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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)

Definitions

• the present invention relates to a construct for the synthesis of fatty alcohols in cyanobacteria, a vector comprising the construct, a cyanobacterium comprising the construct or transformed by the vector, and a method for producing fatty alcohols in cyanobacteria.
• ethanol as a fuel has some drawbacks, namely: (1) low energy density; (2) high volatility; (3) problems caused by its high solubility in water, such as the increased toxicity for microorganisms during fermentation, the high cost for the removal of water phase during distillation separation process and the corrosion of pipelines during transportation.
• biofuel It would be desirable for a biofuel to have properties such as high energy density, low moisture absorption, low volatility, and/or compatibility with existing engines and transport facilities .
• biofuel components prepared from high quality fatty acids such as long chain fatty alcohols and long chain biologic hydrocarbons are drawing more and more attention .
• WO2007/136762 describes the production of fatty acid derivatives by genetically engineered microorganisms such as E. coli and Saccharomyces cerevisiae. It is indicated that the fatty acid derivatives can be useful as biofuels and speciality chemicals.
• microorganism systems used for studying biofuels are primarily heterotrophic microorganisms represented by E. coli and Saccharomyces cerevisiae.
• Pengcheng Fu in his article titled “Genome-scale modeling of Synechocystis sp . PCC 6803 and prediction of pathway insertion", published in the Journal of Chemical
• isobutyraldehyde published in Nature Biotechnology, vol 27, pages 1177 to 1180 describes the use of genetically engineered Synechococcus elongatus PCC7942 to produce isobutyraldehyde and isobutanol directly from C0 2 .
• the inventors of the present invention for the first time, successfully produced fatty alcohols in
• the present invention accordingly provides a construct used for synthesizing fatty alcohols in cyanobacteria, comprising a promoter having activity in cyanobacteria and a fatty acyl-CoA reductase gene under the control of the promoter .
• the present invention provides a vector comprising such a construct; a cyanobacter ium comprising the construct or transformed by the vector; and a method for producing fatty alcohols and/or biologic hydrocarbons in a cyanobacter ium, comprising culturing a cyanobacterium comprising the construct or transformed by the vector under conditions suitable for the synthesis of fatty alcohols; and extracting the desired fatty alcohols from the obtained culture .
• the present invention provides a method of expression of exogenous genes via the use of Synechocystis sp. 6803, the main processes of which are:
• Fig. 1 represents the basic structure of plasmid pFQ9R, in which the Omega fragment of spectinomycin resistance gene, the P rbc promoter and the T rbc terminator are between the upstream and downstream fragments of slr0168 gene of Synechocystis sp. PCC6803; and Xbal and Smal restriction sites are between the promoter and the terminator.
• Fig. 2 represents the basic structure of plasmid pXTl4, which is obtained by cloning far gene ( far_jojoba ) (SEQ ID NO: 1) from Simmondsia chinensis into the plasmid pFQ9R.
• Fig. 3 represents the basic structure of plasmid pXT37a, in which the Omega fragment of spectinomycin resistance gene, the P pe t E promoter and the lacZ gene are between the upstream and downstream fragments of slr0168 gene of Synechocystis sp. PCC6803; and Ndel and EcoRI restriction sites are at the two ends of the lacZ gene.
• Fig. 4 represents the basic structure of plasmid pXT37b, which is similar to plasmid pXT37a, except that the insertion direction of the fragment consisting of Omega fragment, P pe t E promoter and lacZ gene is contrary to that in plasmid pXT37a.
• Fig. 5 represents the basic structure of plasmid pXT34, which is obtained by cloning at3gll980 gene (SEQ ID NO: 2) from Arabidopsis thaliana into the plasmid pXT37a, wherein the at3gl 1980 gene is located downstream of the Ppet E promoter .
• Fig. 6 represents the basic structure of plasmid pXT51, which is obtained by cloning far gene ( far_jojoba ) (SEQ ID NO: 1) from Simmondsia chinensis Into the plasmid ⁇ 3 ⁇ 1 ⁇ , wherein the far gene is located downstream of the P pe t E promoter .
• Fig. 7 represents the basic structure of plasmid pLY2, which is obtained by inserting the Omega fragment of spectinomycin resistance gene between the upstream and downstream fragments of slr0168 gene of Synechocystis sp . PCC6803, and cloning the entire construct into the vector pUC9.
• Fig. 8 illustrates the production of fatty alcohols in the cells of the genetically engineered strain Syn-LY2 after 8 days of culturing (the determination results of GC-MS), wherein C15-OH represents 1 -pentadecanol (used as internal standard), C16-OH represents 1 -hexadecanol , and C18-OH represents 1-octadecanol .
• Fig. 9 illustrates the production of fatty alcohols in the cells of the genetically engineered strain Syn-XT14 after 8 days of culturing (the determination results of GC-MS), wherein C15-OH represents 1 -pentadecanol (used as internal standard), C16-OH represents 1 -hexadecanol , and C18-OH represents 1-octadecanol.
• Fig. 10 illustrates the production of fatty alcohols in the cells of the genetically engineered strain Syn-XT34 after 8 days of culturing (the determination results of GC-MS), wherein C15-OH represents 1 -pentadecanol (used as internal standard), C16-OH represents 1 -hexadecanol , and
• C18-OH represents 1-octadecanol.
• Fig. 11 illustrates the production of fatty alcohols in the cells of the genetically engineered strain Syn-XT51 after 8 days of culturing (the determination results of GC-MS), wherein C15-OH represents 1 -pentadecanol (used as internal standard), C16-OH represents 1 -hexadecanol , and C18-OH represents 1-octadecanol.
• Fig. 12 is a photo of genetically engineered strains cultivated in a column photo-reactor.
• SEQ ID NO: 1 the sequence of fatty acyl-CoA reductase gene from (Simmondsia chinensis) (artificially
• SEQ ID NO: 2 the artificially synthesized sequence according to at3gll980 gene of Arabidopsis thaliana .
• SEQ ID NO: 3 the sequence of the Rubisco promoter fragment P rbc at the upstream of ribulose-1 , 5-diphosphate carboxylase large-subunit gene rbcL from Synechocyst is sp .
• PCC6803 US National Center for Biotechnology Information (NCBI ) ID : NC_000911 ) .
• SEQ ID NO: 4 the sequence of the terminator fragment T rbc at the downstream of r ibulose-1 , 5-diphosphate carboxylase operator from Synechocyst is sp. PCC6803 (NCBI ID: NC_000911) .
• SEQ ID NO: 5 the sequence of the promoter fragment P pe tE at the upstream of the plastocyanin gene petE from Synechocystis sp. PCC6803 (NCBI ID: NC_000911) .
• SEQ ID NO: 6 the N-terminal sequence (also comprising a part of the upstream sequence of the gene) of slr0168 gene from Synechocystis sp. PCC6803 (NCBI ID: NC_000911) .
• SEQ ID NO: 7 the C-terminal sequence (also comprising a part of the downstream sequence of the gene) of slr0168 gene from Synechocystis sp. PCC6803 (NCBI ID: NC_000911) .
• SEQ ID NO: 8 the sequence of the Omega fragment cloned into the plasmid pRL57 (NCBI ID: L05082) .
• SEQ ID NO: 9 the sequence of the lacZ gene cloned into the plasmid pHBl567 (NCBI ID: AP009048) .
• SEQ ID NO : 10 the sequence of the primer alrl524-l
• SEQ ID NO : 11 the sequence of the primer alrl524-2
• SEQ ID NO : 12 the sequence of the primer PI .
• SEQ ID NO : 13 the sequence of the primer P2.
• SEQ ID NO : 14 the sequence of the primer P3.
• SEQ ID NO : 15 the sequence of the primer P4.
• SEQ ID NO : 16 the sequence of the primer XP-1.
• SEQ ID NO : 17 the sequence of the primer XP-2.
• SEQ ID NO : 18 the sequence of the primer XP-3.
• SEQ ID NO : 19 the sequence of the primer XP-4.
• SEQ ID NO : 20 the sequence of the primer lacZ -ml .
• SEQ ID NO : 21 the sequence of the primer lacZ-m2.
• SEQ ID NO : 22 the sequence of the primer lacZ -m3.
• SEQ ID NO : 23 the sequence of the primer Ml3-Rev.
• SEQ ID NO: 24 the sequence of the primer far-1.
• SEQ ID NO: 25 the sequence of the primer far-2.
• SEQ ID NO: 26 the sequence of the Synechocystis sp. 6803 rbcL promoter (Prbcl, 1.3 kb) .
• Cyanobacter ium is understood a member from the group of photoautotrophic prokaryotic microorganisms, which can utilize solar energy and fix carbon dioxide. Cyanobacteria are sometimes also referred to as blue-green algae .
• a “construct” is herein understood a segment comprising one or more nucleic acids, for example a DNA fragment.
• the construct is suitably an artificially constructed segment of one or more nucleic acids.
• the construct can be used to subclone one or more of the nucleic acids, for example a DNA fragment, into a vector.
• a “Fatty acyl-CoA reductase” is understood an enzyme capable of catalyzing the conversion reaction of fatty acyl-CoA to fatty alcohols.
• Rubisco is an enzyme that catalyzes the first reaction of a so-called Calvin cycle in photosynthesis. It may consist of two subunits and the genes encoding the two subunits can be located in one and the same operator in the Synechocystis sp . PCC6803 genome.
• a Rubisco promoter (indicated as P rbc in the embodiments of the present invention) may be cloned to drive the expression of fatty acyl-CoA carboxylase gene in cyanobacteria, and the specific sequence for such a Rubisco promoter P rbc is shown in SEQ ID NO : 3.
• PC is an electron carrier for transferring electron from cytochrome b6/f complex to photosystem I in photosynthesis, and the gene encoding it is abbreviated as “petE” .
• a petE promoter (indicated as P pe t E in the embodiments of the present invention) may be cloned to drive the expression of fatty acyl-CoA carboxylase gene in cyanobacteria, and the specific sequence for such a promoter Ppet E is shown in SEQ ID NO: 5.
• a "slr0168 gene” is a gene in the Synechocystis sp . PCC6803 genome, which codes for a protein with unknown function. Previous studies proved that the deletion of this gene does not affect the physiologic activity of cells, so that the site of this gene has been considered as a neutral site in Synechocystis sp . PCC6803 genome.
• a promoter and a fatty acyl-CoA reductase gene may be integrated at this site by homologous recombination so as to express exogenous fatty acyl-CoA reductase in Synechocystis sp . PCC6803.
• the term "vector” refers to a self-replicating DNA molecule capable of transferring a DNA fragment (for example the gene of interest) into a recipient cell.
• hybridization is intended to mean the process during which, under suitable conditions, two nucleic sequences bond to one another with stable and specific hydrogen bonds so as to form a double strand. These hydrogen bonds can form between the complementary bases adenine (A) and thymine (T) or uracil (U), which may then be referred to as an A-T bond; or between the complementary bases guanine (G) and cytosine (C), which may then be referred to as a G-C bond.
• the hybridization of two nucleic sequences may be total (reference is then made to complementary sequences), i.e. the double strand obtained during this hybridization comprises only A-T bonds and C-G bonds.
• the hybridization may be partial (reference is then made to sufficiently complementary sequences), i.e. the double strand obtained comprises A-T bonds and C-G bonds allowing the double strand to form, but also bases not bonded to a complementary base.
• the hybridization between two complementary sequences or sufficiently complementary sequences depends on the operating conditions that are used, and in particular the stringency.
• the stringency may be understood to denote the degree of homology; the higher the stringency, the higher percent homology between the sequences .
• the stringency may be defined in particular by the base composition of the two nucleic sequences, and also by the degree of mismatching between these two nucleic sequences .
• the stringency can also depend on the reaction parameters, such as the concentration and the type of ionic species present in the hybridization solution, the nature and the concentration of denaturing agents and/or the hybridization temperature. The appropriate conditions can be determined by those skilled in the art.
• Conditions for hybridizing nucleic acid sequences to each other can be described as ranging from low to high stringency.
• Reference herein to hybridization conditions of low stringency includes from at least about 0% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and from at least about 1 M to at least about 2 M salt for washing conditions.
• the temperature for hybridization conditions of low stringency is from about 25°C, more preferably about 30°C to about 42°C.
• Reference herein to hybridization conditions of medium stringency includes from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and from at least about 0.5 M to at least about 0.9 M salt for washing conditions .
• Reference herein to hybridization conditions of high stringency includes from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and from at least about 0.01 M to at least about 0.15 M salt for washing conditions.
• Formamide is optional in these hybridization conditions.
• a particularly preferred non-limiting example of a hybridization condition of low stringency is 6 x SSC (Standard Sodium Citrate) buffer, 1.0% w/v SDS (Sodium Dodecyl Sulfate) at 25-42°C;
• a particularly preferred non-limiting example of a hybridization condition of medium stringency is 2 x SSC (Standard Sodium Citrate) buffer, 1.0% w/v SDS (Sodium Dodecyl Sulfate) at a temperature in the range 20°C to 65°C;
• a particularly preferred non-limiting example of a hybridization conditions of high stringency is 0.1 x SSC (Standard Sodium Citrate) buffer, 0.1% w/v SDS (Sodium Dodecyl Sulfate) at a temperature of at least 65°C.
• a "percent identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base or the identical amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
• Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman (Adv. Appl Math. 2:482, 1970), by the homology alignment algorithm of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), by the search for similarity method of Pearson and Lipman (Proc.
• Percent identities involved in the embodiments of the present invention include at least about 60% or at least about 65% or at least about 70% or at least about 75% or at least about 80% or at least about 85% or at least about 90% or above, such as about 95% or about 96% or about 97% or about 98% or about 99%, such as at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,
• the Cyanobacteria (also known as blue-green algae) in this invention preferably comprise a group of prokaryotic microorganisms capable of performing plant type oxygenic photosynthesis .
• cyanobacter ia may have the following advantages: (1) cyanobacteria are capable of absorbing solar energy and fixing carbon dioxide as carbon source for autotrophic growth, thereby having low cost for culturing; (2) cyanobacteria are ancient microorganisms and have lived on the earth for billions of years, so that they have remarkable adaptability to the environments, and they grow quickly; (3) cyanobacteria are convenient for genetic manipulations, because their genetic background is clear and genomic sequencing of many species of cyanobacteria has been completed which facilitates the genetic engineering of cyanobacteria . Synechocyst is sp . PCC6803 is a preferred unicellular cyanobacter ia, because for Synechocyst is sp . PCC6803 the whole genome sequencing had been completed in 1996.
• the embodiments of the present invention employ a promoter having activity in cyanobacteria .
• This promoter suitably drives the expression of fatty acyl-CoA reductase in cyanobacter ia .
• the characteristics of cyanobacteria as photosynthetic organism can be utilized to absorb solar energy, fix carbon dioxide and synthesize fatty alcohols as biofuels.
• One of the advantages of the present invention is that fatty alcohols are synthesized by using solar energy to fix carbon dioxide in the photosynthetic microorganism cyanobacter ia, wherein the energy for synthesizing fatty alcohols is solar energy and the carbon source is carbon dioxide.
• the production of biofuels utilizing this technology would not be restricted by the lack of raw materials, and the use of such biofuels would not increase carbon emission, i.e., such biofuels are real zero emission biofuels.
• the embodiments of the present invention relate to a construct used for synthesizing fatty alcohols in cyanobacter ia, which may comprise a promoter having activity in cyanobacteria as well as a fatty acyl-CoA reductase gene under the control of the promoter.
• the construct may comprise a marker gene for screening transformants of cyanobacteria, which is located upstream of the promoter having activity in cyanobacter ia .
• such a marker gene comprises the Omega gene as set forth in SEQ ID NO : 8.
• variants of this Omega gene wherein the variant has at least 80% sequence identity, preferably at least 85% sequence identity, more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity and most preferably at least 99% sequence identity, with the Omega gene can be used.
• variants also have marker activity in cyanobacteria .
• the construct may comprise, at the two termini thereof, the N-terminal and C-terminal sequences of slr0168 gene of Synechocystis sp . PCC6803, for homologous recombination.
• the promoter having activity in cyanobacteria is selected from the group consisting of the P r bc promoter and the PpetE promoter.
• variants of these promoters wherein the variant has at least 80% sequence identity, preferably at least 85% sequence identity, more preferably at least 90% sequence identity, even more preferably at least 95% sequence identity and most preferably at least 99% sequence identity, with the P r bc promoter or the PpetE promoter can be used.
• such variants also have promoting activity in cyanobacteria .
• promoter gene can be of great importance in terms of genetic expression.
• the rbc ( r ibulose-1 , 5-bisphosphate carboxylase /oxygenase ) gene promoter gene (P r bc ) is a particularly strong promoter within the cyanobacteria, whilst its product RuBisCO is a main soluble protein within the cyanobacteria which plays an important role in photosynthesis.
• the rbc promoter gene ( P rbc ) advantageously has the effect of being a high efficiency expression gene within cyanobacteria.
• An rbc operon may includes the promoter gene and rbcL, rbcX and rbcS genes.
• the complete operon may further start at a 250bp position upstream on the rbcL gene, and downstream of the rbcS stop codon there may be a 40bp reverse complementary sequence which acts as a termination sequence (as described in more detail in "Construction of a Synechocystis PCC6803 mutant suitable for the study of variant hexadecameric ribulose biphosphate carboxylase/oxygenase enzymes", by Doron Amichay, Ruth Levitz and Michael Gurevitz, Plant Molecular
• the fatty acyl-CoA reductase gene may be selected from the group consisting of: fatty acyl-CoA reductase (far) gene from Simmondsia chinensis , for example as set forth in SEQ ID NO: 1; and at 3gl 1980 gene from Arabidopsis thaliana, for example as set forth in SEQ ID NO: 2.
• the fatty acyl-CoA reductase gene may be farl gene from mouse (see for example National Center for Biotechnology Information (NCBI) ID:
• BC007178 codon-optimized farl gene from mouse; far2 gene from mouse (see for example NCBI ID: BC055759) ; or at3g56700 gene from Arabidopsis thaliana.
• Other suitable fatty acyl-CoA reductase genes include: Francci3_2276 from Frankia sp . Ccl3 (see for example NCBI ID: NC_007777);
• KRH_18580 from Kocuria rhizophila DC2201 (see for example NCBI ID: NC_010617); A20C1_04336 from Act inobacterium PHSC20C1 (see for example NCBI ID: NZ_AAOB01000003 ) ; HCH_05075 from Hahella chejuensis KCTC 2396 (see for example NCBI ID: NC_007645); Maqu_2220 from Marinobacter aquaeolei VT8 (see for example NCBI ID: NC_008740); and RED65_09889 from Oceanobacter sp. RED65 (see for example NCBI ID: NZ_AAQH01000001 ) .
• the embodiments of the present invention may employ the genes having at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity and most preferably at least 99% identity to the above-mentioned genes and coding for a protein having fatty acyl-CoA reductase activity; or the genes capable of hybridizing with the above-mentioned genes under stringent hybridization conditions, preferably
• hybridization conditions of high stringency, and coding for a protein having fatty acyl-CoA reductase activity are hybridization conditions of high stringency, and coding for a protein having fatty acyl-CoA reductase activity.
• the marker gene is the Omega fragment of spectinomycin resistance gene, for example as set forth in SEQ ID NO: 8.
• the cyanobacterium is chosen from the group consisting of Synechococcus PCC 6301, Anabaena sp . strain
• the cyanobacterium is
• the embodiments of the present invention may relate to a vector comprising the construct as defined above.
• this may be for example a shuttle plasmid vector or a genomic integrative plasmid vector. Both types of vector can play a major role in assisting natural blastasis (mainly in single celled blue-green alga) or can introduce one or more of the genes described herein into the cyanobacteria via conjugal transfer.
• Shuttle plasmid vectors can enter the cyanobacteria as a result of conjugal transfer, then duplicating themselves within the cytoplasm.
• Genomic integrative vectors can cause isogenesis of one or more of the genes described herein, or even a whole operon comprising one or more of such genes, within the cyanobacteria genome via isogenic integration, with advangtageous greater stability of expression, overcoming the stability problems encountered with autonomous plasmids .
• the vector is selected from the group consisting of: plasmid pXTl4, which was deposited in China General Microbiological Culture Collection Center under Accession Number of CGMCC 3948 on June 28, 2010, in a form in E. coli (Eco-XTl4) ; plasmid pXT34, which was deposited in China General Microbiological Culture Collection Center under Accession Number of CGMCC 3950 on June 28, 2010, in a form in E. coli (Eco-XT34) ; and plasmid pXT51, which was deposited in China General Microbiological Culture Collection Center under Accession Number of CGMCC 3949 on
• the embodiments of the present invention may relate to a cyanobacterium comprising the construct as defined above, or a cyanobacter ium transformed by the vector as defined above.
• the cyanobacter ium is selected from the group consisting of: cyanobacter ium Syn-XT14, which was deposited in China General Microbiological Culture Collection Center under Accession Number of CGMCC 3894 on June 10, 2010;
• the embodiments of the present invention may relate to a method for producing fatty alcohols in cyanobacteria, comprising: culturing a cyanobacterium comprising the construct as defined above, or a cyanobacter ium transformed by the vector as described above under conditions suitable for the synthesis of fatty alcohols; and extracting the desired fatty alcohols from the obtained culture .
• Fatty alcohols especially long-chain fatty alcohols, such as 1-hexadecanol and 1-octadecanol, were successfully produced in cyanobacteria via the embodiments of the present invention.
• these fatty alcohols may be converted to hydrocarbons by any manner known by the person skilled in the art to be suitable therefore .
• Such hydrocarbons can include alkanes (such as hexadecane or octadecane) and/or alkenes (such as 1-hexadecene or 1-octadecene ) .
• the method for producing fatty alchols in cyanobacteria as described above may therefore further comprise converting the fatty alcohols to hydrocarbons, providing a method for producing
• the fatty alcohols produced via the embodiments of the present invention and/or the hydrocarbons obtained by converting these fatty alcohols can advantageously be used as biofuel components and/or speciality chemicals.
• a biofuel may advantageously have properties such as high energy density, low moisture absorption, low volatility, and/or compatibility with existing engines and transport facilities.
• such a biofuel may be considered a real zero emission biofuel.
• the embodiments of the present invention may relate to a method of expression of exogenous genes via the use of Synechocystis sp . 6803.
• a method of expression of exogenous genes via the use of Synechocystis sp . 6803.
• the Synechocystis sp . 6803 genome integrative plasmid platform used in such a method may be understood to be a kind of genomic integrative vector as described above.
• such a method uses a Synechocystis sp . rbcL promoter (Prbcl, 1.3 kb) , a rbc terminator sequence (Trbc, 0.2 kb) , a 2kb
• spectinomycin resistant marker gene ⁇ and/or a reporter gene lacZ that is obtained via cloning.
• Example 1 Construction of vectors for the transformation of cyanobacteria
• Bacteriol 172 starting page 3138 ) , and the Omega fragment of about 1.9 kb was recovered .
• the plasmid pQLl was digested with Pstl (Takara, Catalog No.: D1073A), and blunt-ended with T4 DNA polymerase (Fermentas, Catalog No. : EP0061) . The two fragments were ligated to obtain the plasmid pQL4.
• PCR was performed by using Pi ( 5 ' -GCGTCGACTCACCATTTGGAC AAAACATCAGG-3 ' ) and P2 ( 5 ' -GCTCTAGACATCTAGGTCAGTCCT CCATAAACATTG-3 ' ) as the primer pair and using the genome of Synechocystis sp. PCC6803 as the template, and the PCR product was cloned into the vector pMDl8-T to obtain the plasmid pFQl; PCR was performed by using P3
• D1078A D1078A were used to cut the T rbc fragment from the plasmid pFQ2; the P r bc and T rbc fragments were inserted at corresponding site of the plasmid pQL4 to obtain the plasmid pFQ6.
• PCR was performed by using the plasmid pHBl536 (GAO Hong, et al, 2007) as the template and using XP-1 ( 5 ' -AGTGGTTCGCATCCTCGG-3 ' ) and XP-2 ( 5 ' -ATGAATCCTTAAT CGGTACCAAATAAAAAAGGGGACCTCTAGG-3 ' ) as well as XP-3
• XP-4 (5 ' -CCAGTGAATCCGTAATCATGGT-3 ' ) as the primer pair, respectively, the PCR product was recovered, and afterwards it was denatured, annealed and extended; then, PCR was performed by using it as the template and using XP-1 and XP-4 as the primer pair, and the PCR product was cloned into the vector pMDl8-T to obtain the plasmid pQLl7.
• the plasmid pQLl7 was digested with Bglll (Takara, Catalog No.: D1021S) and Sphl (Takara, Catalog No.: D1180A), and the recovered fragment was ligated to pHBl536 digested with the same enzymes to obtain the plasmid pQLl8.
• the plasmid pQLl8 was digested with Xbal, the Omega+P pe tE+ i ⁇ 3cZ fragment was recovered and inserted at the same site of the plasmid pXT24a to obtain the plasmid pXT36a.
• PCR was performed by using the plasmid pHBl567 as the template and using lacZ-ml ( 5 ' -ATGGTCAGGTCATGGATGAGCA-3 ' ) and lacZ-m2
• PCR was performed by using it as the template and using lacZ-ml and Ml3-Rev as the primer pair, and the PCR product was cloned into the vector pMDl8-T to obtain the plasmid pXT30.
• the plasmid pXT30 was digested with EcoRI and EcoRV, and the recovered fragment was ligated to pXT36a digested with the same enzymes to obtain the plasmid pXT37b.
• the plasmid pXT37b was digestedwith Xbal , and the two fragment s were recovered, self-ligated and screened to obtain the plasmid pXT37a having an insertion direction contrary to that in pXT37b.
• the plasmid pRL57 was digested with Dral, and the Omega fragment was recovered; the plasmid pKWll88SL was digested with EcoRI, blunt-ended, and the fragment was recovered; the two fragments were ligated to obtain the plasmid pLY2. This plasmid was used as a control plasmid.
• PCR was performed by using he plasmid pXL66 (a gift from
• the PCR product was recovered, digested with Xbal and Smal, and cloned into the same site of the plasmid pFQ9R to obtain the plasmid pXTl4.
• the plasmid pXL66 was digested with Ndel and Xhol, the far gene fragment of Simmondsia chinensis was recovered and inserted into the same site of the plasmid pXT37b to obtain the plasmid pXT51.
• At3gll980 gene of Arabidopsis thai iana was synthesized and cloned into the plasmid pUC57 (the synthesis was conducted by Sangon Biotech (Shanghai) Co., Ltd) to obtain the plasmid pXT31.
• TheplasmidpHBl567 was digested with EcoRI and Xhol , and the 5.4 kb fragment was recovered; the plasmid pHBl536 was digested with Xhol and Ndel, and the 2.4 kb fragment was recovered; the plasmid pXT31 was digested with Ndel+EcoRI, and the at3gll980 fragment was recovered; these three fragments were ligated to obtain the plasmid pXT34.
• Synechocystis sp. PCC6803 is provided in Table 1.
• the mixture of algae cells and DNA was applied onto a nitrocellulose membrane on BGll plate (without antibiotics) and cultivated at 30°C under an illumination condition of 30 ⁇ ⁇ 2 s _1 for 24 hours . Then, the nitrocellulose membrane was transferred to a BGll plate containing 10 ⁇ g mL -1 spectinomycin, and further incubated at 30°C under a condition of 30 ⁇ ⁇ 2 s _1 . After about 5-7 days, the transformants were picked out from the plate, and used to streak the fresh BG11 plate (supplemented with 20 ⁇ g mLT 1 spectinomycin ) . After the cells were enriched, they are inoculated into a liquid BG11 medium (containing 20 ⁇ g mLT 1 spectinomycin) for
• Example 3 Production of fatty alcohols by the genetically engineered cyanobacteria
• Culturing method I shake-flask culturing .
• a normal 500-mL conical flask with 300 mL of liquid BG11 medium (containing 20 ⁇ g mLT 1 spectinomycin) was used for inoculation with an initial concentration (OD 730 of 0.05), and the culturing was performed at 30°C, under an illumination condition of 30 ⁇ ⁇ 2 s _1 and under aeration with air, for 7-8 days.
• Culturing method II column photo-reactor culturing. Normal glass tubes with a height of 575 mm, a diameter of
• the initial inoculation concentration was OD 730 of 0.5, and the culturing was performed at 30°C, under an illumination condition of 100 ⁇ ⁇ 2 s _1 illumination under aeration with air containing 5% C0 2 .
• Hexadecanol and octadecanol were detected in samples of three strains of genetically engineered cyanobacteria : Syn-XT14, Syn-XT34 and Syn-XT51.
• the total yields of intracellular fatty alcohols under normal shake-flask culturing conditions as shown in Table 2 were calculated by referring to the internal standard (pentadecanol ) .
• the results under column photo-reactor culturing conditions also confirmed the ability of the three strains of genetically engineered cyanobacteria for synthesizing fatty alcohols .
• Example 4 Illustration of a method of expression of exogenous genes via the use of Synechocystis sp. 6803
• the rbcL promoter (Prbcl, 1.3 kb) , the termination sequence rbc terminator (Trbc, 0.2 kb) , the 2kb spectinomycin resistant marker gene ⁇ and the reporter gene lacZ of Synechocystis sp . 6803 were cloned separately, being built into a Synechocystis sp . 6803 genome integrative plasmid platform (which relies on the EcoRI restriction enzyme pKWll88 and which connects
• pFQ20 was used to transform the Synechocystis sp . 6803 algal strain, the transformant being obtained by spectinomycin resistant screening, after PCR genotype assay using a specific primer was conducted to confirm that there had been no error, assay of ⁇ - galactosidase activation of the high performance expression platform and exogenous gene genetically engineered Synechocystis sp . 6803 strain was carried out, thus confirming the expression performance of the Synechocystis sp. 6803 exogenous gene expression platform constructed according to this invention .
• Example 4a Cloning of the lazZ gene, Prbcl and Trbc onto a pMDl8T vector
• Example 4b Cloning of the spectinomycin resistant ⁇ gene, Prbcl and Trbc were separately in series with the pMDl8T vector .
• pQL4 is a vector originating in pMDl8T which contains the spectinomycin resistant ⁇ gene.
• restriction enzyme pFQ2 gel extraction of the 200bp Trbc gene fragment [s] was carried out; at the same time using the Smal-Sacl restriction enzyme pFQ5 and a PCR product purification test kit, purification was carried out yielding a double enzyme resistant pFQ5EH gene fragment; using T4 ligase, Trbc was inserted downstream in pFQ5, converting the E.Coli, then spectinomycin and ampicillin were used to perform double-resistance transformant screening, after which plasmid restriction enzyme assay was carried out to ensure that there were no errors, yielding the pFQ6 plasmid;
• pFQ6 contained the ⁇ , Prbcl and Trbc gene fragments;
• Example 4c Implanting the spectinomycin resistant ⁇ gene, Prbcl, Trbc and LacZ genes serially into a cyanobacterial vector .
• pKWll88 is a plasmid platform used in
• Synechocystis sp . 6803 genetic assay and detection of ⁇ - galactosidase activity of the genetically expressed product .
• Synechocystis PCC6803 FQ20 was cultured in liquid until after the growth period, then the algal solution was harvested and tested for - galactosidase activity.
• the wild strain of Synechocystis PCC6803 was used as the negative comparison 1
• Synechocystis PCC6803(LY2) was used as negative comparison 2
• the Synechocystis PCC6803 ( HB1567 ) strain that exhibits galactosidase expression activity was used as the positive comparison .
• CGMCC General Microbiological Culture Collection Center

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