fatty acid biosynthesis is performed in chloroplasts and endoplasmic cells as well as in higher plants. Also, genes that are thought to be orthologs of the fatty acid biosynthesis-related genes known in higher plants have been reported to be present in the clamidomonas genome (Chi et al., 2011 Plant Mol Biol Rep , 29 , 769-783).

U.S. Patent Publication No. 2004-0049807 discloses fatty acid desaturase genes and proteins that regulate the activation of the plant defense signaling pathway, Korean Patent Publication No. 2011-0125576 discloses a lipid-producing microalgae Clamidomonas cell line and its use have been disclosed, but a method of mass-producing fatty acids using the transformed microalgae using the FAB2 gene as in the present invention has not been disclosed.

The present invention has been made in view of the above-described needs. The present inventors prepared a vector for transformation by cloning the FAB2 gene of microalgae, Clamidomonas, and introduced it into Clamidomonas and overexpressed the FAB2 gene, Which is important for the production of biodiesel using oleic acid and linoleic acid, and the increase of the composition of palmitic acid. And confirming that the total fatty acid content was increased, thereby completing the present invention.

In order to solve the above problems, the present invention provides a method for increasing the total fatty acid content and the useful fatty acid ratio in a microalgae including overexpressing a gene encoding a microarray-derived stearoyl-acyl carrier protein desaturase (FAB2) .

The present invention also relates to a method for producing a microalgae, which comprises transforming microbial algae with a recombinant vector containing a gene encoding a microalgae-derived FAB2 protein, thereby over-expressing the FAB2 protein coding gene, Thereby providing a method for producing the increased transformed microalgae.

The present invention also provides a transformed microalgae having increased total fatty acid content and useful fatty acid ratio as compared to wild-type microalgae produced by the above method.

The present invention also provides a composition for increasing total fatty acid content and useful fatty acid content in microalgae, which comprises a gene encoding a microalgae-derived FAB2 protein.

The present invention also provides a method for producing biodiesel comprising the step of overexpressing a gene encoding a microalgae-derived FAB2 protein in a microalgae to produce a fatty acid in a microalgae.

The present invention provides a novel use of the FAB2 gene capable of controlling the fatty acid biosynthetic pathway of all microalgae. The microalgae transformed with this gene have an increased total fatty acid content. In particular, oleic acid, which is an unsaturated fatty acid and has a relatively high oxidation stability, Linoleic acid, and palmitic acid, and thus can be very useful as microalgae for producing biodiesel excellent in quality / quantity.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of a microalgae transformation vector containing the FAB2 gene of the present invention. FIG. CrFAB2 represents the cDNA of the gene encoding the FAB2 protein derived from Cladidomonas reinhardtii.
Figure 2 shows the insertion of a transfection vector in the transformed microalgae of the present invention. Southern blot analysis using the DNA of the transformed microalgae indicates that a transfection vector has been introduced. Indicating that the transformed lines have one or more CrFAB2 DNA fragments of different sizes compared to the control cc-125. (transfection line), CrFAB2-ov-3 (transfection line), CrFAB2-ov-2 (transfection line)
FIG. 3 shows the results of RT-PCR (reverse transcription PCR) analysis of the expression of mRNA of the introduced FAB2 gene. a is a photograph showing mRNA of FAB2 expressed in the transfection vector. It is not expressed in the control cc-125 but it is expressed in the transformation line. Tub-α shows that the same amount of cDNA was used for each lane. b shows quantitatively the amount of mRNA expression of whole FAB2 in the cells, showing that the transformed microalga line has a higher expression level of FAB2 than the control. (transformation line), CrFAB2-ov-3 (transformation line), CrFAB2-ov-3 (transformation line)
Fig. 4 shows changes in the fatty acid composition ratio and the total amount of fatty acids of the transformants into which FAB2 was introduced according to the present invention. a shows the fatty acid composition ratio of the transformant, showing that the composition of oleic acid, linoleic acid and palmitic acid is increased. b shows the total fatty acid content of the transformants in which FAB2 was inserted, indicating that the total fat mass of the three transfection lines was increased by 28% on average.

In order to accomplish the object of the present invention, the present invention provides a method for producing microalgae, comprising the step of overexpressing a gene encoding a microarray-derived stearoyl-acyl carrier protein desaturase (FAB2) protein in a microalgae, Is increased.

In the method according to one embodiment of the present invention, the FAB2 protein may be derived from microalgae, preferably from the genus Chlamydomonas , more preferably from Chlamydomonas reinhardtti ), most preferably, but not exclusively, the amino acid sequence of SEQ ID NO: 2.

In addition, the gene encoding the FAB2 protein of the present invention may comprise the nucleotide sequence of SEQ ID NO: 1. Variants of the above base sequences are also included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 . "% Of sequence homology to polynucleotides" is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

In the method according to one embodiment of the present invention, the microalgae is selected from the group consisting of chlorella ellipsoidadea ellipsoidea), Chlorella Thoreau Kearney Ana (Chlorella sorokiniana) and Chlorella vulgaris (Chlorella Chlorella living in sea water and fresh water, such as vulgaris), Chlamydomonas (Chlamydomonas), see carboxamide (Volvox), Kane ATO three Ross (Cheatoceros), L ohdak tilrum (Phaeodactylum), skeletal rekto nematic (Skelectonema), butterfly Kula (Navicula ), Carlo Naples three (Caloneise), Chemnitz Skiathos (Nitzschia), Tala Please Syrah (Thalassiosira), amphorae (Amphora), nanno keulroriseu (Nannochloris), nanno claw Rob sheath (Nannochloropsis), tetra-cell Miss (Tetraselmis), based Canary Ella (Dunaliella), Spirulina (Spirulina), micro between seutiseu (Microcystis), come la thoria (Oscillatoria), tricot des US Taunus (Tricodesminus), isopropyl keurioh sheath (Isochryosis), Pavlova (Dinophyseae a (Pavlova) or Dino pisae ), Preferably a chlamydomonas spp., More preferably a Chlamydomonas reinhardtii , but is not limited thereto Do not.

In the method according to an embodiment of the present invention, the useful fatty acid may be oleic acid or linoleic acid, but is not limited thereto.

The present invention also relates to a method for producing a microalgae comprising the step of over-expressing a FAB2 protein coding gene by transforming a microalgae with a recombinant vector comprising a gene encoding a microarray-derived stearoyl-acyl carrier protein desaturase (FAB2) The present invention provides a method for producing transformed microalgae having increased total fatty acid content and useful fatty acid ratio.

The FAB2 protein, microalgae and useful fatty acids in the method for producing the transformed microalgae have increased total fatty acid content and useful fatty acid ratio compared to the wild-type microalgae of the present invention, as described above.

The term "recombinant" refers to a cell in which a cell replicates a new, heterologous or homologous recombinant nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

The term "vector" is used to refer to a DNA fragment (s), nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA, and the expression cassette (promoter, enhancer, termination signal, and polyadenylation signal) in the host cell can be independently regenerated. The term "carrier" is often used interchangeably with "vector ". The term "expression vector" means a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotic cells are known.

The expression vector will preferably comprise one or more selectable markers. The marker is typically a nucleic acid sequence having a property that can be selected by a chemical method, and includes all genes capable of distinguishing a transformed cell from a non-transformed cell. Examples include, but are not limited to, antibiotic resistance genes such as Kanamycin, G418, Bleomycin, hygromycin, chloramphenicol, paromomycin.

In the recombinant vector of the present invention, the promoter is selected from the group consisting of a pHsp70A gene designating a thermal shock protein of Clamidomonas, a small subunit gene rbcS2 of ribulose-5-phosphate synthase, a beta-tubulin gene And promoters of the pSaD gene constituting the photosystem I complex, but are not limited thereto. The term "promoter " refers to the region of DNA upstream from the structural gene and refers to a DNA molecule to which an RNA polymerase binds to initiate transcription. A "microalgae promoter" is a promoter capable of initiating transcription in microalgae cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental conditions or cell differentiation. Constructive promoters may be preferred in the present invention because the choice of transformants can be made by various tissues at various stages. Thus, constitutive promoters do not limit selectivity.

In the recombinant vector of the present invention, a conventional terminator can be used. Examples of the terminator include a photosystem I subunit gene (PsaD) and a terminator of the Octopine gene of Agrobacterium tumefaciens. But is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in microalgal cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

The present invention also provides a transformed microalgae having increased total fatty acid content and useful fatty acid ratio as compared to wild-type microalgae produced by the above method.

Since microalgae have similar characteristics to plants, they can be transformed into microbial algae by particle bombardment, electroporation, glass beads or silicon fibers. Agrobacterium, Agrobacterium, etc. Preferably, microorganism protoplasts are isolated and mixed with a vector, followed by transformation by glass bead agitation. However, , But is not limited thereto.

In the transgenic microalga according to an embodiment of the present invention, the microalgae may be preferably a Chlamydomonas spp., And more preferably, it may be a clamidomonas hardhide, but is not limited thereto.

In the transgenic microalgae according to one embodiment of the present invention, the useful fatty acid may be oleic acid or linoleic acid, but is not limited thereto.

In addition, the present invention provides a composition for increasing total fatty acid content and useful fatty acid content in microalgae, comprising a gene encoding a microarray-derived stearoyl-acyl carrier protein desaturase (FAB2) protein.

The composition comprises an active ingredient derived from microalgae, preferably from the genus Chlamydomonas , more preferably from Chlamydomonas reinhardtii , most preferably from the amino acid sequence of SEQ ID NO: 2 And a gene coding for FAB2 protein. By transforming the gene into microalgae, the total fatty acid content and the ratio of the useful fatty acid can be increased in the microalgae.

In addition,

Overexpressing a gene encoding a microalgae-derived FAB2 (stearoyl-acyl carrier protein desaturase) protein in a microalgae to produce a fatty acid in a microalgae; And

And converting the produced fatty acid into biodiesel. The present invention also provides a method for producing biodiesel.

In the process according to one embodiment of the invention, the FAB2 proteins derived from microalgae, preferably from Chlamydomonas (Chlamydomonas) in origin, and more preferably from Chlamydomonas lane hard Ti (Chlamydomonas reinhardtti ), most preferably the amino acid sequence of SEQ ID NO: 2.

In the method according to one embodiment of the present invention, the method of converting the produced fatty acid into biodiesel can use any method known in the art, and is not particularly limited to a specific method.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example  One: FAB2  Securing genes

The cDNA of the microalgae, Chlamydomonas reinhardtii , was extracted and amplified by PCR to obtain FAB2 cDNA corresponding to SEQ ID NO: 1. The RNA extraction method is as follows. Total RNA was isolated from the transformed clamidomonas using TRI solution (Molecular Research Center, Cincinnati, OH, USA) and 2 ug of RNA was used for cDNA synthesis.

Example  2: FAB2  Production of transgenic vector with gene

PCr102 (Kim et al., 2011 J Phycol, 47 (4), 821-828) in which 1206 bp FAB2 DNA obtained by simultaneously digesting the FAB2 gene of Example 1 with restriction enzymes NcoI and EcoRV was simultaneously digested with NcoI and EcoRV, Vector to prepare a clamidomonas transformant vector pCr102-FAB2 (Fig. 1).

Example  3: Microalgae Clamidomonas FAB2  Transformation of genes

The Chlamydomonas reinhardtti ) wild-type strain CC-125 was cultured in TAP-acetate-phosphate at 25 ° C for 16 hours for 3 days and used for transformation. For transformation, pCr102-FAB2 vector and clomidomonas protoplasts isolated by autolysin treatment were mixed and transformed with glass beads method (Kindle, 1990 Proc Natl Acad Sci USA, 87 (3), 1228-1232) Respectively. After transformation, lines with hygromycin resistance were selected. In order to confirm the introduction of the pCr102-FAB2 vector of the antibiotic resistant line, the Southern blot analysis method was performed to select the line into which the vector was inserted. Standard methods were used for genomic DNA isolation, PCR, electrophoresis, and Southern blot analysis (Sambrook and Russell 2001, Cold Spring Harbor: Cold Spring Harbor Laboratory Press). 10 [mu] g of genomic DNA isolated from the transformed clamidomonas was treated with KpnI restriction enzyme and used for Southern blot analysis. The FAB2 gene was amplified by PCR and labeled with ³² P and used as a probe. The probe signal was analyzed using an imaging plate (Fujifilm, Japan). As a result, various copies of the FAB2 gene were inserted in each line (FIG. 2). RT-PCR was performed to confirm the expression of the FAB2 gene in the transformation line. RNA extraction and cDNA synthesis were carried out in the same manner as described in Example 1 above. It was confirmed that the FAB2 mRNA derived from the pCr102-FAB2 vector introduced from the transformant was expressed normally (Fig. 3A), and the total expression amount of the FAB2 mRNA of the transformant was higher than that of the control (cc-125) ).

Example  4. From Clermido Monas FAB2  Functional testing of genes

No studies have been performed on FAB2 in Clamidomonas. Once the increase in oleic acid in the clomidomonas FAB2 overexpressing transformant is confirmed, it can be considered that the climidomonas FAB2 genetic material catalyzes the synthesis of oleic acid. Therefore, in this example, GC analysis was performed to extract total fat from the transformed Clamidomonas and to observe changes in fatty acid composition (FIG. 4). Total lipid separation was performed using 20 mg of lyophilized sample and the extraction method was Sasser (Sasser 1990 USFCC Newsletter, 20 , 1-6). The analysis equipment is as follows; Gas chromatography (YL-6100GC, Younglin Sci., Korea), INNOWAX capillary column (Agilent, USA, 30 m × 0.32 mm × 0.5 μm). Each fatty acid was analyzed in comparison with the standard Supelco 37 Component FAME Mix (Sigma, USA). Increase in oleic acid was observed in all three transformation lines used in the analysis and increase in palmitic acid and linoleic acid was observed (FIG. 4A). An increase in total fatty acid content was also observed (Figure 4b). These results demonstrate that FAB2 is involved in the synthesis of oleic acid in microalgae, Clamidomonas, and it also demonstrates that this gene is an important gene involved in the regulation of palmitic acid, linoleic acid and total fatty acid content.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Method for increasing total fatty acid content and useful fatty          acid ratio in transgenic microalgae overexpressing FAB2 gene <130> PN12149 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 1206 <212> DNA <213> Chlamydomonas reinhardtti <400> 1 atggctctgg gccagcaggc gatgcagcgc aagggcgccc ttaacgccaa cagggcctcg 60 cgcaaggctt gcgtcgtccg cgcgcaggcg gttgcctcgg ctccccaaca gccggccact 120 gcttcgcagt atgttcctca cgtccagggc ccgatcatca tgaatggtca ggtgctgcac 180 agcatcacgg ctgagcgcct ggatgtggtg cgcagcctgg aggacggcta cctgcagagc 240 caggtggtgc ctctgctgaa gcccgtggag aagtgctggc agcccgccga cttcctgccg 300 ccctcggagg accccgactt cctggacaag gtgcgcgagc tgcgcaagcg cgctgccaac 360 ctgcccgatg actacctggt ggtcttcacc ggcgacatga tcaccgagga ggcgctgccc 420 acctacatga ccatgctcaa caccctggac ggcgttcgcg atgagaccgg cgccagccag 480 accccctggg ccaagtggac gcgcgagtgg accgccgagg agaaccgcca cggcgacgtc 540 atgaaccgct acatgtacct gactggccgc gtcaacatga aggcggtgga ggtgaccgtg 600 cagaacctga ttggctccgg catggacccc aagaccgaga acaaccccta cctgggcttc 660 tgctacacct ccttccagga gcgcgccacc aaggtgtccc acggcaacac cgcccgccac 720 gctctggagc acggcgacga cgtgctggcc aagatctgcg gctccatcgc ctcggacgag 780 ggccgccacg agatcgccta ctgcaagatc atggacggcc tgtttgagcg cgaccccagc 840 ggcgccatga ttgcgttcgg cgacatgatg aagaagcaga tcgtgatgcc cgcccacctc 900 atgaacgaca acgtgcacca cgccaacacc ggccgcaacc tgttcgcgga cttctccgct 960 gtggctgaga acacgggcac ctacaccgcc atggactacg ctgacatcat ggagcacctg 1020 gtgggccgct ggaacgtcaa gaacctgacc ggcctcaacg gcgacgccgc cgccatgcag 1080 gagtacgtca tcaagctgcc cgaccgcatc cgcaagctgg cggagaaggc caccgcccgc 1140 cggaagaagg gcaaggtcgt gcacgcgccc ttcagctggg tgttcaaccg cgaggtggcc 1200 ctgtaa 1206 <210> 2 <211> 401 <212> PRT <213> Chlamydomonas reinhardtti <400> 2 Met Ala Leu Gly Gln Gln Ala Met Gln Arg Lys Gly Ala Leu Asn Ala   1 5 10 15 Asn Arg Ala Ser Arg Lys Ala Cys Val Val Arg Ala Gln Ala Val Ala              20 25 30 Ser Ala Pro Gln Gln Pro Ala Thr Ala Ser Gln Tyr Val Pro His Val          35 40 45 Gln Gly Pro Ile Ile Met Asn Gly Gln Val Leu His Ser Ile Thr Ala      50 55 60 Glu Arg Leu Asp Val Val Arg Ser Leu Glu Asp Gly Tyr Leu Gln Ser  65 70 75 80 Gln Val Val Pro Leu Leu Lys Pro Val Glu Lys Cys Trp Gln Pro Ala                  85 90 95 Asp Phe Leu Pro Pro Ser Glu Asp Pro Asp Phe Leu Asp Lys Val Arg             100 105 110 Glu Leu Arg Lys Arg Ala Asn Leu Pro Asp Asp Tyr Leu Val Val         115 120 125 Phe Thr Gly Asp Met Ile Thr Glu Glu Ala Leu Pro Thr Tyr Met Thr     130 135 140 Met Leu Asn Thr Leu Asp Gly Val Arg Asp Glu Thr Gly Ala Ser Gln 145 150 155 160 Thr Pro Trp Ala Lys Trp Thr Arg Glu Trp Thr Ala Glu Glu Asn Arg                 165 170 175 His Gly Asp Val Met Asn Arg Tyr Met Tyr Leu Thr Gly Arg Val Asn             180 185 190 Met Lys Ala Val Glu Val Thr Val Gln Asn Leu Ile Gly Ser Gly Met         195 200 205 Asp Pro Lys Thr Glu Asn Asn Pro Tyr Leu Gly Phe Cys Tyr Thr Ser     210 215 220 Phe Gln Glu Arg Ala Thr Lys Val Ser His Gly Asn Thr Ala Arg His 225 230 235 240 Ala Leu Glu His Gly Asp Asp Val Leu Ala Lys Ile Cys Gly Ser Ile                 245 250 255 Ala Ser Asp Glu Gly Arg His Glu Ile Ala Tyr Cys Lys Ile Met Asp             260 265 270 Gly Leu Phe Glu Arg Asp Pro Ser Gly Ala Met Ile Ala Phe Gly Asp         275 280 285 Met Met Lys Lys Gln Ile Val Met Pro Ala His Leu Met Asn Asp Asn     290 295 300 Val His His Ala Asn Thr Gly Arg Asn Leu Phe Ala Asp Phe Ser Ala 305 310 315 320 Val Ala Glu Asn Thr Gly Thr Tyr Thr Ala Met Asp Tyr Ala Asp Ile                 325 330 335 Met Glu His Leu Val Gly Arg Trp Asn Val Lys Asn Leu Thr Gly Leu             340 345 350 Asn Gly Asp Ala Ala Met Gln Glu Tyr Val Ile Lys Leu Pro Asp         355 360 365 Arg Ile Arg Lys Leu Ala Glu Lys Ala Thr Ala Arg Arg Lys Lys Gly     370 375 380 Lys Val Val His Ala Pro Phe Ser Trp Val Phe Asn Arg Glu Val Ala 385 390 395 400 Leu    

Claims (12)

A method for increasing total fatty acid content and useful fatty acid ratio in microalgae comprising over-expressing a gene encoding a stearoyl-acyl carrier protein desaturase (FAB2) protein derived from Chlamydomonas reinhardtii in a microalgae . 2. The method according to claim 1, wherein the FAB2 protein comprises the amino acid sequence of SEQ ID NO: 2. The method according to claim 1, wherein the microalgae are of the genus Chlamydomonas . 2. The method of claim 1, wherein the microalgae are of the genus Chlamydomonas . The method of claim 3, wherein the microalgae is Chlamydomonas reinhardtii. The method of claim 1, wherein the microalgae are Chlamydomonas reinhardtii. The method according to claim 1, wherein the useful fatty acid is oleic acid or linoleic acid, wherein the total fatty acid content and the ratio of the useful fatty acid are increased in the microalgae. Comprising the step of transfecting a microalgae with a recombinant vector comprising a gene encoding a stearoyl-acyl carrier protein desaturase (FAB2) protein derived from Chlamydomonas reinhardtii to overexpress the FAB2 protein coding gene A method for producing transformed microalgae having increased total fatty acid content and useful fatty acid ratio compared to transgenic microalgae. Transgenic microalgae having increased total fatty acid content and useful fatty acid ratio compared to non-transformed microalgae produced by the method of claim 6. The transformed microalgae of claim 7, wherein the microalgae are of the genus Chlamydomonas . The transformed microalgae of claim 8, wherein the microalgae are Chlamydomonas reinhardtii. The transformed microalgae according to claim 7, wherein the useful fatty acid is oleic acid or linoleic acid. A composition for increasing total fatty acid content and useful fatty acid content in microalgae, comprising a gene encoding a stearoyl-acyl carrier protein desaturase (FAB2) protein derived from Chlamydomonas reinhardtii. Overexpressing a gene encoding a stearoyl-acyl carrier protein desaturase (FAB2) protein derived from Chlamydomonas reinhardtii in a microalgae to produce a fatty acid in a microalgae; And
And converting the produced fatty acid to biodiesel.

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