EP2524035A2 - Constructs, vectors and cyanobacteria for the synthesis of fatty alcohols, and methods for producing fatty alcohols in cyanobacteria - Google Patents

Abstract

The present invention relates to constructs, vectors and cyanobacteria for the synthesis of fatty alcohols, and methods for producing fatty alcohols in cyanobacteria. Specifically, the present disclosure 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.

Description

CONSTRUCTS, VECTORS AND CYANOBACTERIA FOR THE SYNTHESIS OF FATTY ALCOHOLS, AND METHODS FOR PRODUCING FATTY ALCOHOLS

IN CYANOBACTERIA

Technical field of the invention

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.

Background of the invention

Currently, the sustainable development of economy and society is increasingly restricted by energy and environment related problems . Renewable biofuels are considered as an effective way to solve said problems.

Technical routes for the production of bio-ethanol are relatively well developed. However, 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.

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 .

Recently, 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 .

S.K. Lee et. al . in their article titled "Metabolic engineering of microorganisms for biofuels production: from bugs to synthetic biology to fuels", published in Current Opinion in Biotechnology, volume 19, issue 6, December 2008 pages 556 to 563 provide a review concerning the status and prospective of such biofuels.

Steen, E. J., et . al . in their article titled "Microbial production of fatty-acid-derived fuels and chemicals from plant biomass", published in Nature , volume 463, 28 January 2010, pages 559 to 562 describe the engineering of Escherichia coli to produce structurally tailored fatty esters (biodiesel), fatty alcohols, and waxes directly from simple sugars.

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.

At present, the microorganism systems used for studying biofuels are primarily heterotrophic microorganisms represented by E. coli and Saccharomyces cerevisiae.

S.A. Angermayr et . al. in their article titled "Energy biotechnology with cyanobacteria" , published in Current Opinion in Biotechnology, volume 20, issue 3, June 2009, pages 257 to 263, describes the possibility to fortify photosynthetic organisms with the ability to produce biofuels. The article describes an approach to redirect cyanobacterial intermediary metabolism by channeling intermediates into fermentative metabolic pathways.

J. Dexter et . al . in their article titled "Metabolic engineering of cyanobacter ia for ethanol production", published in Energy & Environmental Science, volume 2, issue 8, 2009, pages 857 to 864 describe the conversion from solar energy to bioethanol (yield of 5.2 mmol /OD₇₃₀ /L/d) by co-expressing the genes of pyruvate decarboxylase and ethanol dehydrogenase derived from Zymomonas mobilis in Synechocystis sp . PCC6803.

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

Technology & Biotechnology volume 84, issue 4, April 2009, pages 473 to 483, describes a reconstruction of a genome-scale Synechocyst is sp . PCC 6803 metabolic network, including 633 genes, 704 metabolites and 831 metabolic reactions . Heterotrophic, photoautotrophic and

mixotrophic growth conditions were simulated and the Synechocystis model was used for in silico predictions for the ethanol fermentation pathway.

P. Lindberg et al. in their article titled "Engineering a platform for photosynthetic isoprene production in cyanobacteria, using Synechocystis as the model organism", published in Metab Eng. volume 12, issue 1, October 2009, pages 70-79 describe the genetic engineering of the cyanobacterium synechocystis, conferring the ability to generate volatile isoprene hydrocarbons from CO(2) and

H(2)0. Heterologous expression of the Pueraria montana (kudzu) isoprene synthase (IspS) gene in Synechocystis enabled photosynthetic isoprene generation in these cyanobacteria.

S. Atsumi et al . , in their article titled "Direct photosynthetic recycling of carbon dioxide to

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 C0₂.

Productivity was increased by overexpres sion of ribulose 1 , 5-bi sphosphate carboxylase/oxygenase (Rubisco) .

X. Liu et al . , in their article titled "Production and secretion of fatty acids in genetically engineered cyanobacteria . " published in Proceedings of the National Academy of Sciences of the USA, 29 March 2010, describe the production and secretion of free fatty acids in genetically modified Synechocystis sp. PCC6803.

It would be an advancement in the art to provide a method for producing fatty alcohols and/or long chain biologic hydrocarbons in cyanobacteria .

It may further be advantageous to construct a route for synthesizing fatty alcohols in cyanobacteria so as to achieve the in vivo synthesis of fatty alcohols in microorganisms.

In addition, it would be an advancement in the art to provide a method that allows for an improved exogenous gene expression efficiency in cyanobacter ia . Such a method allowing highly efficient expression of exogenous genes within cyanobacter ia could be very helpful in any method for producing fatty alcohols and/or long chain biologic hydrocarbons in cyanobacteria .

Summary of the invention

The inventors of the present invention, for the first time, successfully produced fatty alcohols in

cyanobacteria.

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 .

In addition, 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 .

Further the present invention provides a method of expression of exogenous genes via the use of Synechocystis sp. 6803, the main processes of which are:

a) separately constructing a Synechocystis sp. 6803 rbcL promoter (Prbcl, 1.3 kb) and a rbc terminator sequence (Trbc, 02 kb), a 2kb spectinomycin resistant marker gene

Ω and a reporter gene lacZ on a Synechocystis sp. 6803 genome integrative plasmid platform, yielding a platform plasmid pFQ20 which already contains the reporter gene for use in the expression of exogenous genes and which allows genetic integration using Synechocystis sp. 6803;

b) Assay of the effectiveness of exogenous gene expression by the platform via submitting the transformed pFQ20 Synechocystis sp . 6803 algal strain of which the transformant was obtained after spectinomycin resistance screening, to PCR genotype assay using a specific primer, which allows detection of β- galactosidase activity of the transformant expression platform and the exogenous gene engineered strain of Synechocystis sp . 6803, in order to carry out assay of the expression performance of the Synechocystis sp . 6803 exogenous gene expression platform.

Brief description of the drawings

The invention has been illustrated by the non-limiting following figures :

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_pet_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_pet_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 ρΧΊ3Ί¹ο , wherein the far gene is located downstream of the P_pet_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.

Description of the sequences

SEQ ID NO: 1: the sequence of fatty acyl-CoA reductase gene from (Simmondsia chinensis) (artificially

synthesized gene) .

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_petE 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) .

Definition of Terms

The following terms will be understood as defined herein unless otherwise stated. Such definitions include without recitation those meanings associated with these terms known to those skilled in the art.

By a " 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 .

By 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.

By a "Fatty acyl-CoA reductase" is understood an enzyme capable of catalyzing the conversion reaction of fatty acyl-CoA to fatty alcohols.

"Ribulose-1, 5-bisphosphate carboxylase/oxygenase" (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. In the embodiments of the present invention, 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.

"Plastocyanin" (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" . In the embodiments of the present invention, a petE promoter (indicated as P_pet_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. In the embodiments of the present invention 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.

In the embodiments of the present invention, 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.

The term "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. Or 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. Preferably, 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. In general, washing is carried out at T_m = 69.3 + 0.41 (G+C)%, where T_m is in degree s Centigrade and ( G+C ) % refers to the mole percentage of guanine plus cytosine; in line with the article of J. Marmur et al . titled

"Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature", published in Journal of Molecular Biology volume 5, issue 1, July 1962, pages 109-118. However, the T_m of a duplex DNA may decrease by 1°C with every increase of 1% in the number of mismatch base pairs in line with the article of W.M. Bonner et al . titled " A Film Detection Method for

Tritium-Labelled Proteins and Nucleic Acids in

Polyacrylamide Gels", published in the European Journal of Biochemistry, volume 46, issue 1, 1974, pages 83-88.

Formamide is optional in these hybridization conditions. Accordingly, 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; and 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. An extensive guide to the hybridization of nucleic acids can be found in Tijssen (1993) "Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes", Part I, Chapter 2 (Elsevier, New York); Ausubel et al . , eds . (1995) "Current Protocols in Molecular Biology", Chapter

2 (Greene Publishing and Wiley-Inter science , New York); and/or Sambrook et al. (1989) "Molecular Cloning: A Laboratory Manual " (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York) .

The term "identity" or "percent identity" refers to the sequence identity between two amino acid sequences or between two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical positions /total number of positions (e.g., overlapping positions) x 100) . For example, 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. Natl. Acad. Sci. USA 85:2444, 1988), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), and/or by manual alignment and visual inspection (see, e.g., Ausubel et al, Current Protocols in Molecular Biology (1995 supplement ) ) .

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%,

74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

Detailed Description of the Invention

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 .

The use of 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 . In this manner 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. Thus, 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.

In one aspect, 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.

Further, the construct may comprise a marker gene for screening transformants of cyanobacteria, which is located upstream of the promoter having activity in cyanobacter ia .

Preferably such a marker gene comprises the Omega gene as set forth in SEQ ID NO : 8. However, also 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. Suitably such variants also have marker activity in cyanobacteria . Further, 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.

In a most preferred embodiment, the promoter having activity in cyanobacteria is selected from the group consisting of the P_rbc promoter and the PpetE promoter.

However, also 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_rbc promoter or the PpetE promoter can be used. Suitably such variants also have promoting activity in cyanobacteria .

The choice of promoter gene can be of great importance in terms of genetic expression. For example the rbc ( r ibulose-1 , 5-bisphosphate carboxylase /oxygenase ) gene promoter gene (P_rbc ) 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. Especially for Synechocystis PCC6803, 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

Biology 23: 465-476, 1993, incorporated herein by reference ) .

In a further preferred embodiment, 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. In addition, 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 ) . In addition, 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.

In a further preferred embodiment, the marker gene is the Omega fragment of spectinomycin resistance gene, for example as set forth in SEQ ID NO: 8.

Preferably the cyanobacterium is chosen from the group consisting of Synechococcus PCC 6301, Anabaena sp . strain

PCC 7120, Synechococcus PCC 7002 , Synechococcus elongatus sp. strain PCC 7942 and Synechocystis sp. PCC6803. In a most preferred embodiment, the cyanobacterium is

Synechocystis sp . PCC6803.

In another aspect, the embodiments of the present invention may relate to a vector comprising the construct as defined above.

Depending on the state in which the vector exists within the cyanobacteria, 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 .

Preferably, 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

June 28, 2010, in a form in E. coli (Eco-XT51) .

In another aspect, 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. Preferably, 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;

cyanobacterium Syn-XT34, which was deposited in China

General Microbiological Culture Collection Center under Accession Number of CGMCC 3895 on June 10, 2010; and cyanobacterium Syn-XT51, which was deposited in China General Microbiological Culture Collection Center under Accession Number of CGMCC 3896 on June 10, 2010.

In a further aspect, 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.

If desired, 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 ) . In a preferred embodiment 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

hydrocarbons .

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. Such 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. In addition such a biofuel may be considered a real zero emission biofuel.

In another aspect, the embodiments of the present invention may relate to a method of expression of exogenous genes via the use of Synechocystis sp . 6803. Such a method can be helpful in any method for producing fatty alcohols and/or long chain biologic hydrocarbons in cyanobacteria as described above. 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. In a preferred embodiment 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 Ω and/or a reporter gene lacZ that is obtained via cloning.

Examples

The invention is further illustrated by the following non-limiting examples.

Example 1: Construction of vectors for the transformation of cyanobacteria

1. Construction of the plasmid pFQ9R (figure 1)

PCR was performed by using alrl524-l

( 5 ' -ACCTCCAGCCATTAGCG AAAC-3 ' ) and alrl524-2

( 5 ' -CTCTCACAATTGCCCTACCT-3 ' ) as the primer pair and using the genome of Anabaena PCC7120 as the template, and the PCR product was cloned into the vector pMDl8-T (Takara, CatalogNo. : D101A) to obtain the plasmid pQLl . Dral (Takara, Catalog No. : D1037A) was used to digest the plasmid pRL57 (Cai Y. and Wolk C. (1990) "Use of a conditionally lethal gene in Anabaena sp . strain PCC 7120 to select for double recombinants and to entrap insertion sequences." J.

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

( 5 ' -CCCCCGGGGTTACAGTTTTGGCAATTACT-3 ' ) and P4

(5 '-CGAGCTCTTCCCCACTTAGATAAAAAATCCG-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 pFQ2. Sail (Takara, Catalog No.: D1080A) and Xbal (Takara, Catalog No.: D1093A) were used to cut the P_rbc fragment from the plasmid pFQl; Xmal (New England BioLabs, Catalog No . : R018 OS ) and Sacl (Takara, Catalog No. : D1078A) were used to cut the T_rbc fragment from the plasmid pFQ2; the P_rbc and T_rbc fragments were inserted at corresponding site of the plasmid pQL4 to obtain the plasmid pFQ6.

The plasmid pKWll88 (Williams J. G. K. (1988)

"Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803" Methods in Enzymology 167:pages 766-778) was digested with EcoRI and

self -ligated, then blunt-ended with Xmal, and then it was self-ligated to obtain the plasmid pKWH88SL. Hindlll (Takara, Catalog No. : D1060A) and EcoRI (Takara, Catalog No. : D1040A) were used to digest the plasmid pFQ6, and the Omega+Prbc+Trbc fragment was recovered; EcoRI was used to digest the plasmid pKWH88SL; and the two fragments were ligated to obtain the plasmid pFQ9R.

2. Construction of the plasmids pXT37a and pXT37b (figure 3 respectively figure 4)

The plasmid pHBl567 (Gao Hong, et al, (2007)

"Construction of Copper-Induced Gene Expression Platform in Synechocystis sp . PCC6803", Acta Hydrobiologica Sinica, Vol. 31, No. 2, pages 240-244) was digested with Xbal, the 5.4 kb fragment was recovered and self-ligated to obtain the plasmid pXT24. The plasmid pXT24 was digested with Ndel (Takara, Catalog No. : D1161A), blunt-ended with T4 DNA polymerase and self-ligated; then, it was digested with EcoRI, blunt-ended with T4 DNA polymerase and self-ligated to obtain the plasmid pXT24a. 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

( 5 ' -CCCTTTTTTATTTGGTACCGATTAAGGATTCATAGCGGTTGCC-3 ' ) and

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_petE+ 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

( 5 ' -AATCCCCATGTGGAAACCGT-3 ' ) as well as lacZ-m3

( 5 ' -ACGGTTT CCACATGGGGATT-3 ' ) and Ml3-Rev

( 5 ' -AGCGGATAACAATTTCACAC AGGA-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 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.

3. Construction of the plasmid pLY2 (Figure 7)

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.

4. Construction of the plasmid pXTl4 (figure 2)

PCR was performed by using he plasmid pXL66 (a gift from

Professor Chaitan Khosla of Standford University) as the template and using far-1

( 5 ' -GGGTCTAGAATGGAAGAGATGGGCAGCATC-3 ' ) and far-2 ( 5 ' -AAA CCCGGGATCAATTCAGGACATGTTCCACGA-3 ' ) as the primer pair, 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.

5. Construction of the plasmid pXT51 (figure 6)

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.

6. Construction of the plasmid pXT34 (figure 5)

According to the sequence of SEQ ID No: 2, 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.

A summary of the information on the plasmids and strains used for expressing fatty acyl-CoA reductase in

Synechocystis sp. PCC6803 is provided in Table 1.

Example 2 : Transformation of cyanobacter ia and screening of transformants

1. 10 mL of algae cells in logarithmic growth phase (OD₇₃₀ of about 0.5-1.0) was taken, and centrifuged to collect the cells; the cells were washed twice with fresh BGll medium, and then resuspended in 1 mL BGll medium (1.5 g IT¹ NaN0₃, 40 mg IT¹ Κ₂ΗΡ0 ·3Η₂0, 36 mg IT¹ CaCl₂ · 2H₂0, 6 mg LT¹ citric acid, 6 mg LT¹ ferric ammonium citrate, 1 mg LT¹ EDTA disodium salt, 20 mg LT¹ NaC0₃, 2.9 mg LT¹ H₃B0₃, 1.8 mg LT¹ MnCl₂-4H₂0, 0.22 mg LT¹ ZnS0 -7H₂0, 0.39 mg LT¹ NaMo0 -2H₂0, 0.079 mg LT¹ CuS0 -5H₂0 and 0.01 mg LT¹

CoCl₂ · 6H₂0) .

2. 0.2 mL of cell suspension was placed in a new EP tube, 2-3 μg of the expression plasmid as listed in Table 1 was added, and the resulting mixture was mixed well and incubated at 30°C under an illumination condition of 30 μΕ π ² s^_1 for 5 hours.

3. 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 μΕ π ² s^_1 for 24 hours . Then, the nitrocellulose membrane was transferred to a BGll plate containing 10 μg mL^-1 spectinomycin, and further incubated at 30°C under a condition of 30 μΕ π 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 μg mLT¹ spectinomycin ) . After the cells were enriched, they are inoculated into a liquid BG11 medium (containing 20 μg mLT¹ spectinomycin) for

cultivation .

5 After the cells were transferred twice in a liquid medium, the yield of fatty alcohols was measured.

Example 3: Production of fatty alcohols by the genetically engineered cyanobacteria

1. Experimental steps:

(1) Culturing method I : shake-flask culturing . A normal 500-mL conical flask with 300 mL of liquid BG11 medium (containing 20 μg mLT¹ spectinomycin) was used for inoculation with an initial concentration (OD₇₃₀ of 0.05), and the culturing was performed at 30°C, under an illumination condition of 30 μΕ π 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

50 mm and a liquid volume of 500 mL (loading capacity of about 1 L) were used. The initial inoculation concentration was OD₇₃₀ of 0.5, and the culturing was performed at 30°C, under an illumination condition of 100 μΕ π ² s^_1 illumination under aeration with air containing 5% C0₂.

(2) 200 mL of medium was taken, algae cells were collected by centrifugation, and resuspended in 10 mL TE (pH8.0) buffer, and then the cells were disrupted via ultrasonication.

(3) 40 μg pentadecanol (as internal standard) was added to the sonicated cells, and an equivalent volume of chloroform : methanol (v/v 2:1) was added, the resulting mixture was mixed well and kept at room temperature for 0.5 hour .

(4) After centrifuging at 3,000 g for 5 minutes, the organic phase was recovered, and dried at 55°C under blowing with nitrogen gas .

( 5 ) 1 mL n-hexane was added to dissolve the precipitate . After filtration with 0.45 μηι filter membrane, GC-MS analysis was performed.

2. Experimental results :

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 .

The results indicate that the genetically engineered cyanobacteria Syn-XT14, Syn-XT34 and Syn-XT51 were capable of producing fatty alcohols, and this process for producing fatty alcohols can be enlarged in small scale.

1. Information on the plasmids and strains used for expressing fatty acyl-CoA reductase in Synechocystis sp . PCC6803

Table 2. Yields of fatty alcohols of the genetically engineered strains under normal shake-flask culturing conditions

N.D = Not detectable

Table 3. Yields of fatty alcohols of the genetically engineered strains under column photo-reactor culturing conditions

Example 4: Illustration of a method of expression of exogenous genes via the use of Synechocystis sp. 6803 1) The rbcL promoter (Prbcl, 1.3 kb) , the termination sequence rbc terminator (Trbc, 0.2 kb) , the 2kb spectinomycin resistant marker gene Ω 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

automatically after removal of the C.K2 fragment, yielding pKWll88SL), thus yielding the pFQ20 plasmid platform

Prbcl

-term ner already containing the reporter gene designed for the purposes of exogenous gene expression by Synechocystis sp.

6803 and capable of genomic integration.

2) 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 β- 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

1) PCR cloning of the Prbcl and Trbc gene fragments was carried out on Synechocystis sp. 6803; the pFQl plasmid

Prbcl

Sail Xbal

was obtained after Prbcl linked to the pMDl8T vector; the pFQ2 plasmid

was obtained after Trbc linked to the pMDl8T vector. The E.Coli (BL21 DE3 ) cloned LacZ gene (3.1 kb) was inserted into the pUC19 plasmid, yielding the pQLl2 plasmid. lacZ

2) It was confirmed that there were no errors in the nucleotide sequencing after all the plasmid fragments were subjected to sequence analysis.

Example 4b: Cloning of the spectinomycin resistant Ω gene, Prbcl and Trbc were separately in series with the pMDl8T vector .

1) pQL4 is a vector originating in pMDl8T which contains the spectinomycin resistant Ω gene.

Using the Xbal and Sail resistant enzyme plasmid pQL4, linear pQL4EH gene fragments were recovered; the Xbal and Sail resistant enzyme plasmid pFQl was also used to recover

DNA Prbcl fragments; the pQL4EH segments and the segmented Prbcl were connected using T4 ligase, causing conversion of the E.Coli; spectinomycin and ampicillin were used to perform double-resistance transformant screening, then plasmid restriction enzyme assay was carried out to ensure that there were no errors, finally yielding the pFQ5 plasmid;

2) An alternative method: Taking the Smal-Sacl

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;

^Xbal Smal

pFQ6 contained the Ω , Prbcl and Trbc gene fragments; Example 4c: Implanting the spectinomycin resistant Ω gene, Prbcl, Trbc and LacZ genes serially into a cyanobacterial vector .

1) Structure of the expression vector and method of processing: pKWll88 is a plasmid platform used in

Synechocystis sp . 6803 genomic integration. Using EcoRI resistant enzyme pKWll88, linking occurred after removal of the CK2 fragment, yielding pKWl!88SL.

After removal of the Smal locus from pKWll88SL the pFQ15 plasmid was obtained:

2) Processing of target gene: Using Hindlll-Sacl resistant enzyme pFQ6 gel recovery, the Ω, Prbcl and Trbc fragments were obtained, reinforcement with T4 ligase was carried out at 37°C for 30 minutes; the fragments obtained after reinforcement were then subjected to purification using a PCR product purification kit; after the pFQ15 vector expression was cut open using EcoRI further reinforcement and purification was carried out using the same methods; after the Ω, Prbcl and Trbc fragments and pFQ15 fragments had undergone reinforcement they were linked using T4 ligase, converting E.Coli, the transformant was then subjected to PCR assay using specific primers to confirm the direction of insertion of the serially linked fragments, allowing screening of cloned correctly inserted fragments confirming that there were no errors regarding the extracted plasmid resistant enzymes, the product then being named pFQ9Forward.

3) Using Xbal and Smal restricted pQLl2 and gel recovered lacZ gene fragments, these were inserted into pFQ9Forward, converting E.Coli, the lacZ fragments within the transformant then being subjected to PCR assay using specific primers, after it had been confirmed that there were no errors in the extracted plasmid restricted enzyme

Taking PCR amplified 1.3kb Prbcl, Sail and Xbal with identical loci were used to replace the 0.3kb Prbc yielding pFQ20.

Prbcl, 1.3kb

Example 4d: Conversion of the expression vector to

Synechocystis sp . 6803, genetic assay and detection of β- galactosidase activity of the genetically expressed product .

1) A natural method was used to cause pFQ20 conversion of the wild variant of the Synechocystis sp. 6803 cell. The transformant developed around a week later; after the transformant was amplified in BG11 liquid containing spectinomycin for one week the algal solution was harvested and the genomic DNA extracted. PCR assay relying on specific primers was used to detect whether the transformant contained the target LacZ gene. After assay confirmed that there were no errors, the mutated algal strain was named Synechocystis PCC6803 (FQ20 ) .

2) 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, Synechocystis

PC6803(QL15) was used as negative comparison 2.

The Synechocystis PCC6803 ( HB1567 ) strain that exhibits galactosidase expression activity was used as the positive comparison .

PpetE

Of these, the Synechocystis PCC6803(LY2) transgenic algal strain possessed the resistant Ω gene.

3) The results demonstrate that, the galactosidase activity of the constructed genetically engineered Synechocystis 6803FQ20 exceeded that of the known positive comparison under identical conditions, whilst no activity was detected in the negative comparisons.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described . The present embodiments are , therefore, to be considered in all respects as illustrative and not restrictive .

Deposition information of the samples of biological materials :

All of the above strains were deposited at the China General Microbiological Culture Collection Center (CGMCC) , Institute of Microbiology, Chinese Academy of Sciences, No. 1 West Beic en Road, Chaoyang District, Beijing 100 101, China.

Claims

C L A I M S

1. A construct, useful 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.

2. The construct according to claim 1, further comprising a marker gene for screening trans formant s of cyanobacteria, which is located upstream of said promoter having activity in cyanobacteria.

3. The construct according to claim 1 or 2, further comprising, at the two termini thereof, the N-terminal and

C-terminal sequences of slr0168 gene of Synechocyst is sp. PCC6803, for homologous recombination.

4. The construct according to any one of claims 1 to 3, wherein the promoter having activity in cyanobacteria is selected from the group consisting of: a P_rbc promoter comprising a sequence set out in SEQ ID NO : 3 ; a P_petE promoter comprising a sequence set out in SEQ ID NO : 5 ; and variants of these promoters, wherein the variant has at least 80% sequence identity with a sequence set out in SEQ ID NO : 3 or SEQ ID NO : 5.

5. The construct according to any one of claims 1 to 4, wherein said fatty acyl-CoA reductase gene is a gene selected from the group consisting of: fatty acyl-CoA reductase gene from Simmondsia chinensis comprising a sequence as set forth in SEQ ID NO : 1 ; at3gll980 gene from

Arabidopsis thaliana comprising a sequence as set forth in SEQ ID NO : 2 ; variants of these fatty acyl-CoA reductase genes, wherein the variant has at least 80% sequence identity with a sequence set out in SEQ ID NO : 1 or SEQ ID NO : 2 and encodes for a protein having fatty acyl-CoA reductase activity; and genes capable of hybridizing with the above mentioned genes and coding for a protein having fatty acyl-CoA reductase activity.

6. The construct according to any one of claims 1 to 5, wherein said marker gene is the Omega fragment of spectinomycin resistance gene comprising a sequence set forth in SEQ ID NO : 8 ; or a variant thereof that has at least 80% sequence identity with a sequence set out in SEQ ID NO : 8.

7. The construct according to any one of claims 1 to 6, wherein said cyanobacterium is Synechocystis sp. PCC6803.

8. A vector, comprising a construct according to any one of claims 1 to 7.

9. The vector according to claim 8, which 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; plasmid pXT34, which was deposited in China General Microbiological Culture Collection Center under Accession Number of CGMCC 3950 on June 28, 2010; and plasmid pXT51, which was deposited in China General Microbiological

Culture Collection Center under Accession Number of CGMCC 3949 on June 28, 2010.

10. A cyanobacter ium comprising a construct according to any one of claims 1 to 7.

11. A cyanobacterium which is transformed with a vector according to claim 8 or 9.

12. The cyanobacterium according to claim 10 or 11, which is selected from the group consisting of: cyanobacterium Syn-XT14, which was deposited in China General

Microbiological Culture Collection Center under Accession

Number of CGMCC 3894 on June 10, 2010; cyanobacterium Syn-XT34, which was deposited in China General

Microbiological Culture Collection Center under Accession Number of CGMCC 3895 on June 10, 2010; and cyanobacterium Syn-XT51, which was deposited in China General

Microbiological Culture Collection Center under Accession Number of CGMCC 3896 on June 10, 2010.

13. A method for producing fatty alcohols in a

cyanobacterium, comprising:

culturing the cyanobacter ium according to any one of claims 10 to 12 under conditions suitable for the synthesis of fatty alcohols; and

extracting the desired fatty alcohols from the obtained culture .

14. The method of claim 13 further comprising converting the fatty alcohols to hydrocarbons.

15. A biofuel comprising one or more fatty alcOhols produced by the method according to claim 13 or comprising one or more hydrocarbons derived from one or more fatty alcohols produced by the method according to claim 14.

16. A method of expression of exogenous genes via the use of Synechocystis sp . 6803, the main processes of which are: a) separately constructing a Synechocystis sp. 6803 rbcL promoter (Prbcl, 1.3 kb) and a rbc terminator sequence (Trbc, 02 kb), a 2kb spectinomycin resistant marker gene Ω and a reporter gene lacZ on a Synechocystis sp. 6803 genome integrative plasmid platform, yielding a platform plasmid pFQ20 which already contains the reporter gene for use in the expression of exogenous genes and which allows genetic integration using Synechocystis sp. 6803;

b) Assay of the effectiveness of exogenous gene expression by the platform via submitting the transformed pFQ20 Synechocystis sp . 6803 algal strain of which the transformant was obtained after spectinomycin resistance screening, to PCR genotype assay using a specific primer, which allows detection of β- galactosidase activity of the transformant expression platform and the exogenous gene engineered strain of Synechocystis sp . 6803, in order to carry out assay of the expression performance of the Synechocystis sp . 6803 exogenous gene expression platform.