Abstract:
The present specification discloses a transformed  Synechococcus elongatus  strain which may directly produce squalene from carbon dioxide, and a method for producing squalene and a method for removing carbon dioxide, using the same. In an aspect, the strain may produce squalene using carbon dioxide as a carbon source. The  Synechococcus elongatus  strain is economically efficient because a high-value added squalene is produced using light and carbon dioxide present in the atmosphere as a carbon source, and the method for producing squalene is eco-friendly because the strain may be utilized to remove or reduce carbon dioxide in the atmosphere by using microorganisms. The strain of the present disclosure may produce only squalene, which is a desired target material with high purity, and has an advantage in that squalene may be continuously mass-produced.

Description:
CROSS-REFERENCE TO RELATED APPLICATION 
       [0001]    This application claims priority to Korean Patent Application No. 10-2016-0041774, filed on Apr. 5, 2016, and all the benefits accruing therefrom under 35 U.S.C. §119, the contents of which in its entirety are herein incorporated by reference. 
       BACKGROUND 
     Field 
       [0002]    The present specification discloses a transformed  Synechococcus elongatus  strain which may mass-produce squalene from carbon dioxide, and a method for producing squalene and a method for removing carbon dioxide, using the same. 
       DESCRIPTION ABOUT NATIONAL SUPPORT RESEARCH AND DEVELOPMENT 
       [0003]    This study is made by the support of KOREA CCS 2020 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 (Subject Identification No.: 2015030270). 
       DESCRIPTION OF THE RELATED ART 
       [0004]    Squalene is a triterpenoid-based unsaturated hydrocarbon in which 30 carbon atoms and 50 hydrogen atoms are linked by 6 double bonds, and is usually contained in the human body and animal and vegetable fats and oils. Squalene has been used for various uses in the industries, such as an acid-fast functional supplement food, a cosmetic raw material, a vaccine support raw material, and a feed raw material. According to a report (Global Trends &amp; Forecasts to 2019), the market of squalene is expected to grow to 177.06 million dollars, the average annual growth rate of the market is 10.3%, and the market tends to grow every year. Squalene has been generally obtained by extraction from the liver of deep-water sharks, but this method is against animal protection policies, and squalene may be also extracted from vegetables, but the method for extracting squalene from vegetables is inefficient because wide lands are required for cultivation. Recently, starting from yeast strains, various attempts such as a method for producing squalene from microalgae, and the like have been continued, and methods using yeast need a lot of sugars during the production process, and thus are economically inefficient, and when microalgae are used, the limitation thereof is clear because other impurities in addition to squalene, which is a desired target material, are produced. Therefore, there is a need for studies on a method which may economically and stably mass-produce squalene. 
       SUMMARY 
       [0005]    In an aspect, an object of the present disclosure is to produce squalene by an eco-friendly method using microorganisms. 
         [0006]    In another aspect, an object of the present disclosure is to provide a  Synechococcus elongatus  strain having a capability of producing squalene. 
         [0007]    In still another aspect, an object of the present disclosure is to continuously mass-produce squalene using the  Synechococcus elongatus  strain. 
         [0008]    In yet another aspect, an object of the present disclosure is to produce squalene using carbon dioxide to be discarded as a carbon source. 
         [0009]    In an exemplary embodiment, the present disclosure provides a  Synechococcus elongatus  strain including: a gene encoding an enzyme producing 1-deoxy-D-xylulose 5-phosphate from pyruvate and D-glyceraldehyde 3-phosphate; a gene encoding an enzyme producing dimethylallyl diphosphate from isopentenyl diphosphate; a gene encoding an enzyme producing dimethylallyl diphosphate from isopentenyl diphosphate; a gene encoding an enzyme producing farnesyl diphosphate from dimethylallyl diphosphate; and a gene encoding an enzyme producing squalene from farnesyl diphosphate. 
         [0010]    In another exemplary embodiment, the present disclosure provides a method for preparing squalene, the method including: culturing the strain. 
         [0011]    In another exemplary embodiment, the present disclosure provides a method for removing carbon dioxide, the method including: culturing the strain. 
         [0012]    In an aspect of the present disclosure, a transformed  Synechococcus elongatus  strain may mass-produce squalene using carbon dioxide as a carbon source. The  Synechococcus elongatus  strain is economically efficient because a high-value added squalene is produced using light and carbon dioxide present in the atmosphere as a carbon source, and the method for producing squalene is eco-friendly because the strain may be utilized to remove or reduce carbon dioxide in the atmosphere by using microorganisms. The strain of the present disclosure may produce only squalene, which is a desired target material with high purity, and has an advantage in that squalene may be continuously mass-produced. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0013]      FIG. 1  is a view illustrating a production pathway for a transformed  Synechococcus elongatus  strain (CDP-ME is an acronym for 4-diphosphocytidyl-2-C-methyl-D-erythritol, CDP-MEP is an acronym for 4-diphosphocytidyl-2-C-methyl-D-erythritol-2-phosphate, ME-cPP is an acronym for 2-C-Methyl-D-erythritol-2,4-cyclopyrophosphate, and HMBPP is an acronym for 4-hydroxy-3-methyl-but-2-enyl pyrophosphate). 
           [0014]      FIG. 2 a    is a schematic view of a first vector according to an exemplary embodiment. 
           [0015]      FIG. 2 b    is a schematic view of a second vector according to an exemplary embodiment. 
           [0016]      FIG. 3  is a view illustrating the growth of a wide-type strain and a transformed strain. 
           [0017]      FIG. 4 a    is a view illustrating a gas chromatography result confirming that a material produced by the transformed strain is squalene. 
           [0018]      FIG. 4 b    is a photograph confirming squalene produced by the transformed strain by the unaided eye. 
           [0019]      FIG. 5  is a view illustrating a result confirming that the transformed strain has a capability of producing squalene. 
       
    
    
       [0020]    In  FIGS. 3 and 5 , WT means a wild-type strain, SeSC33S means a mutant strain of a  Synechococcus elongatus  strain, in which a pSe1Bb1s-dxs-idi-ispA vector at an NSI site and a pSe2Bb1k-sqs vector (including a squalene synthase gene derived from  Saccharomyces cerevisiae ) at an NSII site are transformed, and SeSC34S means a mutant strain of a  Synechococcus elongatus  strain, which is transformed with a pSe1 Bb1 s-dxs-idi-ispA vector at an NSI site and a pSe2Bb1k-sqs vector (including a squalene synthase gene derived from  Methylococcus capsulatus ) at an NSII site. Further, SeSC41S means a mutant strain of a  Synechococcus elongatus  strain, which is transformed with a pSe1Bb1s-dxs-dxr-idi-ispA vector at an NSI site and a pSe2Bb1k-sqs vector (including a squalene synthase gene derived from  Saccharomyces cerevisiae ) at an NSII site, and SeSC42S means a mutant strain of a  Synechococcus elongatus  strain, in which a pSe1Bb1s-dxs-dxr-idi-ispA vector at an NSI site and a pSe2Bb1k-sqs vector (including a squalene synthase gene derived from  Methylococcus capsulatus ) at an NSII site are transformed. 
       DETAILED DESCRIPTION 
       [0021]    Cyanobacteria are microorganisms which may produce energy through photosynthesis and fix carbon dioxide to produce metabolites. Cyanobacteria being prokaryotes are easy to be genetically modified compared to microalgae being eukaryotes, and thus are advantageous for altering metabolic pathways or artificially regulating metabolites. Recently, various biofuel substitutes or chemical products have been produced by introducing a synthetic biological/metabolic engineering technique based on the genetic modification technology to use metabolic pathways that have not existed. 
         [0022]    The present inventors genetically modified a  Synechococcus elongatus  strain, one of cyanobacteria, thereby constructing a new mutant strain which may directly produce a squalene material from carbon dioxide. 
         [0023]    Hereinafter, the present disclosure will be described in detail. 
         [0024]    In an aspect, the present disclosure is a  Synechococcus elongatus  strain including: a gene encoding an enzyme producing 1-deoxy-D-xylulose 5-phosphate (DXP) from pyruvate and D-glyceraldehyde 3-phosphate (G3P); 
         [0025]    a gene encoding an enzyme producing dimethylallyl diphosphate (DMAPP) from isopentenyl diphosphate (IPP); a gene encoding an enzyme producing farnesyl diphosphate (FPP) from dimethylallyl diphosphate (DMAPP); and a gene encoding an enzyme producing squalene from farnesyl diphosphate (FPP). 
         [0026]    The gene encoding the enzyme producing 1-deoxy-D-xylulose 5-phosphate (DXP) from pyruvate and D-glyceraldehyde 3-phosphate (G3P) may be a deoxyxylulose-5-phosphate synthase. Further, the gene encoding the enzyme producing dimethylallyl diphosphate (DMAPP) from isopentenyl diphosphate (IPP) may be an isopentenyl diphosphate delta isomerase. In addition, the enzyme producing farnesyl diphosphate (FPP) from dimethylallyl diphosphate (DMAPP) may be a geranyl diphosphate synthase, and the enzyme producing squalene from farnesyl diphosphate (FPP) may be a squalene synthase. 
         [0027]    The strain may mass-produce farnesyl diphosphate (FPP) which is a precursor of squalene, and thus may mass-produce squalene therefrom. The strain may synthesize squalene from two molecules of farnesyl diphosphate. 
         [0028]    For example, the gene encoding the enzyme producing 1-deoxy-D-xylulose 5-phosphate (DXP) from pyruvate and D-glyceraldehyde 3-phosphate (G3P) may be derived from  E. coli . Furthermore, the gene encoding the enzyme producing dimethylallyl diphosphate (DMAPP) from isopentenyl diphosphate (IPP) may be derived from  E. coli , and the gene encoding the enzyme producing farnesyl diphosphate (FPP) from dimethylallyl diphosphate (DMAPP) may also be derived from  E. coli . Meanwhile, the gene encoding the enzyme producing squalene from farnesyl diphosphate (FPP) may be derived from  Saccharomyces cerevisiae  or may be derived from  Methylococcus capsulatus.    
         [0029]    In an exemplary embodiment, the gene encoding the enzyme producing 1-deoxy-D-xylulose 5-phosphate (DXP) from pyruvate and D-glyceraldehyde 3-phosphate (G3P) may include a sequence of SEQ ID NO. 1. 
         [0030]    Further, the gene encoding the enzyme producing dimethylallyl diphosphate (DMAPP) from isopentenyl diphosphate (IPP) may include a sequence of SEQ ID NO. 2. In addition, in an exemplary embodiment, the gene encoding the enzyme producing farnesyl diphosphate (FPP) from dimethylallyl diphosphate (DMAPP) may include a sequence of SEQ ID NO. 3. 
         [0031]    In an exemplary embodiment, the gene encoding an enzyme producing squalene from farnesyl diphosphate (FPP) may include a sequence of SEQ ID NO. 4 or 5. The sequence of SEQ ID NO. 4 includes a squalene synthase gene derived from  Saccharomyces cerevisiae , and the sequence of SEQ ID NO. 5 includes a squalene synthase gene derived from  Methylococcus capsulatus.    
         [0032]    In an exemplary embodiment, the strain may further include a gene encoding an enzyme producing 2-C-methyl-D-erythritol-4-phosphate (MEP) from 1-deoxy-D-xylulose 5-phosphate (DXP). The gene encoding the enzyme producing 2-C-methyl-D-erythritol-4-phosphate (MEP) from 1-deoxy-D-xylulose 5-phosphate (DXP) may be derived from  E. coli . The gene encoding the enzyme producing 2-C-methyl-D-erythritol-4-phosphate (MEP) from 1-deoxy-D-xylulose 5-phosphate (DXP) may be a 1-deoxy-D-xylulose-5-phosphate reductase. Meanwhile, in an exemplary embodiment, the gene encoding the enzyme producing 2-C-methyl-D-erythritol-4-phosphate (MEP) from 1-deoxy-D-xylulose 5-phosphate (DXP) may include a sequence of SEQ ID NO. 6. 
         [0033]    In the present specification, the gene encoding an enzyme producing 1-deoxy-D-xylulose 5-phosphate (DXP) from pyruvate and D-glyceraldehyde 3-phosphate (G3P) is referred to as ‘dxs gene’. Furthermore, in the present specification, the gene encoding the enzyme producing dimethylallyl diphosphate (DMAPP) from isopentenyl diphosphate (IPP) is referred to as ‘idi gene’, the gene encoding the enzyme producing farnesyl diphosphate (FPP) from dimethylallyl diphosphate (DMAPP) is referred to as ‘ispA gene’, the gene encoding the enzyme producing squalene from farnesyl diphosphate (FPP) is referred to as ‘sqs gene’, and the gene encoding the enzyme producing 2-C-methyl-D-erythritol-4-phosphate (MEP) from 1-deoxy-D-xylulose 5-phosphate (DXP) is also referred to as ‘dxr gene’. 
         [0034]    In an exemplary embodiment, the strain may be transformed with a first vector and/or a second vector. The expression ‘the first or the second’ is used only to differentiate the type of vector, and does not limit the order or method of transformation. 
         [0035]    The first vector may include: the gene encoding the enzyme producing 1-deoxy-D-xylulose 5-phosphate (DXP) from pyruvate and D-glyceraldehyde 3-phosphate (G3P); 
         [0036]    the gene encoding the enzyme producing dimethylallyl diphosphate (DMAPP) from isopentenyl diphosphate (IPP); and the gene encoding the enzyme producing farnesyl diphosphate (FPP) from dimethylallyl diphosphate (DMAPP). The first vector may include a sequence of SEQ ID NO. 7. 
         [0037]    Further, the first vector further include the gene encoding the enzyme producing 2-C-methyl-D-erythritol-4-phosphate (MEP) from 1-deoxy-D-xylulose 5-phosphate (DXP). The first vector may include a sequence of SEQ ID NO. 8. 
         [0038]    In an exemplary embodiment, the strain may be transformed with only the first vector, and in this case, the strain mass-produces farnesyl diphosphate (FPP) which is a precursor of squalene, and thus may separately produce squalene by using the same. 
         [0039]    In an exemplary embodiment, the second vector may include the gene encoding the enzyme producing squalene from farnesyl diphosphate (FPP). The second vector may include a sequence of SEQ ID NO. 9 or 10. A second vector including the sequence of SEQ ID NO. 9 includes a squalene synthase gene derived from  Saccharomyces cerevisiae , and a second including the sequence of SEQ ID NO. 10 includes a squalene synthase gene derived from  Methylococcus capsulatus.    
         [0040]    The first vector may be inserted into a neutral site I (NSI) of  Synechococcus elongatus  which is a parent strain. 
         [0041]    In addition, the second vector may be inserted into a neutral site II (NSII) of  Synechococcus elongatus  which is a parent strain. 
         [0042]    The first vector may sequentially include: a spectinomycin-resistant gene as selection marker; a lacI repressor; a trc promoter; and a target gene. The target gene may be the gene encoding the enzyme producing 1-deoxy-D-xylulose 5-phosphate (DXP) from pyruvate and D-glyceraldehyde 3-phosphate (G3P); the gene encoding the enzyme producing dimethylallyl diphosphate (DMAPP) from isopentenyl diphosphate (IPP); and the gene encoding the enzyme producing farnesyl diphosphate (FPP) from dimethylallyl diphosphate (DMAPP). The first vector may include a sequence of SEQ ID NO. 7. 
         [0043]    Furthermore, the first vector may further include the gene encoding the enzyme producing 2-C-methyl-D-erythritol-4-phosphate (MEP) from 1-deoxy-D-xylulose 5-phosphate (DXP) as the target gene. The first vector further including the gene encoding the enzyme producing 2-C-methyl-D-erythritol-4-phosphate (MEP) from 1-deoxy-D-xylulose 5-phosphate (DXP) may include a sequence of SEQ ID NO. 8. 
         [0044]    The target genes to be inserted into the first vector may be each derived from  E. coli.    
         [0045]    In the present specification, the target gene may mean a gene which is expressed in a strain and inserted into a vector so as to exhibit the function of the corresponding gene. 
         [0046]    The second vector may sequentially include: a kanamycin-resistant gene as selection marker; 
         [0047]    a lacI repressor; a trc promoter; and a target gene. The target gene may be a gene encoding an enzyme producing squalene from farnesyl diphosphate (FPP). The gene encoding the enzyme producing squalene from farnesyl diphosphate (FPP) may be derived from  Saccharomyces cerevisiae  or  Methylococcus capsulatus . The second vector may include a sequence of SEQ ID NO. 9 or 10. 
         [0048]    The target genes to be inserted into the first vector and the second vector may be located between the BglII site and the BamHI site, which are restriction enzyme sites. 
         [0049]    In the vector disclosed in the present specification, all the genes are linked operably to each other. The term “operably” means that the target genes may be expressed normally. 
         [0050]    The strain may be a strain in which the first vector and/or the second vector are/is transformed with  Synechococcus elongatus  PCC7942 (Accession number: ATCC 33912), which is a parent strain. Into the parent strain, only the first vector may be introduced, and both the first vector and the second vector may also be introduced. 
         [0051]    The strain may be a strain belonging to accession number KCTC 12966BP. The accession number KCTC 12966BP strain may mean a strain of a  Synechococcus elongatus  strain, in which a pSe1 Bb1 s-dxs-idi-ispA vector at an NSI site and a pSe2Bb1k-sqs vector (including a squalene synthase gene derived from  Saccharomyces cerevisiae ) at an NSII site are transformed. 
         [0052]    In another aspect, the present disclosure is a method for producing squalene, the method including: culturing the transformed  Synechococcus elongatus  strain. 
         [0053]    The culturing may be performed under conditions of 0.1% to 10% CO 2  and a temperature of 10° C. to 40° C. For example, the strain may be cultured under conditions of a 5% CO 2  concentration and 30° C. 
         [0054]    Further, In another aspect, the present disclosure is a method for removing carbon dioxide, the method including: culturing the transformed  Synechococcus elongatus  strain. 
       EXAMPLES 
       [0055]    Hereinafter, the present disclosure will be described in more detail through the Examples. However, the following Examples are provided only for illustrative purposes to facilitate the understanding of the present disclosure, and the purview and scope of the present disclosure is not limited thereto. 
       Example 1 
       [0056]    Referring to a prior paper (Kim, S. W., Keasling, J. D., 2001. Metabolic engineering of the nonmevalonate isopentenyl diphosphate synthesis pathway in  Escherichia coli  enhances lycopene production. Biotechnol. Bioeng. 72, 408-415), a methylerythritol phosphate pathway (MEP pathway) and a metabolic pathway from pyruvic acid and D-glyceraldehyde 3-phosphate to farnesyl diphosphate were newly created. The DNA sequences of dxs gene, dxr gene, idi gene, and ispA gene of  E. coli  was subjected to codon optimization, and then the sequences were custom synthesized and constructed from Genescript®. 
       Example 2 Construction of Four Squalene-Producing Strains Using SyneBrick Vectors pSe1Bb1s-GFP Vector and pSe2Bb1k-GFP Vector 
       [0057]    A first vector was constructed by using a pSe1Bb1s-GFP vector. The pSe1Bb1s-GFP vector was constructed by using a pBbE1c-RFP vector (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12) and a pSeBb1k-GFP vector (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression. J Biol Eng 5:12). 
         [0058]    The GFP portion of the SyneBrick vector pSe1Bb1s-GFP was removed by using the EcoRI-BglII restriction enzyme, and then a DNA sequence of the ispA gene treated with the custom synthesized EcoRI-BamHI restriction enzyme was inserted into the site. The pSe1Bb1s-ispA vector thus completed was treated with the EcoRI-BglII restriction enzyme, and then a DNA sequence of the idi gene treated with the EcoRI-BamHI restriction enzyme was inserted thereinto. In the same manner, the dxs gene or the dxs gene-dxr gene was sequentially inserted thereinto, thereby finally constructing ‘pSe1 Bb1 s-dxs gene-idi gene-ispA gene’ and ‘pSe1 Bb1s-dxs gene-dxr gene-idi gene-ispA gene’ vectors ( FIG. 2 a   ). The completed vector was inserted into the Neutral Site-I of a wide-type  S. elongatus  PCC7942 strain. A transformed  S. elongatus  PCC7942 strain, in which the intermediate flow of the MEP metabolic pathway was increased, was constructed. Transformation was confirmed through PCR (5′-&gt;3′ Primer Sequence: Forward: CCAGCAGCGGCTGCCTGCCCAAAAG (SEQ ID NO. 11))/Reverse: GAAAGCGTGACGAGCAGGGA (SEQ ID NO. 12). Meanwhile, the second vector was constructed by using pSe2Bb1k-GFP. Referring to the document information, the GFP portion of the SyneBrick vector pSe2Bb1k-GFP was removed by using the EcoRI-BamHI restriction enzyme, and then a custom synthesized DNA sequence of a squalene synthase gene of  Methylococcus capsulatus  or  Saccharomyces cerevisiae  was inserted thereinto. The completed pSe2Bb1k-sqs gene vector ( FIG. 2 b   ) was inserted into the Neutral Site-II of the transformed  S. elongatus  PCC7942 strain described above, in which the intermediate flow of the MEP metabolic pathway was increased. Transformation was confirmed through PCR (5′-&gt;3′ Primer Sequence: Forward: GGCTACGGTTCGTAATGCCA (SEQ ID NO. 13))/Reverse: GAGATCAGGGCTGTACTTAC (SEQ ID NO. 14). 
       Example 3 Confirmation of Transformed Strain&#39;s Capability of Producing Squalene 
       [0059]    The transformed strains prepared in Example 2 were cultured for a predetermined time to test whether squalene was produced from 5% carbon dioxide which was directly supplied. As a specific culturing condition, 100 ml of a BG-11 medium including a 10 mM MOPS buffer was put into a 100 ml-bottle, the constructed squalene producing strain was diluted at an O.D of 0.6 when initially cultured, and the diluted solution was put into the medium. Further, 10 ug/ml of a spectinomycin antibiotic and 5 ug/ml of kanamycin were put into the medium, and then the resulting medium was cultured under conditions continuously supplying 100 uE m−2 s−1 and 5% CO 2  at 30° C. in a stationary incubator. An inducer 1 mM IPTG required for expression of genes was put into the medium 1 day after the initiation of culturing, the optical density of cells was measured at a wavelength of 730 nm until 8 days after culturing, and the amount of squalene produced was also measured during the culturing for 8 days. 
         [0060]    The growth curves of four transformed strains and the wild-type strain are as illustrated in  FIG. 3 . Further, through a gas chromatography analysis method, it was confirmed that the material produced from the strain was squalene ( FIGS. 4 a  and 4 b   ). The transformed strain into which ‘pSe1Bb1k-dxs gene-dxr gene-idi gene-ispA gene’ was inserted produced up to 0.41 to 0.12 mg/L/OD730 of squalene for the culture time. Moreover, the strain into which the pSe1 Bb1 k-dxs gene-idi gene-ispA gene was inserted produced up to 4.98 to 1.36 mg/L/OD730 of squalene for the culture time ( FIG. 5 ). 
       ACCESSION NUMBER 
       [0061]    Depositary Institution: Korea Research Institute of Bioscience &amp; Biotechnology 
         [0062]    Accession number: KCTC12966BP 
         [0063]    Commissioned date: 2015-Dec. 18