Abstract:
Disclosed herein are a transformed  Synechococcus elongatus  strain having improved capability of producing acetone and a method for producing acetone and a method for removing carbon dioxide using the same. In an aspect, the transformed  Synechococcus elongatus  strain of the present disclosure can produce acetone with high selectivity using carbon dioxide as a carbon source. The present disclosure is economical because the  Synechococcus elongatus  strain can economically produce high value-added acetone using carbon dioxide existing in the atmosphere as a carbon source without requiring an additional catalytic reaction. Also, the present disclosure is environment-friendly because carbon dioxide in the atmosphere can be removed or reduced using the microorganism.

Description:
DESCRIPTION ABOUT NATIONAL SUPPORT RESEARCH AND DEVELOPMENT 
       [0001]    This work is supported by business of the National Research Foundation of Korea grant-funded by the Korean Government (Ministry of Science, ICT and Future Planning) under the supervision of Korea Institute of Science and Technology, and the subject number thereof is 2015U00023(2N40353). Also, This work is supported by the support of KCRC CCS2020 business of Korea Ministry of Science, ICT and Future Planning under the supervision of 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. 2014M1A8A1049277). 
     
    
     FIELD OF THE INVENTION 
       [0002]    Disclosed herein are a transformed  Synechococcus elongatus  strain having improved capability of producing acetone and a method for producing acetone and a method for removing carbon dioxide using the same. 
       CROSS-REFERENCE TO RELATED APPLICATION 
       [0003]    This application claims priority to Korean Patent Application No. 10-2015-0030616, filed on Mar. 4, 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. 
       BACKGROUND OF THE INVENTION 
       [0004]    Recently, concerns about depletion of fossil fuel resources and environmental pollution are increasing globally and the energy problem is becoming an important social issue due to change in oil prices, effectuation of the UN Framework Convention on Climate Change, etc. In particular, with the prediction of depletion of petroleum which is used in all industrial fields, the energy problem is becoming an important threat to national security and survival. For this reason, interests in biofuels that can replace the fossil fuel are increasing consistently. 
         [0005]    A biofuel refers to a fuel obtained from biomass, including not only living organisms but also byproducts of metabolic activities such as excrement of animals. It is a renewable source of energy, unlike the fossil fuel. Recently, also in Korea, development of biofuels using microorganisms is being studied actively with the advance in biotechnology and attempts are made to extend its scope to bioethanol, biobutanol, biodiesel, etc. 
       SUMMARY 
       [0006]    In an aspect, the present disclosure is directed to providing a  Synechococcus elongatus  strain having improved capability of producing acetone. 
         [0007]    In another aspect, the present disclosure is directed to producing acetone on a large scale using a  Synechococcus elongatus  strain. 
         [0008]    In another aspect, the present disclosure is directed to removing a carbon dioxide using a  Synechococcus elongatus  strain. 
         [0009]    In an aspect, the present disclosure relates to a  Synechococcus elongatus  strain having an acetone selectivity, defined as the molar ratio of acetone in the total product produced by the strain, of 0.8 or greater under a condition of 30° C. and 5% carbon dioxide. 
         [0010]    In another aspect, the present disclosure relates to a method for producing acetone, including a step of culturing a  Synechococcus elongatus  strain. 
         [0011]    In another aspect, the present disclosure relates to a method for removing carbon dioxide, including a step of culturing a  Synechococcus elongatus  strain. 
         [0012]    In an aspect, the transformed  Synechococcus elongatus  strain of the present disclosure can produce acetone with high selectivity using carbon dioxide as a carbon source. The present disclosure is economical because the  Synechococcus elongatus  strain can economically produce high value-added acetone using carbon dioxide existing in the atmosphere as a carbon source without requiring an additional catalytic reaction. Also, the present disclosure is environment-friendly because carbon dioxide in the atmosphere can be removed or reduced using the microorganism. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0013]      FIG. 1  shows the acetone metabolic pathway of a transformed  Synechococcus elongatus  strain. 
           [0014]      FIGS. 2A  to 2C show the optical density ( FIG. 2A ), acetone production ( FIG. 2B ) and acetate production ( FIG. 2C ) of a  Synechococcus elongatus  strain transformed with a pSe1Bb1s-atoB-atoDA-adc vector. 
           [0015]      FIGS. 3A to 3C  show the optical density ( FIG. 3A ), acetone production ( FIG. 3B ) and acetate production ( FIG. 3C ) of a  Synechococcus elongatus  strain transformed with a pSe1Bb1s-atoB-ctfAB-adc vector. 
           [0016]      FIGS. 4 a  to 4 c    show the optical density ( FIG. 4A ), acetone production ( FIG. 4B ) and acetate production ( FIG. 4C ) of a  Synechococcus elongatus  strain transformed with a pSe1Bb1s-nphT7-atoDA-adc vector. 
           [0017]      FIGS. 5A to 5C  show the optical density ( FIG. 5A ), acetone production ( FIG. 5B ) and acetate production ( FIG. 5C ) of a  Synechococcus elongatus  strain transformed with a pSe1Bb1s-nphT7-ctfAB-adc vector. 
           [0018]      FIG. 6  shows the sequence of an atoB-derived gene (SEQ ID NO.: 1). 
           [0019]      FIG. 7  shows the sequence of an nphT7-derived gene (SEQ ID NO.: 2). 
           [0020]      FIG. 8  shows the sequence of an atoDA-derived gene (SEQ ID NO.: 3). 
           [0021]      FIG. 9  shows the sequence of a ctfAB-derived gene (SEQ ID NO.: 4). 
           [0022]      FIG. 10  shows the sequence of an adc-derived gene (SEQ ID NO.: 5). 
           [0023]      FIGS. 11A to 11D  show the sequence of a pSe1Bb1s-atoB-atoDA-adc vector, in sequence (SEQ ID NO.: 6). 
           [0024]      FIGS. 12A to 12D  show the sequence of a pSe1Bb1s-atoB-ctfAB-adc vector, in sequence (SEQ ID NO.: 7). 
           [0025]      FIGS. 13A to 13D  show the sequence of a pSe1Bb1s-nphT7-atoDA-adc vector(SEQ ID NO.: 8), in sequence. 
           [0026]      FIGS. 14A to 14D  show the sequence of a pSe1Bb1s-nphT7-ctfAB-adc vector (SEQ ID NO.: 9), in sequence. 
           [0027]      FIG. 15  shows the sequence of a forward primer(SEQ ID NO.: 10) used in polymerase chain reaction (PCR) for identifying the transformation of the strain. 
           [0028]      FIG. 16  shows the sequence of a reverse primer(SEQ ID NO.: 11) used in polymerase chain reaction (PCR) for identifying the transformation of the strain. 
       
    
    
     DETAILED DESCRIPTION 
       [0029]    Hereinafter, the present disclosure is described in detail. 
         [0030]      Synechococcus elongatus  is a species of cyanobacteria. The prokaryotic cyanobacteria are useful in altering metabolic pathways or artificially regulating metabolites because genetic modification is easy. The inventors of the present disclosure have completed the present disclosure based on this characteristic of cyanobacteria using the techniques of synthetic biology and metabolic engineering. 
         [0031]    In an aspect, the present disclosure relates to a  Synechococcus elongatus  strain having an acetone selectivity of 0.8 or greater under a condition of 30° C. and 5% carbon dioxide. 
         [0032]    In the present disclosure, the acetone selectivity is defined as the molar ratio of acetone in the total product produced by the  Synechococcus elongatus  strain. 
         [0033]    Specifically, the acetone selectivity may be 0.7 or greater, 0.75 or greater, 0.8 or greater, 0.81 or greater, 0.83 or greater, 0.85 or greater, 0.87 or greater, 0.89 or greater, 0.91 or greater, 0.93 or greater, 0.95 or greater, 0.97 or greater, 0.98 or greater, 0.99 or greater or 1. And, the temperature may be, for example, 10-50° C., 10-45° C., 10-40° C., 10-35° C., 15-50° C., 20-45° C., 25-40° C. or 30-40° C. And, the carbon dioxide concentration may be 1-10%. But, without being limited thereto, the concentration may be 0.01% or higher, 0.05% or higher, 0.07% or higher, 0.09% or higher, 1% or higher, 2% or higher, 4% or higher, 5% or higher, 6% or higher, 8% or higher, 9% or higher, 11% or higher, 13% or higher, 15% or higher, 17% or higher, 20% or higher, 25% or higher, 30% or higher, 40% or higher, 50% or higher, 60% or higher, 70% or higher, 80% or higher or 90% or higher, and may be 91% or lower, 85% or lower, 80% or lower, 76% or lower, 71% or lower, 66% or lower, 61% or lower, 56% or lower, 51% or lower, 46% or lower, 41% or lower, 36% or lower, 31% or lower, 25% or lower, 19% or lower, 15% or lower, 13% or lower, 12% or lower, 9% or lower, 7% or lower, 5% or lower, 4% or lower, 3% or lower, 2% or lower or 1% or lower. 
         [0034]    In this aspect, the strain may contain: one or more selected from a group consisting of an acetyl-CoA transferase gene and an acetyl-CoA synthase gene; an acetoacetyl-CoA transferase gene; and an acetoacetate decarboxylase gene. 
         [0035]    In the present disclosure, the acetyl-CoA transferase gene refers to a gene that encodes acetyl-CoA transferase. For example, it may be one derived from an atoB gene of an  E. coli  K-12 MG1655 strain. 
         [0036]    In the present disclosure, the acetyl-CoA synthase gene refers to a gene that encodes acetyl-CoA synthase. For example, it may be one derived from an nphT7 gene of a  Streptomyces  sp. strain. 
         [0037]    And, in the present disclosure, the acetoacetyl-CoA transferase gene refers to a gene that encodes acetoacetyl-CoA transferase. For example, it may be one derived from an atoDA gene of an  E. coli  K-12 MG1655 strain. Or, it may be one derived from a ctfAB gene of a  Clostridium acetobutylicum  strain. 
         [0038]    Also, in the present disclosure, the acetoacetate decarboxylase gene refers to a gene that encodes the enzyme acetoacetate decarboxylase. For example, it may be one derived from an adc gene of a  Clostridium acetobutylicum  strain. 
         [0039]    In the  Synechococcus elongatus  strain according to an aspect of the present disclosure, the acetyl-CoA transferase gene may contain a sequence of SEQ ID NO 1, the acetyl-CoA synthase gene may contain a sequence of SEQ ID NO 2, the acetoacetyl-CoA transferase gene may contain a sequence of SEQ ID NO 3 or SEQ ID NO 4, and the acetoacetate decarboxylase gene may contain a sequence of SEQ ID NO 5. 
         [0040]    In this aspect, in the present disclosure, the sequence of SEQ ID NO 1 contains a sequence derived from an atoB gene, which encodes the acetyl-CoA transferase gene, and the sequence of SEQ ID NO 2 contains a sequence derived from an nphT7 gene, which encodes the acetyl-CoA synthase gene. And, the sequence of SEQ ID NO 3 contains a sequence derived from an atoDA gene, which encodes the acetoacetyl-CoA transferase gene, and the sequence of SEQ ID NO 4 contains a sequence derived from a ctfAB gene, which encodes the acetoacetyl-CoA transferase gene. And, the sequence of SEQ ID NO 5 contains a sequence derived from an adc gene, which encodes the acetoacetate decarboxylase gene. 
         [0041]    In this aspect, the  Synechococcus elongatus  strain may be one transformed with a vector containing: a gene containing a sequence derived from an atoB gene or an nphT7 gene; a gene containing a sequence derived from an atoDA gene or a cftAB gene; and a gene containing a sequence derived from an adc gene. 
         [0042]    The genes in all the vectors disclosed in the present disclosure are linked operably. The expression operable means that a target gene can be expressed normally. 
         [0043]    Also, the transformed  Synechococcus elongatus  strain may be  Synechococcus elongatus  PCC7942 (ATCC® 33912™) transformed with the vector. 
         [0044]    In this aspect, the vector may further contain: a pUC replication origin as a replication origin; neutral sites located upstream and downstream of the replication origin; a spectinomycin resistance gene as a selection marker; a repressor selected from a group consisting of a lac I repressor, a tetR repressor and an AraC repressor; a promoter selected from a group consisting of a trc promoter, a tetA promoter or a modified tetA promoter, a BAD promoter and a cbbL promoter; and a BglII site, a BamHI site, an EcoRI site and an XhoI site as restriction enzyme sites. 
         [0045]    The neutral site may be one derived from  Synechococcus elongatus  PCC 7942. For example, the neutral site may include NSIa and NSIb. The vector may be inserted into the genome of  Synechococcus elongatus  PCC 7942 through the neutral sites. 
         [0046]    In this aspect, the vector may contain a target gene. For example, the target gene may be a gene containing a sequence derived from an atoB gene (hereinafter, an atoB-derived gene), a gene containing a sequence derived from an nphT7 gene (hereinafter, an nphT7-derived gene), a gene containing a sequence derived from an atoDA gene (hereinafter, an atoDA-derived gene), a gene containing a sequence derived from a cftAB gene (hereinafter, a cftAB-derived gene) or a gene containing a sequence derived from an adc gene (hereinafter, an adc-derived gene). These genes may be derived from different vectors. The different vectors may have a BglII site and a BamHI site on both sides of the target gene, and two target genes may be contained in one vector through complementary binding between the BglII site of one vector and the BamHI site of another vector upon treatment with a restriction enzyme. For example, a vector containing an atoB-derived gene (hereinafter, a pSe1Bb1s-atoB vector) or a vector containing an nphT7-derived gene (hereinafter, a pSe1Bb1s-nphT7 vector) may be prepared by removing the GFP portion of a SyneBrick vector pSe1Bb1s-GFP using EcoRI-BamHI restriction enzymes and then inserting the DNA sequence of an atoB-derived gene or an nphT7-derived gene. After treating each vector with BamHI-XhoI restriction enzymes, a vector containing ‘an atoB-derived gene and an atoDA-derived gene’ (hereinafter, a pSe1Bb1s-atoB-atoDA vector), a vector containing ‘an atoB-derived gene and a ctfAB-derived gene’ (hereinafter, a pSe1Bb1s-atoB-ctfAB vector), a vector containing ‘an nphT7-derived gene and an atoDA-derived gene’ (hereinafter, a pSe1Bb1s-nphT7-atoDA vector) and a vector containing ‘an nphT7-derived gene and a ctfAB-derived gene’ (hereinafter, a pSe1Bb1s-nphT7-ctfAB vector) may be prepared by inserting the DNA sequence of an atoDA-derived gene or a ctfAB-derived gene treated with BglII-XhoI restriction enzymes. Then, by treating each vector with BamHI-XhoI restriction enzymes and inserting the DNA sequence of an adc-derived gene treated with BglII-XhoI restriction enzymes, a vector containing ‘an atoB-derived gene, an atoDA-derived gene and an adc-derived gene’ (hereinafter, a pSe1Bb1s-atoB-atoDA-adc vector), a vector containing ‘an atoB-derived gene, a ctfAB-derived gene and an adc-derived gene’ (hereinafter, a pSe1Bb1s-atoB-ctfAB-adc vector), a vector containing ‘an nphT7-derived gene, an atoDA-derived gene and an adc-derived gene’ (hereinafter, a pSe1Bb1s-nphT7-atoDA-adc vector) and a vector containing ‘an nphT7-derived gene, a ctfAB-derived gene and an adc-derived gene’ (hereinafter, a pSe1Bb1s-nphT7-ctfAB-adc vector) may be obtained. 
         [0047]    In this aspect, the BglII site and the BamHI site may be located on both sides of the target gene. In another aspect, the order of the target gene and the restriction enzyme sites may be: EcoRI site-&gt;BglII site-&gt;target gene-&gt;BamHI site-&gt;XholI site. 
         [0048]    In the  Synechococcus elongatus  strain according to an aspect of the present disclosure, the vector may contain a sequence from SEQ ID NOS 6-9. Specifically, SEQ ID NO 6 is the sequence of a vector containing an atoB-derived gene, an atoDA-derived gene and an adc-derived gene, SEQ ID NO 7 is the sequence of a vector containing an atoB-derived gene, a ctfAB-derived gene and an adc-derived gene, SEQ ID NO 8 is the sequence of a vector containing an nphT7-derived gene, an atoDA-derived gene and an adc-derived gene, and SEQ ID NO 9 is the sequence of a vector containing an nphT7-derived gene, a ctfAB-derived gene and an adc-derived gene. 
         [0049]    The  Synechococcus elongatus  strain according to an aspect of the present disclosure may be a KCTC12758BP strain, a KCTC12759BP strain, a KCTC 12760BP strain or a KCTC12761BP strain. Specifically, the KCTC12758BP strain is one transformed with the vector of SEQ ID NO 6, the KCTC12759BP strain is one transformed with the vector of SEQ ID NO 7, the KCTC 12760BP strain is one transformed with the vector of SEQ ID NO 8, and the KCTC12761BP strain is one transformed with the vector of SEQ ID NO 9. 
         [0050]    The strain may absorb and fix carbon dioxide. 
         [0051]    In another aspect, the present disclosure relates to a method for producing acetone, including a step of culturing a  Synechococcus elongatus  strain. 
         [0052]    In this aspect, the method for producing acetone may further include a step of supplying carbon dioxide to the strain and may further include a step of supplying potassium acetate. The transformed  Synechococcus elongatus  strain of the present disclosure may produce a larger amount of acetone when it is further supplied with potassium acetate in addition to carbon dioxide. The potassium acetate may be supplied with a concentration of 1-30 mM, although not being limited thereto. Specifically, the concentration may be 0.5-40 mM, 1-30 mM, 3-25 mM, 5-20 mM, 8-15 mM or 9-13 mM. 
         [0053]    In another aspect, the present disclosure relates to a method for removing carbon dioxide, including a step of culturing a  Synechococcus elongatus  strain. Because the strain uses carbon dioxide as a carbon source, it may be useful in removing or reducing carbon dioxide in the atmosphere. 
         [0054]    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 Acetone 
       [0055]    A metabolic pathway as shown in  FIG. 1  was designed to prepare a  Synechococcus elongatus  strain having excellent capability of producing acetone. 
       EXAMPLE 2 
     Preparation of Vector 
       [0056]    At first, pBbE1c-RFP (Lee T S, Krupa R A, Zhang F, Hajimorad M, Holtz W J, Prasad N, Lee S K, Keasling J D (2011 b) BglBrick vectors and datasheets: a synthetic biology platform for gene expression.  J Biol Eng  5:12) and Invitrogen&#39;s pSyn_1 were used. Specifically, the lacI, ptrc and RFP portions of pBbE1c-RFP were subjected to PCR and the spectinomycin resistance gene, NSIa, NS1b and pUC replication origin of pSyn_1 were subjected to PCR. Then, a new vector was completed by joining the two PCR products through the OPEC cloning method (Quan J, Tian J (2009) Circular Polymerase Extension Cloning of Complex Gene Libraries and Pathways.  PLoS ONE  4 (7): e6441. doi:10.1371/journal.pone.0006441) (CPEC Ref. http://j5.jbei.org/j5manual/pages/22.html). In order to replace the RFP portion of the vector with GFP, RFP was removed using EcoRI/XhoI restriction enzymes and the GFP portion of another BglBrick vector pBbB5k-GFP (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) was inserted using EcoRI/XhoI restriction enzymes and a ligase. After transforming the vector into  E. coli  HIT-DH5a (Cat# RH617-J80, RBC Bioscience), the vector was extracted by mini-prep. Because the assembled vector, prepared through PCR, might have been mutated, the entire sequence was investigated through plasmid sequencing. The resulting vector was named as pSe1Bb1s-GFP. Then, after removing the GFP portion using EcoRI-BamHI restriction enzymes, the DNA sequence of an atoB-derived gene or an nphT7-derived gene was inserted. Thus prepared pSe1Bb1s-atoB-derived gene vector and pSe1Bb1s-nphT7-derived gene vector were treated with BamHI-XhoI restriction enzymes and then the DNA sequence of an atoDA-derived gene or a ctfAB-derived gene treated with BglII-XhoI restriction enzymes was inserted. The atoB-derived gene was derived from an  E. coli  K-12 MG1655 strain, the nphT7-derived gene was derived from a  Streptomyces  sp. strain, the atoDA-derived gene was derived from an  E. coli  K-12 MG1655 strain, and the ctfAB-derived gene and the adc-derived gene were derived from a  Clostridium acetobutylicum  strain. All the genes introduced into the vectors were prepared by GENSCRIPT R . 
         [0057]    As a result, four vectors, i.e., a pSe1Bb1s-atoB-atoDA vector, a pSe1Bb1s-atoB-ctfAB vector, a pSe1 Bb1 s-nphT7-atoDA vector and a pSe1Bb1s-nphT7-ctfAB vector, were prepared and finally four acetone-producing vectors were prepared by treating with BamHI-XhoI and then inserting the DNA sequence of an adc gene treated with BglII-XhoI restriction enzymes: a pSe1Bb1s-atoB-atoDA-adc vector (SEQ ID NO 6), a pSe1Bb1s-atoB-ctfAB-adc vector (SEQ ID NO 7), a pSe1Bb1s-nphT7-atoDA-adc vector (SEQ ID NO 8) and a pSe1Bb1s-nphT7-ctfAB-adc vector (SEQ ID NO 9). 
       EXAMPLE 3 
     Preparation of Transformed  Synechococcus elongatus  Strain 
       [0058]    The vectors of SEQ ID NOS 6-9 prepared in Example 2 were inserted into the neutral site I of a wild-type  Synechococcus elongatus  ( S. elongatus ) PCC7942 strain (PCC7942 (ATCC® 33912™)) by natural transformation (Golden et al. 1987, Grigorieva and Shestakov 1982). The transformation was confirmed by PCR (5′-&gt;3′ primer sequence: forward (SEQ ID NO 10): CTGATTGTTCTAGGCGCTG/reverse (SEQ ID NO 11): TTTGGCAATCTGAAGACCCG). 
       EXAMPLE 4 
     Confirmation of Capability of Transformed  Synechococcus elongatus  Strain of Producing Acetone 
       [0059]    The transformed strain obtained in Example 3 was cultured under a carbon dioxide environment and it was investigated whether acetone was produced. Specifically, 100 mL of a BG-11 medium containing a 10 mM MOPS (3-morpholinopropane-1-sulfonic acid) buffer was added to a 100-mL bottle and the acetone-producing strain diluted to an optical density (0.D) of 0.6 initially was added. Then, after adding 10 μg/mL spectinomycin and 10 mM potassium acetate, incubation was performed under a condition of 30° C., 100 μE·m −2 ·s −1  and continuous supply of 5% CO 2 . Since day 1 after the start of the incubation, 0.1 mM IPTG was added as an inducer necessary for gene expression. Optical density at 730 nm, acetate production, pH and acetone production were measured until day 5. 
         [0060]    The strain transformed with the pSe1Bb1s-atoB-atoDA-adc vector (SEQ ID NO 6) produced 3.6 mg/L of acetone ( FIG. 2 b   ). The strain transformed with the pSe1Bb1s-atoB-ctfAB-adc vector (SEQ ID NO 7) produced 134 mg/L of acetone ( FIG. 3 b   ). The strain transformed with the pSe1Bb1s-nphT7-atoDA-adc vector (SEQ ID NO 8) produced 152 mg/L of acetone ( FIG. 4 b   ), and the strain transformed with the pSe1Bb1s-nphT7-ctfAB-adc vector (SEQ ID NO 9) produced 116 mg/L of acetone ( FIG. 5 b   ).