Abstract:
The present disclosure discloses a transformed  Synechococcus elongatus  strain capable of producing biodiesel directly from carbon dioxide and a method for producing biodiesel and a method for removing carbon dioxide using the same. In an aspect, the transformed  Synechococcus elongatus  strain of the present disclosure can produce biodiesel in large scale using carbon dioxide as a carbon source. The  Synechococcus elongatus  strain is environment-friendly because it can be used to remove or reduce carbon dioxide in the atmosphere. The strain of the present disclosure is advantageous in that it can produce biodiesel in large scale because it grows faster and exhibits excellent carbon dioxide fixation capability as compared to other photosynthetic microorganisms.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    This application claims priority to Korean Patent Application No. 10-2015-0147216, filed on Oct. 22, 2015, and all the benefits accruing therefrom under 35 U.S.C. §119, the contents of which in its entirety are herein incorporated by reference. 
       DESCRIPTION ABOUT NATIONAL SUPPORT RESEARCH AND DEVELOPMENT 
       [0002]    This study is made by the support of Development of Technique for Climate Change Response Business of the Korean Ministry of Science, ICT and Future Planning under the supervision of the Korea Institute of Science and Technology, and the subject name thereof is Development of original technology of using recombinant cyanobacteria for continuous direct production of biodiesel (2N38970) (Subject Identification No.: 2014049277). 
       BACKGROUND 
       [0003]    1. Field 
         [0004]    The present disclosure relates to a transformed  Synechococcus elongatus  strain capable of producing biodiesel directly from carbon dioxide and a method for producing biodiesel and a method for removing carbon dioxide using the same. 
         [0005]    2. Description of the Related Art 
         [0006]    With the crisis of depletion of limited energy resources due to the mass consumption of fossil fuels, oil prices remain high consistently and the problems of global warming and environmental pollution persist. For this reason, researches are actively ongoing worldwide on various types of energy sources that can replace fossil fuels. Many researches and efforts are being made also in Korea, which is heavily dependent on imports of energy resources. Until now, the development of bioenergy as a renewable alternative energy source has been quite successful. In particular, biodiesel which can be produced from recyclable animal or plant oil is drawing attentions as a clean alternative fuel. 
         [0007]    Biodiesel is mostly produced from vegetable oils such as soybean oil, palm oil, etc. However, Korea relies mainly on imported soybean oil or palm oil, as the raw materials of biodiesel, and their prices are high and unstable because they are food-related resources greatly affected by the change in international environment. In this situation, Korea&#39;s energy security is threatened not only be petroleum but also by alternative fuels and, therefore, it is urgently needed to provide measures to utilize inexpensive raw materials or domestic resources. 
       SUMMARY 
       [0008]    In an aspect, the present disclosure is directed to providing a  Synechococcus elongatus  strain having capability of producing biodiesel. 
         [0009]    In another aspect, the present disclosure is directed to producing biodiesel by an environment-friendly method using microorganisms. 
         [0010]    In another aspect, the present disclosure is directed to producing biodiesel in large scale using the  Synechococcus elongatus  strain. 
         [0011]    In another aspect, the present disclosure is directed to producing biodiesel while removing carbon dioxide in the atmosphere. 
         [0012]    In an aspect, the present disclosure provides a  Synechococcus elongatus  strain containing: a gene encoding an enzyme which produces acetaldehyde from pyruvate; a gene encoding an enzyme which produces ethanol from acetaldehyde; and gene encoding an enzyme which produces biodiesel from acyl-coenzyme A and ethanol. 
         [0013]    In another aspect, the present disclosure provides a method for producing biodiesel, which includes a step of culturing the  Synechococcus elongatus  strain. 
         [0014]    In another aspect, the present disclosure provides a method for removing carbon dioxide, which includes a step of culturing the  Synechococcus elongatus  strain. 
         [0015]    In an aspect, the transformed  Synechococcus elongatus  strain of the present disclosure can produce biodiesel in large scale using carbon dioxide as a carbon source. The  Synechococcus elongatus  strain can economically produce biodiesel using carbon dioxide present in the atmosphere as a carbon source and the produced biodiesel is excreted extracellularly and can be used conveniently without an additional process. In addition, the present disclosure is environment-friendly because it can be used to remove or reduce carbon dioxide in the atmosphere using the microorganism. The strain of the present disclosure is advantageous in that it can produce biodiesel in large scale because it grows faster and exhibits excellent carbon dioxide fixation capability as compared to other photosynthetic microorganisms. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0016]      FIG. 1  schematically shows the fatty acid ethyl ester (FAEE) production pathway of a transformed  Synechococcus elongatus  strain. 
           [0017]      FIGS. 2A-2C  are schematics of vectors ( FIG. 2A : pSe2Bb1k-pdc,adh vector,  FIG. 2B : pSe1 Bb1 s-atfA vector,  FIG. 2C : pSe[FadE]Bb1c-tesA vector). 
           [0018]      FIGS. 3A-3B  show the cell growth ( FIG. 3A ) of and ethanol production ( FIG. 3B ) by a strain transformed with a pSe2Bb1k-pdc,adh vector. 
           [0019]      FIGS. 4A-4B  show the cell growth ( FIG. 4A ) of and ethanol production ( FIG. 4B ) by a strain transformed with a pSe2Bb1 k-pdc,adh vector and a pSe1Bb1s-atfA vector (indicated by squares) and a strain transformed with a a pSe2Bb1k-pdc,adh vector and a pSe1 Bb1 s-Ws2 vector (indicated by circles). 
           [0020]      FIGS. 5A-5B  show the fatty acid ethyl ester production by a strain transformed with a pSe2Bb1k-pdc,adh vector and a pSe1 Bb1 s-atfA vector ( FIG. 5A ) and a strain transformed with a pSe2Bb1k-pdc,adh vector and a pSe1Bb1s-Ws2 vector ( FIG. 5B ). 
           [0021]      FIGS. 6A-6B  show the cell growth ( FIG. 6A ) of and ethanol production ( FIG. 6B ) by a strain transformed with a pSe2Bb1k-pdc,adh vector, a pSe1Bb1s-atfA vector and a pSe[FadE]Bb1c-tesA vector (indicated by squares) and a strain transformed with a pSe2Bb1k-pdc,adh vector, a pSe1Bb1s-Ws2 vector and a pSe[FadE]Bb1c-tesA vector (indicated by circles). 
           [0022]      FIGS. 7A-7B  show the fatty acid ethyl ester production by a strain transformed with a pSe2Bb1 k-pdc,adh vector, a pSe1Bb1s-atfA vector and a pSe[FadE]Bb1c-tesA vector ( FIG. 7A ) and a strain transformed with a pSe2Bb1 k-pdc,adh vector, a pSe1Bb1s-Ws2 vector and a pSe[FadE]Bb1c-tesA vector ( FIG. 7B ). 
       
    
    
     DETAILED DESCRIPTION 
       [0023]    Hereinafter, the present disclosure is described in detail. 
         [0024]      Synechococcus elongatus  is a species of cyanobacteria. The prokaryotic cyanobacteria are useful in changing metabolic pathways or controlling metabolites artificially because genetic manipulation is easy. The inventors of the present disclosure have completed the present disclosure by utilizing these characteristics of cyanobacteria and synthetic biological/metabolic engineering techniques. 
         [0025]    In an aspect, the present disclosure provides a  Synechococcus elongatus  strain containing: a gene encoding an enzyme which produces acetaldehyde from pyruvate; a gene encoding an enzyme which produces ethanol from acetaldehyde; and gene encoding an enzyme which produces biodiesel from acyl-coenzyme A and ethanol. 
         [0026]    In the present disclosure, biodiesel may refer to an ester converted from a triglyceride, which is the main component of plant oil or animal fat, through reaction with an alcohol. For example, one glycerin molecule and three biodiesel molecules may be produced from the reaction of one triglyceride molecule and three alcohol molecules in the presence of a catalyst. For another example, biodiesel can be produced from the reaction of ethanol with acyl-coenzyme A. 
         [0027]    In an exemplary embodiment, the strain may further contain a gene encoding an enzyme which produces a free fatty acid from an acyl-acyl carrier protein (acyl-ACP). The produced free fatty acid may be used to produce biodiesel. 
         [0028]    In this aspect, the enzyme which produces acetaldehyde from pyruvate may be pyruvate decarboxylase, the enzyme which produces ethanol from acetaldehyde may be alcohol dehydrogenase and the enzyme which produces biodiesel from acyl-coenzyme A and ethanol may be wax-ester synthase. 
         [0029]    And, the enzyme which produces a free fatty acid from an acyl-acyl carrier protein (acyl-ACP) may be thioesterase. 
         [0030]    In the present disclosure, a module may refer to a functional unit having a particular function, such as a set of genes capable of expressing a particular gene. In the present disclosure, a module having capability of producing ethanol from pyruvate is referred to as a module A, a module having capability of producing a free fatty acid and acyl-coenzyme A from an acyl-acyl carrier protein is referred to as a module C and a module having capability of producing biodiesel from ethanol and acyl-coenzyme A is referred to as a module B. Accordingly, a gene encoding an enzyme which produces acetaldehyde from pyruvate and a gene encoding an enzyme which produces ethanol from acetaldehyde may be called genes constituting the module A, a gene encoding an enzyme which produces a free fatty acid from an acyl-acyl carrier protein (acyl-ACP) may be called a gene constituting the module C, and a gene encoding an enzyme which produces biodiesel from acyl-coenzyme A and ethanol may be called a gene constituting the module B. 
         [0031]    In this aspect, a gene encoding pyruvate decarboxylase may contain a sequence of SEQ ID NO: 1, a gene encoding alcohol dehydrogenase may contain a sequence of SEQ ID NO: 2, and a gene encoding wax-ester synthase may contain a sequence of SEQ ID NO: 3 or SEQ ID NO: 4. And, a gene encoding thioesterase may contain a sequence of SEQ ID NO: 5. 
         [0032]    For example, the gene encoding pyruvate decarboxylase may be derived from a pdc gene of  Zymomonas mobilis , the gene encoding alcohol dehydrogenase may be derived from an adh gene of  Zymomonas mobilis , and the gene encoding wax-ester synthase may be derived from an atfA gene of  Acinetobacter  sp. or a Ws2 gene of  Marinobacter hydrocarbonoclasticus  ATCC49840. These genes may be obtained by consulting the following literature: Microbial production of fatty-acid-derived fuels and chemicals from plant biomass (2010).  Nature,  463(7280), 559-562. Steen, E. J., Kang, Y., Bokinsky, G., Hu, Z., Schirmer, A., McClure, A., Del Cardayre SB &amp; Keasling, J. D. 
         [0033]    And, the gene encoding thioesterase may be derived from a tesA gene of  E. coll.    
         [0034]    pdc is a gene encoding pyruvate decarboxylase of  Zymomonas mobilis  strain, adh is a gene encoding alcohol dehydrogenase of  Zymomonas mobilis  strain, atfA of  Acinetobacter  sp. and Ws2 of  Marinobacter hydrocarbonoclasticus  ATCC 49840 are genes encoding wax-ester synthase, and tesA is a gene encoding thioesterase of  E. coli.    
         [0035]    In an exemplary embodiment, the sequence derived from pdc may be a sequence of SEQ ID NO: 1, the sequence derived from adh may be a sequence of SEQ ID NO: 2, the sequence derived from atfA may be a sequence of SEQ ID NO: 3, the sequence derived from Ws2 may be a sequence of SEQ ID NO: 4, and the sequence derived from tesA may be a sequence of SEQ ID NO: 5. 
         [0036]    These genes are codon-optimized for stable expression in the parent strain  Synechococcus elongatus.    
         [0037]    In an exemplary embodiment, the strain may be a  Synechococcus elongatus  strain transformed with a first vector containing a sequence derived from the pdc gene of  Zymomonas mobilis  and a sequence derived from the adh gene of  Zymomonas mobilis  and a second vector containing a sequence derived from the atfA gene of  Acinetobacter  or a sequence derived from the Ws2 gene of  Marinobacter hydrocarbonoclasticus . For example, the first vector may be represented by a pSe2Bb1 k-pdc,adh vector (SEQ ID NO: 6) and the second vector may be represented by a pSe1Bb1 s-atfA vector (SEQ ID NO: 7) or a pSe1Bb1 s-Ws2 vector (SEQ ID NO: 8). 
         [0038]    The sequence listing submitted together with the present disclosure is incorporated herein in its entirety. 
         [0039]    In another exemplary embodiment, the strain may be a  Synechococcus elongatus  strain transformed with the first vector and the second vector and further transformed with a third vector containing a sequence derived from the tesA gene of  E. coli . The third vector may be represented by a pSe[FadE]Bb1c-tesA vector (SEQ ID NO: 9). 
         [0040]    In all the vectors disclosed in the present disclosure, the genes are linked operably. “Operable” means that the target gene can be expressed normally. 
         [0041]    In this aspect, the strain may be a transformed  Synechococcus elongatus  strain wherein the first vector is inserted at the neutral site II (NSII) of the parent strain  Synechococcus elongatus  and the second vector is inserted at the neutral site I (NSI) of the parent strain  Synechococcus elongatus . Also, in this aspect, the third vector may be inserted at the FadE site of the parent strain  Synechococcus elongatus.    
         [0042]    In this aspect, the first vector may contain, in sequence, a kanamycin resistance gene, a lacI repressor, a trc promoter, the gene encoding pyruvate decarboxylase and the gene encoding alcohol dehydrogenase. 
         [0043]    Also, the second vector may contain, in sequence, a spectinomycin resistance gene, a lacI repressor, a trc promoter and the gene encoding wax-ester synthase. 
         [0044]    Also, the third vector may contain, in sequence, a chloramphenicol resistance gene, a lacI repressor, a trc promoter and the gene encoding thioesterase. The genes contained in the vectors are the same as described above. 
         [0045]    In an aspect, the first vector may contain a sequence of SEQ ID NO: 6 and the second vector may contain a sequence of SEQ ID NO: 7 or SEQ ID NO: 8. And, the third vector may contain a sequence of SEQ ID NO: 9. 
         [0046]    In an exemplary embodiment, the strain transformed with the vectors is derived from the parent strain  Synechococcus elongates  PCC7942 (ATCC 33912). 
         [0047]    In an aspect of the present disclosure, the biodiesel may be a fatty acid ethyl ester (FAEE), although not being limited thereto. In particular, the fatty acid ethyl ester may be hexadecanoic acid ethyl ester or octadecanoic acid ethyl ester, although not being limited thereto. 
         [0048]    In an exemplary embodiment, the strain of the present disclosure may be a  Synechococcus elongatus  strain with an accession number KCTC 12883BP or an accession number KCTC 12884BP. 
         [0049]    The strain with an accession number KCTC 12883BP may be a strain transformed the pSe2Bb1k-pdc,adh vector and the strain with an accession number KCTC 12884BP strain may be a strain transformed the pSe2Bb1k-pdc,adh vector, the pSe1 Bb1 s-atfA vector and the pSe[FadE]Bb1c-tesA vector. 
         [0050]    The strain of the present disclosure may absorb and fix carbon dioxide. 
         [0051]    In another aspect, the present disclosure provides a method for producing biodiesel, which includes a step of culturing the  Synechococcus elongatus  strain. 
         [0052]    In this aspect, the step of culturing the strain may include supplying carbon dioxide. 
         [0053]    In this aspect, the method may further include a step of separating and obtaining the biodiesel dissolved in a hydrophobic solvent. Any known hydrophobic solvent may be used without limitation and the biodiesel produced by the strain may be accumulated in the hydrophobic solvent. The  Synechococcus elongatus  strain of the present disclosure excretes the produced biodiesel extracellularly and the excreted biodiesel is dissolved in the hydrophobic solvent. 
         [0054]    In another aspect, the present disclosure provides a method for removing carbon dioxide, which includes a step of culturing the  Synechococcus elongatus  strain. 
         [0055]    Hereinafter, the present disclosure will be described in detail through examples. However, the following examples are for illustrative purposes only and it will be apparent to those of ordinary skill in the art that the scope of the present disclosure is not limited by the examples. 
       [Example 1] Establishment of Strategy for Producing Fatty Acid Ethyl Ester (FAEE) from  Synechococcus elongatus  PCC7942 Strain 
       [0056]    A new metabolic pathway from pyruvate to FAEE via glycolysis was designed referring to the literature (Steen et al., 2010). After codon optimization of the DNA sequences of the pdc gene and the adh gene of  Zymomonas mobilis , the atfA gene of  Acinetobacter  sp., the Ws2 gene of  Marinobacter hydrocarbonoclasticus  ATCC 49840 and the tesA gene of  E. coli , the sequences were synthesized by GenScript®.  Synechococcus elongatus  as a parent strain was transformed sequentially with three modules using a module in which the pdc and adh genes were introduced at the neutral site II (NSII) (module A), a module in which the atfA or Ws2 gene was introduced at the neutral site I (NSI) (module B) and a module in which the tesA gene was introduced at the FadE deletion site (module C) ( FIG. 1 ). 
       [Example 2] Construction of Strain Producing Fatty Acid Ethyl Ester (FAEE) 
     [Example 2-1] Construction of Vectors 
       [0057]    A pSe2Bb1k-GFP vector as a SyneBrick vector, a pUC57-pdc vector wherein the synthesized pdc gene was inserted and a pUC57-adh vector wherein the adh gene was inserted were prepared. In order to replace the GFP region of the SyneBrick vector with pdc and adh, respectively, GFP was removed using EcoRI/BamHI restriction enzymes and then each of the pdc and adh genes cleaved from pUC57-pdc and pUC57-adh using EcoRI/BamHI was inserted using a ligase. As a result, pSe2Bb1 K-pdc and pSe2Bb1 K-adh vectors were constructed. In order to insert the adh gene downstream of the pdc gene, the pSe2Bb1k-pdc vector was treated with BamHI-XhoI restriction enzymes and pSe2Bb1k-adh was treated with BglII-XhoI restriction enzymes. Then, the sequence of the adh gene was inserted downstream of the pdc gene using a ligase. As a result, a pSe2Bb1 k-pdc,adh vector (first vector) was constructed ( FIG. 2A ). 
         [0058]    Also, a pSe1Bb1 s-atfA vector and a pSe1Bb1 s-Ws2 vector (second vector) was constructed by removing the GFP region of pSe1Bb1s-GFP using EcoRI-BamHI restriction enzymes and then inserting the DNA sequence of the synthesized atfA or Ws2 gene ( FIG. 2B ). 
         [0059]    Finally, a pSe[FadE]Bb1c-tesA vector (third vector) was constructed by removing the RFP region of pSe[FadE]Bb1c-RFP using EcoRI-BamHI restriction enzymes and then inserting the DNA sequence of the synthesized tesA gene ( FIG. 2C ). 
         [0060]    All the genes inserted into these vector were constructed by GeneScript®. 
       [Example 2-2] Construction of Transformed Strains 
       [0061]    The constructed pSe2Bb1 k-pdc,adh vector was inserted at the neutral site II of a wild-type  Synechococcus elongatus  PCC7942 strain. Then, a strain in which the modules A and B are introduced together was constructed by inserting the pSe1Bb1 s-atfA or pSe1Bb1 s-Ws2 vector at the neutral site I of the strain in which the module A is introduced. Finally, a biodiesel-producing strain in which the modules A, B and C are introduced together was constructed by inserting the pSe[FadE]Bb1c-tesA vector at the FadE site of the strain in which the module A and B are introduced. 
       [Example 3] Fatty Acid Ethyl Ester (FAEE) Production Capability of Transformed Strains 
       [0062]    In order to investigate the capability of producing biodiesel of the transformed strains constructed in Example 2, the strains were cultured under the following conditions. Specifically, 100 mL of BG-11 medium containing 10 mM MOPS buffer was added to a 100-mL bottle and then the transformed strain was added after diluting to O.D 730 =0.6. Then, after adding 10 μg/mL spectinomycin, 5 μg/mL kanamycin and 5 μg/mL chloramphenicol, the strain was cultured in an incubator of 30° C. and 100 μE·m −2 ·s −1  while continuously supplying 5% CO 2 . After culturing for one day, the strain was treated with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducer necessary for gene expression and with 20% hexadecane to reduce cytotoxicity by the produced fatty acid ethyl ester (FAEE). After culturing for 7 days, optical density at 730 nm and the amount of the produced fatty acid ethyl ester in the hexadecane layer were measured. 
         [0063]    As a result, it was confirmed that up to 250 mg/L ethanol was produced by the transformed strain into which the pdc gene and the adh gene were introduced ( FIG. 3B ). For the strain into which the pdc, adh and atfA genes were inserted and the strain into which the pdc, adh and Ws2 genes were inserted, ethanol production was decreased at 100-150 mg/L and fatty acid ethyl ester (FAEE) peak was observed ( FIGS. 4A and 4B ). From mass spectroscopic analysis, the peak 1 was identified as hexadecanoic acid ethyl ester (C 18 H 36 O 2 ) and the peak 2 was identified as octadecanoic acid ethyl ester (C 20 H 40 O 2 ). Agilent 6890 and Leco&#39;s Time-Of-Flight mass spectrometer (LECO PEGASUSIII) were used for the analysis. 
         [0064]    The hexadecanoic acid ethyl ester peak increased remarkably for the strain into which the pdc, adh, atfA and tesA genes were inserted, and both the hexadecanoic acid ethyl ester (C 18 H 36 O 2 ) and octadecanoic acid ethyl ester (C 20 H 40 O 2 ) peaks increased for the strain into which the pdc, adh, Ws2 and tesA genes were inserted. 
         [0065]    [Accession Numbers] 
         [0066]    Depository authority: Korea Research Institute of Bioscience and Biotechnology 
         [0067]    Accession number: KCTC12883BP 
         [0068]    Date of deposition: 20150827 
         [0069]    Depository authority: Korea Research Institute of Bioscience and Biotechnology 
         [0070]    Accession number: KCTC12884BP 
         [0071]    Date of deposition: 20150827