The present invention relates to methods for producing aikanes of a defined chain length. The invention also provides expression vectors, genetically modified ceils and reaction vessels suitable for use in such methods.

BACKGROUND

As a result of their fully saturated composition, most aikanes have ideal properties for combustion and are the major constituents of gasoline, diesel, and jet fuel being either gaseous (C ₄ ) or liquid (C ₅ -C ₂ o) under standard conditions. Despite widespread biological production (Ladygina, N. et ai. 2006 Process Biochem. 41 , 1001-1014), the pathways for alkane synthesis were relatively unknown until recently. Two enzymes were recently identified that are responsible for alkane production in cyanobacteria: an acyl-acyl carrier protein reductase (AAR) and an aldehyde decarbonyiase (AAD) (Schirmer, A. et al. 2010 Science 329, 559-562). These enzymes convert fatty acid intermediates to aikanes and alkenes and their genes can be expressed in E, co!i resulting in the production and secretion of a mixed population of compounds (such as oddchain aikanes and alkenes, as well as even-chain fatty aldehydes and fatty alcohols). Genetically modified cyanobacteria that secrete fatty acids, the precursors of biofuei production, have also been described (Liu et ai, 2011 , PNAS, 108, 17, 6899-6904).

Existing methods of biofuei production have several disadvantages. Firstly, an input of energy is required in the form of carbon foodstuffs such as carbohydrates, to enable biofuel-producing biological organisms to grow. Moreover, extraction of fatty acids or oils from ceils, then further processing and fractionation, is necessary to convert the carbon compounds produced by cells into biofuei. Even in the rare cases when aikanes are produced by ceils directly, before they can be used as fuel, hydrocarbon extraction from the cells has to be followed by a fractionation step to separate useful compounds from the many unsuitable and multiple carbon chain lengths present. All of these factors greatly lower yield and increase the expense of biofuei production.

Accordingly, there remains a need for an improved method for producing biofuels. BRIEF SUM MARY OF THE DISCLOSURE

In one aspect, the invention provides an expression vector comprising: (i) a nucleic acid molecule encoding a polypeptide having LuxC activity:

(ii) a nucleic acid molecule encoding a polypeptide having LuxD activity;

(iii) a nucleic acid molecule encoding a polypeptide having LuxE activity; and

(iv) a nucleic acid molecule encoding a polypeptide having decarbonyiase activity.

In a further aspect, the invention provides a genetically modified fatty acid biogenic ceil comprising:

(i) a nucleic acid molecule encoding a polypeptide having LuxC activity:

(ii) a nucleic acid molecule encoding a polypeptide having LuxD activity;

(iii) a nucleic acid molecule encoding a polypeptide having LuxE activity; and

(iv) a nucleic acid molecule encoding a polypeptide having decarbonyiase activity, wherein said cell is transformed with at least one of said nucleic acid molecules of (i) to (iv). Preferably, the genetically modified fatty acid biogenic cell is transformed with at least two, three or four of said nucleic acid molecules of (i) to (iv).

Preferably, the genetically modified fatty acid biogenic cell is transformed with a single expression vector comprising said at least one, two, three or four nucleic acid molecules.

Preferably, the genetically modified fatty acid biogenic cell is transformed with the expression vector of the invention.

In a further aspect, the invention provides a method of producing an aikane of a defined chain length comprising culturing a fatty acid biogenic cell comprising a nucleic acid molecule encoding a polypeptide having LuxC activity, a nucleic acid molecule encoding a polypeptide having LuxD activity, and a nucleic acid molecule encoding a polypeptide having LuxE activity under conditions that allow expression of each of said polypeptides having LuxC, LuxD, and LuxE activity respectively, in the presence of a carbon source and a decarbonyiase.

Preferably, the fatty acid biogenic ceil is the genetically modified fatty acid biogenic cell of the invention. Preferably, the fatty acid biogenic cell is a bacterial eel Preferably, the bacterial cell is a photosyniheiic ceil. Even more preferably, the

phoiosynthetic cell is a cyanobacterium. Preferably, the cyanobacterium is of the genus Synechocystis, for example Synechocystis sp. PCC6803, or of the genus Synechococcus, for example Synechococcus sp.PCC7002.

Alternatively, the bacterial cell is of the genus Rhodobacter, for example Rhodobacter sphaeroides.

Alternatively, the bacterial cell is of the genus Escherichia, for example E.coii, or of the genus Acinetobacter, for example A. bayiyi ADP1 ,

Preferably, the ceil comprises a nucleic acid molecule encoding a decarbonyiase.

Preferably, said nucleic acid molecule comprises;

a) a nucleic acid molecule having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:9, wherein said nucleotide sequence encodes a polypeptide having decarbonyiase activity;

b) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:9; or

c) a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:9.

Preferably, said nucleic acid molecule encodes a polypeptide selected from the group consisting of:

a) a polypeptide having at least 70% sequence identity to the amino acid sequence of SEQ ID NQ:2 or SEQ ID NO: 10, wherein said polypeptide has decarbonyiase activity;

b) a polypeptide comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 10; or

c) a polypeptide consisting of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 10.

Optionally, the decarbonyiase is provided in the culture medium. Preferably, the decarbonyiase is provided by co-culturing the fatty acid biogenic ceil with a decarbonyiase producing cell.

Preferably, the decarbonyiase is a polypeptide selected from the group consisting of: a) a polypeptide having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 10, wherein said polypeptide has decarbonyiase activity;

b) a polypeptide comprising the amino acid sequence of SEQ ID NQ:2 or SEQ ID NO: 0; or

c) a polypeptide consisting of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO: 10.

Preferably, the nucleic acid molecule encoding a polypeptide having LuxC activity comprises a nucleic acid molecule having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO:3, wherein said nucleotide sequence encodes a polypeptide having LuxC activity.

Preferably, the polypeptide having LuxC activity is a polypeptide having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:4, wherein said polypeptide has LuxC activity.

Preferably, the nucleic acid molecule encoding a polypeptide having LuxC activity encodes a LuxC polypeptide, Preferably, the nucleic acid molecule encoding a LuxC polypeptide is selected from the group consisting of:

a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:3; and b) a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:3, Preferably, the LuxC polypeptide is selected from the group consisting of:

a) a polypeptide comprising the amino acid sequence of SEQ ID NO:4; and

b) a polypeptide consisting of the amino acid sequence of SEQ ID NO:4,

Preferably, the nucleic acid molecule encoding a polypeptide having LuxD activity comprises a nucleic acid molecule having at least 70% sequence identity to the nucleotide sequence of SEQ ID NO:5, wherein said nucleotide sequence encodes a polypeptide having LuxD activity.

Preferably, the polypeptide having LuxD activity is a polypeptide having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:6, wherein said polypeptide has LuxD activity. Preferably, the nucleic acid molecule encoding a polypeptide having LuxD activity encodes a LuxD polypeptide.

Preferably, the nucleic acid molecule encoding a LuxD polypeptide is selected from the group consisting of:

a) a nucleic acid molecule comprising the nucleotide sequence of SEQ I D NO:5; and b) a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:5.

Preferably, the LuxD polypeptide is selected from the group consisting of:

a) a polypeptide comprising the amino acid sequence of SEQ I D NO:6; and

b) a polypeptide consisting of the amino acid sequence of SEQ I D NO:6.

Preferably, the nucleic acid molecule encoding a polypeptide having LuxE activity comprises a nucleic acid molecule having at least 70% sequence identity to the nucleotide sequence of SEQ ID NQ:7, wherein said nucleotide sequence encodes a polypeptide having LuxE activity.

Preferably, the polypeptide having LuxE activity is a polypeptide having at least 70% sequence identity to the amino acid sequence of SEQ I D NO:8, wherein said polypeptide has LuxE activity.

Preferably, the nucleic acid molecule encoding a polypeptide having LuxE activity encodes a LuxE polypeptide. Preferably, the nucleic acid molecule encoding a LuxE polypeptide is selected from the group consisting of:

a) a nucleic acid molecule comprising the nucleotide sequence of SEQ I D NO:7; and b) a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:7. Preferably, the LuxE polypeptide is selected from the group consisting of:

a) a polypeptide comprising the amino acid sequence of SEQ I D NO:8; and

b) a polypeptide consisting of the amino acid sequence of SEQ I D NO:8.

Preferably, the carbon source is carbon dioxide. Alternatively, the carbon source is bicarbonate. Preferably, the alkane of a defined chain length has a carbon chain length in the range of Ci ₃ to C17. Preferably the carbon chain length is selected from the group consisting of d ₃ ,

In a further aspect, the invention relates to the use of a cell according to the invention in the production of an alkane of a defined chain length.

In a further aspect, the invention relates to the use of a LuxCDE expression construct in a recombinant expression system for the production of an alkane of a defined chain length. Preferably, the construct further comprises a nucleic acid molecule encoding a polypeptide having decarbonylase activity.

In a further aspect, the invention provides a reaction vessel containing a fatty acid biogenic ceil according to the invention and medium sufficient to support growth of said cell.

Preferably, the medium provides conditions that allow expression of each of said polypeptides having LuxC, LuxD, and LuxE activity respectively.

Preferably, the reaction vessel is a bioreactor or a fermenter.

Preferably, the method of the invention is performed in the reaction vessel of the invention.

Preferably, the method of the invention further comprises recovering the alkane of a defined chain length from the culture medium, preferably without requiring alkane extraction from the fatty acid biogenic ceil.

In a further aspect, the invention relates to a method of making a biofuel comprising culturing a fatty acid biogenic cell comprising a nucleic acid molecule encoding a polypeptide having LuxC activity, a nucleic acid molecule encoding a polypeptide having LuxD activity, and a nucleic acid molecule encoding a polypeptide having LuxE activity under conditions that allow expression of each of said polypeptides having LuxC, LuxD, and LuxE activity respectively, in the presence of a carbon source and a decarbonylase.

Preferably, the biofuel is recovered from the culture medium, preferably without requiring biofuel extraction from the fatty acid biogenic ceil.

In a further aspect, the invention relates to a biofuel made by the method of the invention. Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of them mean "including but not limited to", and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

Various aspects of the invention are described in further detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

Figure 1 shows the sequence of reactions catalysed by LuxC, LuxD, and LuxE.

Figure 2 shows PGR products from DNA obtained from the pPD-luxCDE-fad construct (A) wild-type Synechocystis (B, D) and iuxCDE-fad Synechocystis strain (C,E) using primers specific for iuxD (A,B,C) and psbAH (D,E). LuxD primers (SEQ ID NO:14 and SEQ ID NO: 15) were used. PsbAli primers (SEQ ID NO:20 and SEQ ID NO: 23) were also used.

Figure 3 shows GC-MS profiles of growth media obtained from wild-type and IuxCDE-fad transformed Synechocystis grown for 4 days under 30 μηιοΙ photons s ^"1 m ^'2 light in the presence of (A) 5m ¹³ C-labeiled glucose and (B) 20 mM ¹³ C-iabelied sodium bicarbonate (IMaHCOa).

Figure 4 shows mass spectra of (A) pure ¹² C heptadecane standard and (B) growth media obtained from IuxCDE-fad transformed Synechocystis grown for 4 days under 30 μηιοΙ photons s ^"1 m ^"2 light in the presence of 5mM ¹³ C-iabelled glucose showing the presence of ¹³ G-iabelled heptadecane.

Figure 5 shows GC- S profiles of growth media obtained from wild-type and !uxCDE-fad transformed E.coii cells grown to log phase and then overnight at 20°C in the presence of 0.4% (w/v) ¹³ C-labelled glucose and 400μ of the inducer isopropyl thiogalactoside (IPTG).

Figure 6 shows mass spectra of (A) pure ¹² C pentadecane standard and (B) growth media obtained from the luxCDE-fad transformed E.coii cells grown to log phase and then overnight at 20°C in the presence of 0.4% (w/v) ¹³ C-labelied glucose and 400μ of the inducer isopropyl thiogalactoside (IPTG) showing the presence of ¹³ C-iabelied

pentadecane. Figure 7 provides the primers used for generation of construct pWH1274: luxCDE-fad. LuxC for (SEQ ID NO: 12); LuxC rev (SEQ ID NO: 13); LuxD for (SEQ ID NO: 14); LuxD rev (SEQ ID NO: 15); LuxE for (SEQ ID NO: 18); LuxE rev (SEQ ID NO: 17); FAD for (SEQ ID NO: 18); FAD rev (SEQ ID NO: 19). Ribosome binding site sequence is shown underlined.

Figure 8 provides the primers used for generation of construct pPD. f1 for (SEQ ID NO: 20); f1 rev (SEQ ID NO: 21); f2 for (SEQ ID NO: 22); f2 rev (SEQ ID NO: 23).

Figure 9 provides the primers used for generation of construct pPD: LuxCDE-fad. f1 LuxC for (SEQ ID NO: 24); f2 FAD rev (SEQ ID NO: 25).

Figure 10 provides the nucleotide sequence for Photorhabdus luminescens luxC (fatty acid reductase) GenBank: 90093.1 {Photorhabdus luminescens iuxCDABE operon) (SEQ ID NO: 3).

Figure 1 provides the protein sequence for Photorhabdus luminescens LuxC (fatty acid reductase) GenBank: AAA27617.1 (SEQ ID NO: 4).

Figure 12 provides the nucleotide sequence for Photorhabdus luminescens luxD (acyl transferase) GenBank: M90093.1 (Photorhabdus luminescens iuxCDABE operon) (SEQ ID NO: 5). Figure 3 provides the protein sequence for Photorhabdus luminescens LuxD (acyl transferase) GenBank: AAA27618.1 (SEQ ID NO: 8).

Figure 4 provides the nucleotide sequence for Photorhabdus luminescens iuxE (acyl- protein synthetase) GenBank: 90093.1 {Photorhabdus luminescens luxCDABE operon) (SEQ ID NO: 7).

Figure 15 provides the protein sequence for Photorhabdus luminescens LuxE (acyl-protein synthetase) GenBank: AAA27621.1 (SEQ ID NO: 8),

Figure 16 provides the nucieotide sequence for Synechococcus elongatus PCC 7942 fatty aldehyde decarbonylase GenBank: CP000100.1 (Synechococcus elongatus PCC 7942 DNA, complete genome), locus Synpcc7942_1593 (SEQ ID NO: 1). Figure 17 provides the protein sequence for Synechococcus elongatus PCC 7942 fatty aldehyde decarbonylase locus Synpcc7942__1593, GenBank: AAB57823.1 (SEQ ID NO: 2).

Figure 18 provides the nucleotide sequence for Synechocystis sp. PCC 6803, locus sll0208 (encodes hypothetical protein that shows homology to Synechococcus elongatus PCC 7942 fatty aldehyde decarbonylase) Genbank: BA000022.2 [Synechocystis sp. PCC 6803 chromosome, complete genome) (SEQ ID NO: 9).

Figure 9 provides the protein sequence for Synechocystis sp. PCC 6803, locus sll0208 (encodes hypothetical protein that shows homology to Synechococcus elongatus PCC 7942 fatty aldehyde decarbonylase) Genbank: BAA10217.1 (SEQ ID NO: 10).

Figure 20 provides the nucleotide sequence for E. coii - Acinetobacter baylyi shuttle plasmid pWH1274 GenBank: JN381 160 (SEQ ID NO: 11). Figure 21 provides the nucleotide sequence for the codon-optimised luxCDE-fad gene cluster inserted into the pET3a vector for expression in E.coii (SEQ ID NO: 26).

Figure 22 provides a general schematic of fatty acid metabolism. DETAILED DESCRIPTION

Fatty acids are essentia! components of cell membranes and are important sources of metabolic energy in ail organisms. The regulation of fatty acid degradation and

biosynthesis is essential to maintain membrane lipid homeostasis.

Fatty acids of various chain lengths may be used as carbon and energy sources within an organism. During fatty acid metabolism, fatty acids are degraded via the β-oxidation pathway. A general schematic of fatty acid metabolism is provided in Figure 22.

The applicants have surprisingly shown that the fatty acid metabolic pathway in bacteria can be reconfigured so as to result in the production of aikanes of a defined chain length where the chain length is not C ₁₃ as expected from the known function of LuxD, but rather Ci5 and C17. Advantageously, the longer-chain aikanes produced by this reconfigured pathway are particularly suitable for use as a biofuel and require minimal subsequent processing for biofuel applications.

The invention is based on the surprising and unexpected finding that elements of the Lux operon can be combined with a polypeptide having decarbonylase activity so as to reroute the fatty acid metabolic pathway to produce longer-chain (e.g. >Ci ₃ ) aikanes of a defined chain length.

The Lux bioluminescent gene cluster has known application in bioluminescent studies, acting as a reporter of bacteria! gene expression in public health and ciinicai laboratories. The Lux bioluminescent gene cluster is found in a number of bioluminescent bacteria, for example Vibrio fischeri, Vibrio harveyi and Photorhabdus iuminescens, Bioiuminescence is caused by transcription of the iux operon, which is induced by population-dependent quorum sensing. Five genes within the lux operon, \uxCDABE, are required for the emission of visible light, and two genes, luxR and /ax/, are involved in regulating the operon. LuxA and iuxB encode the components of a iuciferase, whereas iuxC, iuxD and iuxE encode acyi-reductase, acyi-transferase, and acyl-protein synthetase respectively.

It has been shown that the fatty acid reductase subunits encoded by the iuxC, IuxD, and IuxE genes (for example the IuxC, IuxD, and IuxE genes of V. harveyi and V, fischeri) have a high specificity for C ₁ acyl ACP chains, resulting in the production of a C ₁₄ aldehyde, tetradecanal (Li et a!. , Biochimica et Biophysica Acta (2000) 237-246; Uiifzer et al., PNAS USA 1978, 266-269). The LuxC, LuxD and LuxE proteins synthesise an aldehyde of defined chain length (C _i4 ) as a substrate for the LuxAB luciferase, which catalyses light-emitting reaction:

FM H ₂ + 0 ₂ + RCHO (aldehyde)→ F N + H ₂ 0 + RCOOH + light

In the present invention, the LuxC, LuxD and LuxE proteins are used to synthesise aldehydes of defined chain lengths that differ from d ₄ , including aldehydes with chain lengths comprising 16 or more carbon atoms. This unprecedented discovery allows aldehydes to be used as substrates for a decarbonylase reaction to produce C13-C17 alkanes of a defined chain length. The reaction steps are as follows: Firstly, an acyl-ACP thioesterase (LuxD) cleaves a fatty acyi group from an acyl-acyl carrier protein (ACP) formed during fatty acid biosynthesis, and forms a fatty acid with a defined carbon chain length acid as illustrated in Figure 1. Next, acyi-protein synthetase (LuxE) and acyi-CoA reductase (LuxC) activate the specific long-chain fatty acid and then catalyse its NADPH- dependent reduction to an aldehyde. In the final step this aldehyde is available for conversion to an alkane via, for example, the fatty aldehyde decarbonylase (FAD). This enzyme can reduce long chain aldehydes to the corresponding alkane. Other enzymes may also be utilised to convert aldehydes to alkanes, in reactions not involving carbon loss.

The invention is exemplified above using the LuxC, LuxD, LuxE and FAD proteins. However, the invention may also be achieved using other polypeptides with LuxC, LuxD, LuxE and decarbonylase activity respectively. The invention includes controlled modification of alkane chain length using LuxD proteins from a variety of bacteria, and the use of native and engineered LuxD modules within the LuxC- LuxD- LuxE- FAD ensemble of enzymes.

The invention overcomes a number of the disadvantages of the prior art The invention enables bacteria that require minimal nutrient requirements to be utilised to produce biofuel. In particular, the invention enables photosynthetic bacteria to directly convert C0 ₂ to biofueis consisting of defined and fixed C _3- i ₇ alkanes.

Surprisingly, the alkane biofuel is not sequestered within the bacterial cell, and instead i excreted into the growth medium, removing the requirement for extraction of the ceils using potentially toxic and expensive solvents. The defined carbon chain length of the alkane biofuel removes the need for subsequent fractionation. The length of the

hydrocarbon chain makes it ideal for direct use as a biodiesel. Biofuei

As used herein, the term 'biofuei' refers to any fuel that is derived from a biological source. Biofuei can refer to one or more hydrocarbons, one or more alcohols, or one or more fatty esters or a mixture thereof. Biofuei includes but is not limited to bio-diesei, bio-gasoline and biofuei for jets.

As used herein, the term "hydrocarbon" refers to a chemical compound that contains the elements carbon (C), hydrogen (H), and optionally oxygen (O). There are essentially three types of hydrocarbons, e.g. aromatic hydrocarbons, saturated hydrocarbons (e.g. aikanes) and unsaturated hydrocarbons. The term includes fuels, biofueis, plastics, waxes, solvents, and oils.

As used herein, the term "aikane", refers to chemical compounds that consist only of the elements carbon (C) and hydrogen (H) (i.e. hydrocarbons) wherein these atoms are linked together exclusively by single bonds (i.e. they are saturated compounds) without any cyclic structure. Aikanes are also known as paraffins or saturated hydrocarbons and are the major constituents of diesel, gasoline and jet fuel. As used herein, the term "defined chain length" refers to an aikane of with a fixed number of carbon elements. In the context of the invention, the term "defined chain length" is used to mean that the aikane product does not comprise a mixture of aikanes with different carbon chain lengths, but that the aikanes produced all have the same number of carbons. In one aspect of the invention, the aikane has a carbon chain length of C ₄ , C ₅ , C ₆ , C ₇ , C ₈ ,

9, On), n , l 2, n, l 4, 15, U16, U | 7, 18, 19, U20. 1 , 2> ^-»22, 4> 5> ^26, 7.

C29, C ₃₀ , C31 , C ₃₂ , C _33l C34, C35, or C ₃₆ . More preferably, the aikane has a carbon chain length of C ₁₃ , C ₁₅ , or C ₁₇ . In certain embodiments of the invention, the specific cell used may affect the aikane carbon chain length produced. By way of example, the applicants have shown that cells of the genus Synechocystis are capable of producing C ₁₇ aikanes, whereas E.coii are capable of producing C ₁₅ aikanes. Nucleic acid molecules and polypeptides

As used herein, the term "nucleic acid molecule" includes DNA molecules (e.g. , a cDNA or genomic DNA) and RNA molecules (e.g. , a mRNA) and analogs of the DNA or R NA generated, e.g., by the use of nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.

With regard to genomic DNA, the term "isolated" includes nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an "isolated" nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5'- and/or 3'-ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.

As used herein, the term "hybridizes under stringent conditions" describes conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in available references (e.g., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1 -6.3.8). Aqueous and non-aqueous methods are described in that reference and either can be used. A preferred example of stringent hybridization conditions are hybridization in 6x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in G,2x SSC, 0.1 % (w/v) SDS at 50°C. Another example of stringent hybridization conditions are hybridization in 6x SSC at about 45°C, followed by one or more washes in Q.2x SSC, 0.1 % (w/v) SDS at 55°C. A further example of stringent hybridization conditions are hybridization in 8x SSC at about 45°C, followed by one or more washes in 0.2x SSC, 0.1 % (w/v) SDS at 60°C. Preferably, stringent hybridization conditions are hybridization in 6x SSC at about 45°C, followed by one or more washes in 0.2x SSC, 0.1 % (w/v) SDS at 65°C. Particularly preferred stringency conditions (and the conditions that should be used if the practitioner is uncertain about what conditions should be applied to determine if a molecule is within a hybridization limitation of the invention) are 0.5 molar sodium phosphate, 7% (w/v) SDS at 65°C, followed by one or more washes at 0.2x SSC, 1 % (w/v) SDS at 65°C. Preferably, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence of SEQ ID NO:1 , 3, 5, 7 or 9.

As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). As used herein, the terms "gene" refers to nucleic acid molecules which include an open reading frame encoding protein, and can further include non-coding regulatory sequences and introns. As used herein, the term "recombinant" refers to a biomolecuie, for example a gene or a protein that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a nucleic acid molecule as it is found in nature, (3) is operativeiy linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature,

As used herein, "recombinant expression system" refers to one or more expression vectors comprising nucleic acid molecules that are recombinant in respect of the host cell.

A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of (e.g., the sequence of SEQ ID NO:2, 4, 8, 8 or 10) without abolishing or, more preferably, without substantially altering a biological activity, whereas an "essential" amino acid residue results in such a change. For example, amino acid residues that are conserved among the polypeptides of the present invention are predicted to be particularly non-amenable to alteration, except that amino acid residues in transmembrane domains can generally be replaced by other residues having approximately equivalent

hydrophobicity without significantly altering activity.

A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoieucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoieucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in protein is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of coding sequences, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis of SEQ ID NO: 1 , 3, 5, 7 or 9, the encoded proteins can be expressed recombinanfly and the biological activity of the protein can be determined. As used herein, a "biologically active portion" of protein or a protein portion with "biological activity" includes a fragment of protein that participates in an interaction between molecules and non-molecules. Biologically active portions of protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the protein, e.g., the amino acid sequences shown in SEQ ID NO: 2, 4, 6, 8, or 10, which include fewer amino acids than the full length protein, and exhibit at least one activity of the encoded protein. Typically, biologically active portions comprise a domain or motif with at least one activity of the protein, e.g., the biologically active portion may retain one of the following activities (as appropriate); decarbonylase activity, LuxC activity, LuxD activity, or LuxE activity.

A biologically active portion of protein can be a polypeptide that is, for example, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more amino acids in length of SEQ ID NO: 2, 4, 6, 8 or 10. Biologically active portions of protein can be used as targets for developing agents that modulate mediated activities, e.g., biological activities described herein.

Calculations of sequence homology or identity (the terms are used interchangeably herein) between sequences are performed as follows.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred embodiment, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman et al. (1970) J. Μοί Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a BLOSU 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. in yet another preferred embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.C P matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1 , 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of the invention) are a BLOSUM 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

Alternatively, the percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of Meyers et ai. (1989) CABIOS 4: 1 -17) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the

BLAST and XBLAST programs (version 2.0) of Aitschui, et ai. (1990) J. Μοί Biol.

215:403-410). BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be utilized as described in Aitschui et al. (1997, Nuci. Acids Res.

25:3389-3402). When using BLAST and gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See

<http : //www. ncbi.nim.nih.gov>. The polypeptides described herein can have amino acid sequences sufficiently or substantially identical to the amino acid sequences of SEQ ID NO:2, 4, 6, 8 or 10. The terms "sufficiently identical" or "substantially identical" are used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g. with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain or common functional activity. For example, amino acid or nucleotide sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as sufficiently or substantially identical.

Preferably expression vectors and fatty acid biogenic cells of the present invention comprise a nucleic acid sequence encoding a polypeptide having decarbonylase activity. A polypeptide having "decarbonylase activity", as used herein, refers to a polypeptide that retains the functional enzymatic activity of a decarbonylase i.e. is capable of converting a fatty acid aldehyde to a hydrocarbon (in reactions involving or not involving carbon loss). As used herein, polypeptides having "decarbonylase activity" include ADM (aikanal decarboxylative monooxygenase enzyme), fatty aldehyde decarbonylase (FAD), and any other enzymes that are capable of converting a fatty acid aldehyde to a hydrocarbon. As used herein, polypeptides having "decarbonylase activity" also include by way of example pea (Pisum sativum), an octadecanal decarbonylase that converts octadecanai to heptadecane (Cheesbrough, T.M. and, Koiattukudy, P.E, Proc, Natl. Acad. Sci. USA, 1984 81 6613-17); a cobalt requiring decarbonylase in the green colonial algae Botryococcus braunii (Dennis, ., and Koiattukudy, P.E. Proc. Natl. Acad. Sci. USA, 1992 89 5306-10); and the protein encoded by the CER1 gene in Arabidopsis thaliana which is involved in wax biosynthesis (M.G.Aarts et al, P.E. Plant Cell, 1995 7 21215-27).

A nucleic acid molecule encoding a polypeptide having decarbonylase activity may be endogenous to the fatty acid biogenic ceil. Alternatively, or in addition, a nucleic acid molecule encoding a polypeptide having decarbonylase activity may be introduced into the cell by transforming the ceil with an expression vector comprising said nucleic acid molecule. By way of example, the FAD enzyme is naturally present in many, but not ail cyanobacteria, and can reduce long chain aldehydes to the corresponding aikane.

The nucleic acid molecule encoding a polypeptide having decarbonylase activity preferably encodes a decarbonylase polypeptide. Preferably, the nucleic acid molecule encodes a fatty aldehyde decarbonyiase (FAD) derived from the bacterium Synechococcus elongatus, which is encoded by the nucleic acid of SEQ ID NO: 1 or 9 and which has the amino acid sequence of SEQ ID NO:2 or 10 respectively. In one embodiment, the nucleic acid molecule encoding a polypeptide having

decarbonyiase activity is at least about: 80%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 00%, homologous to the entire length of the nucleotide sequence shown in SEQ ID NO: 1 or 9, or portions or fragments thereof. In a further embodiment, the nucleic acid molecule encoding a polypeptide having decarbonyiase activity is a variant nucleic acid molecule that hybridizes to SEQ ID NO: 1 or 9 and encodes a polypeptide that has decarbonyiase activity.

In one embodiment the nucleic acid molecule encoding a polypeptide having

decarbonyiase activity encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 2 or 10. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO: 2 or 10, or substitution, deletion or insertion of non-critical residues in non- critical regions of the protein. Non-functional allelic variants are naturally occurring amino acid sequence variants of SEQ ID NO: 2 or 10 that do not have decarbonyiase activity. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 2 or 10, or a substitution, insertion or deletion in critical residues or critical regions. Nucleic acid molecules corresponding to natural allelic variants and homoiogues of the

decarbonyiase nucleic acid molecules of the invention can be isolated based on their homology to the nucleic acid molecules of the invention using the nucleotide sequences described in SEQ ID NO: 1 or 9 or a portion thereof, as a hybridization probe under stringent hybridization conditions. In another embodiment, the nucleic acid molecule encoding a polypeptide having decarbonyiase activity comprises a nucleotide sequence that encodes the polypeptide of SEQ ID NO: 2 or portions or fragments thereof. In another embodiment, the nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide that is at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, homologous to the entire length the polypeptide of SEQ ID NO: 2, or portions or fragments thereof. Preferably expression vectors and fatty acid biogenic cells of the present invention comprise a nucleic acid sequence encoding a polypeptide having LuxC activity, A polypeptide having "LuxC activity", as used herein, refers to a polypeptide that retains the functional enzymatic activity of LuxC i.e. which catalyses the reduction of a fatty acyl group to a fatty aldehyde by NADPH (nicotinamide adenine dinucleotide phosphate).

Polypeptides having "LuxC activity" include, for example, acyl-reductases, AAR (acyl-ACP reductase) and FAR (fatty acyl CoA reductase).

A nucleic acid molecule encoding a polypeptide having LuxC activity may be endogenous to the fatty acid biogenic ceil. Alternatively, or in addition, a nucleic acid molecule encoding a polypeptide having LuxC activity may be introduced into the ceil by transforming the cell with an expression vector comprising said nucleic acid molecule.

The nucleic acid molecule encoding a polypeptide having LuxC activity preferably encodes the LuxC polypeptide derived from Photorhabdus luminescens, which is encoded by the nucleic acid of SEQ ID NO:3 and which has the amino acid sequence of SEQ ID NO:4.

In one embodiment, the nucleic acid molecule encoding a polypeptide having LuxC activity is at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 98%, 97%, 98%, 99% or 100%, homologous to the entire length of the nucleotide sequence shown in SEQ ID NO:3, or portions or fragments thereof.

In a further embodiment, the nucleic acid molecule encoding a polypeptide having LuxC activity is a variant nucleic acid molecule that hybridizes to SEQ ID NO:3 and encodes a polypeptide that has LuxC activity.

In one embodiment the nucleic acid molecule encoding a polypeptide having LuxC activity encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 4. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:4, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein. Nonfunctional allelic variants are naturally occurring amino acid sequence variants of SEQ ID NO: 4 that do not have LuxC activity. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 4, or a substitution, insertion or deletion in critical residues or critical regions. Nucleic acid molecules corresponding to natural allelic variants and homologues of the LuxC nucleic acid molecules of the invention can be isolated based on their homology to the nucleic acid molecules of the invention using the nucleotide sequences described in SEQ ID NO:3 or a portion thereof, as a hybridization probe under stringent hybridization conditions. In another embodiment, the nucleic acid molecule encoding a polypeptide having LuxC activity comprises a nucleotide sequence that encodes the polypeptide of SEQ ID NO: 4 or portions or fragments thereof. In another embodiment, the nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide that is at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, homologous to the entire length the polypeptide of SEQ ID NO: 4, or portions or fragments thereof.

Preferably expression vectors and fatty acid biogenic cells of the present invention comprise a nucleic acid sequence encoding a polypeptide having LuxD activity. A polypeptide having "LuxD activity", as used herein, refers to a polypeptide that retains the functional enzymatic activity of LuxD i.e. the cleavage of the fatty acyi group from an acyl- acyi carrier protein (acyl-ACP) to form a fatty acid with a defined chain length.

Polypeptides having "LuxD activity" include, for example, acyl transferases. Acyl-ACP thioesterase is an alternative name for LuxD.

A nucleic acid molecule encoding a polypeptide having LuxD activity may be endogenous to the fatty acid biogenic ceil. Alternatively, or in addition, a nucleic acid molecule encoding a polypeptide having LuxD activity may be introduced into the cell by transforming the cell with an expression vector comprising said nucleic acid molecule.

The nucleic acid molecule encoding a polypeptide having LuxD activity preferably encodes the LuxD polypeptide derived from Photorhabdus iuminescens, which is encoded by the nucleic acid of SEQ ID NO:5 and which has the amino acid sequence of SEQ ID NO:6. In one embodiment, the nucleic acid molecule encoding a polypeptide having LuxD activity is at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, homologous to the entire length of the nucleotide sequence shown in SEQ ID NO:5, or portions or fragments thereof. In a further embodiment, the nucleic acid molecule encoding a polypeptide having LuxD activity is a variant nucleic acid molecule that hybridizes to SEQ ID NO:5 and encodes a polypeptide that has LuxD activity. In one embodiment the nucleic acid molecule encoding a polypeptide having LuxD activity encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 6. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NQ:8, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein. Nonfunctional allelic variants are naturally occurring amino acid sequence variants of SEQ ID NO: 6 that do not have LuxD activity. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 6, or a substitution, insertion or deletion in critical residues or critical regions. Nucleic acid molecules corresponding to natural allelic variants and homoiogues of the LuxD nucleic acid molecules of the invention can be isolated based on their homology to the nucleic acid molecules of the invention using the nucleotide sequences described in SEQ ID NO:5 or a portion thereof, as a hybridization probe under stringent hybridization conditions.

In another embodiment, the nucleic acid molecule encoding a polypeptide having LuxD activity comprises a nucleotide sequence that encodes the polypeptide of SEQ ID NO: 8 or portions or fragments thereof, in another embodiment, the nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide that is at least about: 80%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, homologous to the entire length the polypeptide of SEQ ID NO: 6, or portions or fragments thereof. Preferably expression vectors and fatty acid biogenic cells of the present invention comprise a nucleic acid sequence encoding a polypeptide having LuxE activity. A

"polypeptide having LuxE activity", as used herein, refers to a polypeptide that retains the functional enzymatic activity of LuxE i.e. activation of the fatty acyi group with ATP

(adenosine triphosphate) and its transfer to LuxC. Polypeptides having "LuxE activity" include, for example, acyi protein synthases.

A nucleic acid molecule encoding a polypeptide having LuxE activity may be endogenous to the fatty acid biogenic ceil. Alternatively, or in addition, a nucleic acid molecule encoding a polypeptide having LuxE activity may be introduced into the ceil by transforming the cell with an expression vector comprising said nucleic acid molecule. The nucleic acid molecule encoding a polypeptide having LuxE activity preferably encodes the LuxE polypeptide derived from Photorhabdus luminescens, which is encoded by the nucleic acid of SEQ ID NO:7 and which has the amino acid sequence of SEQ ID NO:8. In one embodiment, the nucleic acid molecule encoding a polypeptide having LuxE activity is at least about: 80%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, homologous to the entire length of the nucleotide sequence shown in SEQ ID NO:7, or portions or fragments thereof. In a further embodiment, the nucleic acid molecule encoding a polypeptide having LuxE activity is a variant nucleic acid molecule that hybridizes to SEQ ID NQ:7 and encodes a polypeptide that has LuxE activity.

In one embodiment the nucleic acid molecule encoding a polypeptide having LuxE activity encodes a naturally occurring allelic variant of a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 8. Functional allelic variants will typically contain only conservative substitution of one or more amino acids of SEQ ID NO:8, or substitution, deletion or insertion of non-critical residues in non-critical regions of the protein. Nonfunctional allelic variants are naturally occurring amino acid sequence variants of SEQ ID NO: 8 that do not have LuxE activity. Non-functional allelic variants will typically contain a non-conservative substitution, a deletion, or insertion or premature truncation of the amino acid sequence of SEQ ID NO: 8, or a substitution, insertion or deletion in critical residues or critical regions. Nucleic acid molecules corresponding to natural allelic variants and homologues of the LuxE nucleic acid molecules of the invention can be isolated based on their homology to the nucleic acid molecules of the invention using the nucleotide sequences described in SEQ ID NO:7 or a portion thereof, as a hybridization probe under stringent hybridization conditions.

In another embodiment, the nucleic acid molecule encoding a polypeptide having LuxE activity comprises a nucleotide sequence that encodes the polypeptide of SEQ ID NO: 8 or portions or fragments thereof. In another embodiment, the nucleic acid molecule comprises a nucleotide sequence that encodes a polypeptide that is at least about: 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, homologous to the entire length the polypeptide of SEQ ID NO: 8, or portions or fragments thereof. In another embodiment any of the nucleic acid molecules described previously, comprises specific changes in the nucleotide sequence so as to optimize codons and mRNA secondary structure for translation in the host cell. Preferably, the codon usage of the nucleic acid is adapted for expression in the host cell, for example codon optimisation can be achieved using Calcgene, Hale, RS and Thomas G. Protein Exper, Purif. 12, 185-188 (1998), UpGene, Gao, W et ai. Biotechnol. Prog. 20, 443-448 (2004), or Codon Optimizer, Fugisang, A. Protein Exper, Purif. 31 , 247-249 (2003). Amending the nucleic acid according to the preferred codon optimization can be achieved by a number of different experimental protocols, including, modification of a small number of codons, Vervoort et a/, Nucleic Acids Res. 25: 2069-2074 (2000), or rewriting a large section of the nucleic acid sequence, for example, up to 1000 bp of DNA, Hale, RS and Thomas G. Protein Exper. Purif. 12,185-188 (1998). Rewriting of the nucleic acid sequence can be achieved by recursive PGR, where the desired sequence is produced by the extension of overlapping oligonucleotide primers, Prodromou and Pearl, Protein Eng. 5: 827-829 (1992). Rewriting of larger stretches of DNA may require up to three consecutive rounds of recursive PCR, Hale, RS and Thomas G. Protein Exper. Purif. 12, 185-188 (1998), Te'o et al, FEMS MicrobioL Lett. 190: 13-19, (2000).

Alternatively, the level of cognate tRNA can be elevated in the host cell. This elevation can be achieved by increasing the copy number of the respective tRNA gene, for example by inserting into the host cell the relevant tRNA gene on a compatible multiple copy plasmid, or alternatively inserting the tRNA gene into the expression vector itself.

In another embodiment any of the nucleic acid molecules described previously, comprises specific changes in the nucleotide sequence so as to optimize expression, activity or functional life of the encoded poiypeptide(s). Preferably, the nucleic acids described previously are subjected to genetic manipulation and disruption techniques. Various genetic manipulation and disruption techniques are known in the art including, but not limited to, DNA Shuffling (US 8, 132,970, Punnonen J et al, Science & Medicine, 7(2): 38- 47, (2000), US 8, 132,970), serial mutagenesis and screening. One example of mutagenesis is error-prone PCR, whereby mutations are deliberately introduced during PCR through the use of error-prone DNA polymerases and reaction conditions as described in US 2003152944, using for example commercially available kits such as The GeneMorph ^® I! kit (strstagerie*, us). Randomized DNA sequences are cloned into expression vectors and the resulting mutant libraries screened for altered or improved protein activity. Expression vector

As used herein, the term "vector" or "construct" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. The terms "vector" and "construct" are used interchangeably herein. The vector can be capable of autonomous replication or it can integrate into a host DNA. The vector may include restriction enzyme sites for insertion of recombinant DNA and may include one or more selectable markers. The vector can be a nucleic acid in the form of a plasmid, a bacteriophage or a cosmid. Preferably the vector is suitable for expression in a ceil (i.e. the vector is an "expression vector"). Preferably, the expression vector is suitable for expression in a fatty acid biogenic ceil. Most preferably, the vector is suitable for expression in bacteria, for example in a photosynthetic bacterial cell such as cyanobacferia (most preferably of the genus

Synechocystis or Synechococcus) or, for example, in bacteria of the genus Acinetobacter or Escherichia. Preferably the vector is capable of propagation in a host ceil and is stably transmitted to future generations.

"Operably linked" as used herein, refers to a single or a combination of the below- described control elements together with a coding sequence in a functional relationship with one another, for example, in a linked relationship so as to direct expression of the coding sequence.

"Regulatory sequences" as used herein, refers to, DNA or RNA elements that are capable of controlling gene expression. Examples of expression control sequences include promoters, enhancers, silencers, Shine Dalgarno sequences, TATA- boxes, internal ribosomal entry sites (IRES), attachment sites for transcription factors, transcriptional terminators, polyadenylation sites, RNA transporting signals or sequences important for UV-iight mediated gene response. Preferably the vector includes one or more regulatory sequences operativeiy linked to the nucleic acid sequence to be expressed. Regulatory sequences include those which direct constitutive expression, as well as tissue-specific regulatory and/or inducible sequences.

"Promoter", as used herein, refers to the nucleotide sequences in DNA or RNA to which RNA polymerase binds to begin transcription. The promoter may be inducible or constitutively expressed. Alternatively, the promoter is under the control of a repressor or stimulatory protein. Preferably the promoter is a T7, T3, lac, lac UV5, tac, trc, [iambda]PL, Sp6 or a UV-inducible promoter. "Transcriptional terminator" as used herein, refers to a DNA element, which terminates the function of RNA polymerases responsible for transcribing DNA into RNA. Preferred transcriptional terminators are characterized by a run of T residues preceded by a GC rich dyad symmetrical region.

"Translational control element", as used herein, refers to DNA or RNA elements that control the translation of mRNA. Preferred translational control elements are ribosome binding sites. Preferably, the translational control element is from a homologous system as the promoter, for example a promoter and its associated ribozyme binding site.

Preferred ribosome binding sites are T7 or T3 ribosome binding sites.

"Restriction enzyme recognition site" as used herein, refers to a motif on the DNA recognized by a restriction enzyme.

"Selectable marker" as used herein, refers to proteins that, when expressed in a host cell, confer a phenotype onto the ceil which allows a selection of the cell expressing said selectable marker gene. Generally this may be a protein that confers resistance to an antibiotic such as ampiciilin, kanamycin, chloramphenicol, tetracyclin, hygromycin, neomycin or methotrexate. Further examples of antibiotics are Penicillins; Ampiciilin HCI, Ampiciilin Na, Amoxycillin Na, Carbeniciliin sodium, Penicillin G, Cephalosporins,

Cefotaxim Na, Cefalexin HCI, Vancomycin, Cycloserine. Other examples include

Bacteriostatic inhibitors such as: Chloramphenicol, Erythromycin, Lincomycin, Tetracyclin, Spectinomycin sulfate, Clindamycin HCI, Chlortetracyciine HCI.

The design of the expression vector depends on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, and the like. The expression vectors of the invention can be introduced into host ceils to thereby produce proteins or polypeptides, including fusion proteins or polypeptides, encoded by nucleic acids as described herein (e.g., the decarbonyiase polypeptide encoded by the nucleic acid molecule of SEQ ID NO: 1 or SEQ ID NO: 9, the LuxC polypeptide encoded by the nucleic acid molecule of SEQ ID NO:3, the LuxD polypeptide encoded by the nucleic acid molecule of SEQ ID NO:5, or the LuxE polypeptide encoded by the nucleic acid molecule of SEQ ID NO:7).

Expression of proteins in prokaryotes is most often carried out in a bacterial host cell with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus of the recombinant protein. Such fusion vectors typically serve three purposes: 1 ) to increase expression of recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such vectors are within the scope of the present invention.

Preferably the vector comprises those genetic elements which are necessary for expression of any one, two, three or four of a decarbonylase, LuxC, LuxD and/or LuxE protein(s) in a host cell. The elements required for transcription and translation in the host ceil include a promoter, a coding region for the protein(s) of interest, and a transcriptional terminator.

Expression vectors of the invention can be bacterial expression vectors, for example recombinant bacteriophage DNA, piasmid DNA or cosmid DNA, yeast expression vectors e.g. recombinant yeast expression vectors, vectors for expression in insect cells, e.g. , recombinant virus expression vectors, for example baculovirus, or vectors for expression in plant ceils, e.g. recombinant virus expression vectors such as cauliflower mosaic virus, Ca V, tobacco mosaic virus, T V, or recombinant piasmid expression vectors such as Ti plasmids. Preferably, the vector is suitable for expression in photosynthetic bacterial ceils such as cyanobacteria (most preferably of the genus Synechocystis or Synechococcus). For example, the vector is preferably pPD. Alternatively, and also preferably, the vector is pAQ1 Ex-P, a generic expression vector for the introduction of genes into cyanobacteria of the genus Synechococcus. Alternatively, and also preferably, the vector is pl ND4 or pRKSK , which are expression vectors for the introduction of genes into purple

phototrophic bacteria such as Rhodobacter sphaeroides.

Alternatively, and also preferably, the vector is suitable for expression in bacteria of the genus Acinetobacter. For example, the vector is preferably pWH 1274. Alternatively, the vector is suitable for expression in Escherichia coii. For example, the vector is preferably pET3a (Novagen). Preferably, the vector is a bacterial expression vector (i.e. a vector that promotes expression of one or more nucleic acid molecules it encodes). The terms "expression vector', "expression construct", "construct" and "vector' are used interchangeably herein. "LuxCDE" as used herein, refers to the nucleic acid molecules encoding a LuxC, LuxD and LuxE polypeptides respectively. Similarly, "LuxCDE-fad" as used herein, refers to the nucleic acid molecules encoding a LuxC, LuxD, LuxE and FAD polypeptides respectively.

"LuxCDE expression construct", as used herein, refers to an expression vector that comprises a nucleic acid molecule encoding a polypeptide having LuxC activity, a nucleic acid molecule encoding a polypeptide having LuxD activity, and a nucleic acid molecule encoding a polypeptide having LuxE activity. The nucleic acid molecules may be in any order within the expression vector, including in the order of LuxCED, LuxDEC, LuxDCE, LuxECD, LuxEDC etc. The nucleic acid molecules may be separated by other nucleic acids within the expression vector, or may by contiguous.

In a preferred embodiment, the expression vectors and fatty acid biogenic cells of the present invention comprise at least two, three or four of: a nucleic acid molecule encoding a polypeptide having LuxC activity, a nucleic acid molecule encoding a polypeptide having LuxD activity, a nucleic acid molecule encoding a polypeptide having LuxE activity, and a nucleic acid molecule encoding a polypeptide having decarbonylase activity, as described herein. The nucleic acid molecules in the expression vector may be in any order. The nucleic acid molecules may be separated by other nucleic acids within the expression vector, or may by contiguous.

Preferably, the expression vector is a high-copy-number expression vector; alternatively, the expression vector is a low -copy-number expression vector, for example, a Mini-F plasmid, or it integrates into the genome of the host bacterium. Preparation of Expression Vectors

A person of skill in the art will be aware of the molecular techniques available for the preparation of expression vectors.

The nucleic acid molecule for incorporation into the expression vector of the invention, as described above, can be prepared by synthesizing nucleic acid molecules using mutually priming oligonucleotides and the nucleic acid sequences described herein. A number of molecular techniques have been developed to operably link DNA to vectors via complementary cohesive termini. In one embodiment, complementary homopolymer tracts can be added to the nucleic acid molecule to be inserted into the vector DNA. The vector and nucleic acid molecule are then joined by hydrogen bonding between the complementary homopo!ymeric tails to form recombinant DNA molecules.

In an alternative embodiment, synthetic linkers containing one or more restriction sites are used to operably link the nucleic acid molecule to the expression vector, in one

embodiment, the nucleic acid molecule is generated by restriction endonuciease digestion. Preferably, the nucleic acid molecule is treated with bacteriophage T4 DNA polymerase or E. coll DNA polymerase I, enzymes that remove protruding, 3'-singie-stranded termini with their 3'-5'-exonucleolytic activities, and fill in recessed 3'-ends with their polymerizing activities, thereby generating blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA iigase. Thus, the product of the reaction is a nucleic acid molecule carrying polymeric linker sequences at its ends. These nucleic acid molecules are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the nucleic acid molecule.

Alternatively, a vector comprising ligation-independent cloning (LIC) sites can be employed. The required PGR amplified nucleic acid molecule can then be cloned into the LIC vector without restriction digest or ligation (Aslanidis and de Jong, Nucl. Acid. Res. 18, 6069-8074, (1990), Haun, et al, Biotechniques 13, 515-518 (1992).

In order to isolate and/or modify the nucleic acid molecule of interest for insertion into the chosen plasmid, it is preferable to use PCR. Appropriate primers for use in PCR preparation of the sequence can be designed to isolate the required coding region of the nucleic acid molecule, add restriction endonuciease or LIC sites, place the coding region in the desired reading frame.

In a preferred embodiment a nucleic acid molecule for incorporation into an expression vector of the invention, is prepared by the use of the polymerase chain reaction as disclosed by Saiki et al ( 988) Science 239, 487-491 , using appropriate oligonucleotide primers. The coding region is amplified, whilst the primers themselves become

incorporated into the amplified sequence product, in a preferred embodiment the amplification primers contain restriction endonuclease recognition sites which allow the amplified sequence product to be cloned into an appropriate vector.

Preferably, the nucleic acid molecule of SEQ ID NO: 1 , 3, 5, 7 or 9 is obtained by PGR and introduced into an expression vector using restriction endonuclease digestion and ligation, a technique which is well known in the art. More preferably, the nucleic acid molecule of SEQ ID NO:1 , 3, 5, 7 or 9 is introduced into the expression vector pPD or pWH 274.

Alternatively, the nucleic acid molecule of SEQ ID NO: 1 , 3, 5, 7 or 9 is introduced into an expression vector by yeast homologous recombination (Raymon et a\., Biotechniques. 26(1): 134-8, 140-1 , 1999).

The expression vectors of the invention can contain a single copy of the nucleic acid molecule described previously, or multiple copies of the nucleic acid molecule described previously.

Preferably, the expression vector of the present invention comprises at least one, two, three or four of the decarbonyiase coding sequence of SEQ ID NO: 1 or SEQ ID NQ:9, the LuxC coding sequence of SEQ ID NO:3, the LuxD coding sequence of SEQ ID NQ:5, and the LuxE coding sequence of SEQ ID NO:7.

Host cells

"Purified preparation of cells," as used herein, refers to, in the case of cultured cells or microbial ceils, a preparation of at least 10%, and more preferably, 50% of the subject cells.

"Fatty acid biogenic cell", as used herein refers to any ceil capable of producing fatty acids from a carbon source. The term refers to the particular subject ceil and also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

"Genetically modified fatty acid biogenic cell", as used herein refers to transformed or transfected fatty acid biogenic ceils. The term refers to the particular subject cell and also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

"Host cell" and "recombinant host cell", as used herein, are used interchangeably to refer to the particular subject ceil (i.e. the fatty acid biogenic cell or the genetically modified fatty acid biogenic ceil).

The host ceil may be an aerobic cell or alternatively a facultative anaerobic cell.

Preferably, the ceil is a bacterial ceil. Alternatively, the cell may be a yeast ceil (e.g.

Saccharomyces, Pichia), an algae cell, an insect ceil, or a plant cell.

Bacterial host cells include Gram-positive and Gram-negative bacteria. Suitable bacterial host ceils include, but are not limited to the Gram-negative bacteria, for example a bacterium of the family Enterobacteria, most preferably Escherichia coii. Expression in E. coli offers numerous advantages, particularly low development costs and high production yields. Cells suitable for high protein expression include, for example, E.coii W3110, the B strains of E. coii, E.coii BL21 , BL21 (DE3), and BL21 (DE3) pLysS, pLysE, DH1 , DH4I, DH5, DH5I, DH5IF', DH5I CR, DH10B, DHiOB/p3, DH1 IS, C600, HB101 , JM101 , JM105, JM 109, JM110, K38, RR1 , Y1088, Y1089, CSH18, ER1451 , ER1647 are particularly suitable for expression. E. coii K12 strains are also preferred as such strains are standard laboratory strains, which are non-pathogenic, and include NovaBiue, J 109 and DH5a (Novogen®), E. coii K12 RV308, E. coii K12 C800, E. coii HB101 , see, for example, Brown, Molecular Biology Labfax (Academic Press (1991)). Alternatively, Enterobacteria from the genera Saimoneiia, Shigella, Enterobacter, Serratia, Proteus and Erwinia may be suitable. Other prokaryotic host cells include Serratia, Pseudomonas, Cau!obacter, or Cyanobacteria, for example bacteria from the genus Synechocystis or Synechococcus, more particularly Synechocystis sp. PCC 8803 or Synechococcus sp PCC 6301. Alternatively, the host ceil may be of the genus Bacillus, for example Bacillus brevis or Bacillus subtilis, Bacillus thuringienesis. Alternatively, the host ceil may be of the genus Lactococcus, for example Lactococcus lactis. Alternatively, the bacterial cell is of the actinomycetes family, more particularly from the genus

Streptomyces, Rhodococcus, Corynebacterium, Mycobacterium. More particularly, Streptomyces lividans, Streptomyces ambofaciens, Streptomyces fradiae, Streptomyces griseofuscus, Rhodococcus erythropolis, Corynebacterium gluamicum, Mycobacterium smegmatis. Alternatively, the host cell may be of the genus Acinetobacter, for example A.baylyi ADP1. Alternatively, the host cell may be a purple photofrophic bacterium of the genus Rhodobacter, Rhodopseudomonas, Rhodospirillum or Rubrivivax, more particularly Rhodobacter sphaeroides.

Photosynthetic bacteria, including cyanobacteria, are efficient at converting solar energy and carbon dioxide into biofuels. Any photosynthetic bacterial cells may be suitable host cells in the invention,

Cyanobacteria possess several advantages as organisms for bioindustrial processes, including simple input requirements (light, C0 ₂ , low nutrient media), their natural diversity, which makes them tolerant of poor environments and eliminates competition for vital agricultural land, ease of genetic manipulation, and their ability to act as a carbon sink by removing C0 ₂ from the environment. They are therefore a preferred host cell for the present invention. Standard techniques for propagating vectors in prokaryotic hosts are well-known to those of skill in the art (see, for example, Ausubel et al. Short Protocols in Molecular Biology 3rd Edition (John Wiley & Sons 1995)).

To maximize recombinant protein expression, the expression vectors of the invention may express the nucleic acid molecule incorporated therein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., (1990) Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California, 119-128), Alternatively, the nucleic acid molecule incorporated into an expression vector of the invention, can be altered so that the individual codons for each amino acid are those preferentially utilized in the chosen host cell (Wada et al., (1992) Nucleic Acids Res. 20:21 11-21 18). Such alteration of nucleic acid sequences of the invention can be carried out by standard DNA synthesis techniques.

Preferably, the amount of alkane produced by the cells of the invention is at least 0.1 %, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1 % dry cell weight, and at least two times the amount produced by an otherwise identical cell, cultured under identical conditions, but lacking the expression vector of the invention.

"Percent dry cell weight" refers to a measurement of alkane production obtained as follows: a defined volume of culture is centrifuged to pellet the cells. Cells are washed then dewetted by at least one cycle of microcenfrifugafion and aspiration. Cell pellets are lyophiiized overnight, and the tube containing the dry ceil mass is weighed again such that the mass of the cell pellet can be calculated within ± 0.1 mg, simultaneously a second sample of the culture is harvested, washed and dewetted. The resulting ceil pellet, corresponding to 1-3 mg of dry cell weight, is then extracted by vortexing in approximately 1 mi acetone plus butylated hydroxytolune (BHT) as antioxidant and an international standard e.g. n-hetacosane. Cell debris is then pelleted by centrifugation and the supernatant (extractant) is taken for analysis by GC. For accurate quantitation of alkanes, flame ionization detection (FID) may be used. Alkane concentrations in the biological extracts can be calculated using calibration relationships between GC-FID peak area and known concentrations of authentic alkane standards. Knowing the volume of the extractant, the resulting concentrations of the alkane species in the extractant, and the dry ceil weight of the cell pellet extracted, the percentage of dry cell weight that comprises alkanes can be determined.

Host Cell Transformation

A host ceil transformed or transfected with an expression vector of the invention, comprising a nucleic acid molecule as described previously, can be used to produce (i.e. express) an alkane of a defined chain length.

The expression vector of the present invention can be introduced into fatty acid biogenic ceils by conventional transformation or transfection techniques.

"Transformation" and "transfection", as used herein, refer to a variety of techniques known in the art for introducing foreign nucleic acids into a fatty acid biogenic cell. The terms "transformed" and "transfected" are used interchangeably herein to refer to any technique for introducing foreign nucleic acids into a fatty acid biogenic ceil.

Transformation of appropriate cells with an expression vector of the present invention is accomplished by methods known in the art and typically depends on both the type of vector and ceil. Said techniques include, but are not limited to calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, chemoporation or electroporation.

Techniques known in the art for the transformation of fatty acid biogenic cells are disclosed in for example, Sambrook et ai (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y; Ausubel et al (1987) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY; Cohen et ai (1972) Proc. Natl. Acad. Sci. USA 89, 2110; Luchansky et a! (1988) oL Microbio!, 2, 837-646. All such methods are incorporated herein by reference.

Successfully transformed cells, that is, those cells containing the expression vector of the present invention, can be identified by techniques well known in the art. For example, cells transfected with the expression vector of the present invention can be cultured to produce proteins with decarbonyiase, LuxC, LuxD or LuxE activity. Cells can be examined for the presence of the expression vector DNA by techniques well known in the art. Alternatively, the presence of the decarbonyiase, LuxC, LuxD, or LuxE protein, or portion and fragments thereof can be detected using antibodies which hybridize thereto.

In a preferred embodiment the invention comprises a culture of transformed fatty acid biogenic cells. Preferably the culture is clonally homogeneous. The fatty acid biogenic cell can contain a single copy of the expression vector described previously, or alternatively, multiple copies of the expression vector.

A fatty acid biogenic cell transformed or transfected with an expression vector of the invention, comprising a nucleic acid molecule as described previously, can be used to produce (i.e., express) an alkane of a defined chain length at an increased rate of production relative to a non-transformed or transfected ceil cultured under the same conditions.

Methods of producing an alkane of a defined chain length

The present invention provides a method for the large scale production of an alkane of a defined chain length, by utilizing a fatty acid biogenic ceil comprising a nucleic acid molecule encoding a polypeptide having LuxC activity, a nucleic acid molecule encoding a polypeptide having LuxD activity, and a nucleic acid molecule encoding a polypeptide having LuxE activity. Preferably, the cell is transformed or transfected with an expression vector of the invention.

The fatty acid biogenic cell may comprise an endogenous nucleic acid molecule encoding a polypeptide having decarbonyiase activity. Alternatively, or in addition, the fatty acid biogenic ceil may be transformed with an expression vector comprising a nucleic acid molecule encoding a polypeptide having decarbonyiase activity. In a further alternative, or in addition, the fatty acid biogenic ceil may be cultured with a cell expressing a polypeptide having decarbonyiase activity (i.e. a decarbonyiase producing ceil). In a further alternative, or in addition, or a polypeptide having decarbonylase activity may be provided in the culture medium. For example, a decarbonylase may be added directly to the culture medium prior to or during culture of the host cell. Preferably, the culture conditions are such that allow expression of said polypeptides having LuxC, LuxD, and LuxE activity respectively. Where appropriate, and preferably, the culture conditions are such that allow expression of said polypeptides having LuxC, LuxD, LuxE and decarbonylase activity respectively. Preferably, the cells are provided with the necessary substrate for the production of an alkane of a defined chain length.

Cells are grown or cultured in the manner with which the skilled worker is familiar, depending on the host organism. The culture medium (also called "growth medium", "medium" or "media" herein) to be used must suitably meet the requirements of the strains in question. Preferably, the culture media is sufficient to support the growth of the host cell. Descriptions of suitable culture media for various microorganisms can be found in the textbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA, 1981). By way of example only, bacteria of the genus Synechocystis are preferably cultured under conditions with a light intensity of about 30pmol photons rrf ² s ^"1 (although it can be increased if required up to 150 μηιοΙ photons rrf ² s ¹ ).

Fatty acid biogenic ceils may be grown in a liquid medium comprising one or more of a carbon source, usually in the form of sugars, a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, inorganic salts, trace elements such as salts of iron, manganese and magnesium and, if appropriate, vitamins, at temperatures of between 0°C and 100°C, preferably between 10°C and 60°C, while gassing in oxygen.

Preferred carbon sources are sugars, such as mono-, di- or polysaccharides. Examples of carbon sources are glucose, carbon dioxide, sodium bicarbonate, bicarbonate, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Preferably, the carbon source is carbon dioxide. Alternatively, and preferably, the carbon source is bicarbonate.

Sugars can also be added to the media via complex compounds such as molasses or other by-products from sugar refining. The addition of mixtures of a variety of carbon sources may also be advantageous. Other possible carbon sources are oils and fats such as, for example, soya oil, sunflower oil, peanut oil and/or coconut fat, fatty acids such as, for example, palmitic acid, stearic acid and/or linoleic acid, alcohols and/or polyalcohols such as, for example, glycerol, methanol and/or ethanoi, and/or organic acids such as, for example, acetic acid and/or lactic acid.

Nitrogen sources are usually organic or inorganic nitrogen compounds or materials comprising these compounds. Examples of nitrogen sources comprise ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as cornsteep liquor, soya meal, soya protein, yeast extract, meat extract and others. The nitrogen sources can be used individually or as a mixture.

Inorganic salt compounds which may be present in the media comprise the chloride, phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron. Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or else organic sulfur compounds such as mercaptans and thiols may be used as sources of sulfur for the production of sulfur-containing fine chemicals, in particular of methionine. Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used as sources of phosphorus.

Chelating agents may be added to the medium in order to keep the metal ions in solution. Particularly suitable chelating agents comprise dihydroxyphenols such as catechol or protocatechuate and organic acids such as citric acid.

The culture media used according to the invention may also comprise other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factors and salts are frequently derived from complex media components such as yeast extract, molasses, cornsteep liquor and the like. It is moreover possible to add suitable precursors to the culture medium. The exact composition of the media compounds heavily depends on the particular experiment and is decided upon individually for each specific case. Information on the optimization of media can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (Editors P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 983577 3). Growth media can also be obtained from commercial suppliers, for example Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.

The pH of the liquid medium can either be kept constant, that is to say regulated during the cuituring period, or not. The cultures can be grown batchwise, semi-batchwise or continuously. Batch fermentation may prove particularly useful when large scale

production of alkane is required. Alternatively, a fed batch and/or continuous culture can be used to generate the required yield of alkane. Nutrients can be provided at the beginning of the fermentation or fed in semi-continuously or continuously. The products produced can be isolated from the organisms as described above by processes known to the skilled worker, for example by extraction, distillation, crystallization, if appropriate precipitation with salt, and/or chromatography. To this end, the host cells can

advantageously be disrupted beforehand. In this process, the pH value is advantageously kept between pH 4 and 12, preferably between pH 8 and 9, especially preferably between pH 7 and 8, An overview of known cultivation methods can be found in the textbook by Chmiel (BioprozeBtechnik 1. Einfuhrung in die Bioverfahrenstechnik [Bioprocess technology 1. Introduction to Bioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheral equipment] (Vieweg Verlag, Brunswick/Wiesbaden, 1994)).

All media components are sterilized, either by heat (20 min at 1.5 bar and 121 °C) or by filter sterilization. The components may be sterilized either together or, if required, separately. Ail media components may be present at the start of the cultivation or added continuously or batchwise, as desired.

The culture temperature will vary depending on the particular experiment and the host ceil. The culture temperature is normally between 15°C and 45°C, preferably at from 25°C to 40°C, more preferably at from 25 to 37 °C and may be kept constant or may be altered during the experiment. By way of example only, for bacteria from the genus Synechocystis, the temperature is preferably at from 25 to 35 °C and more preferably at 30°C. The pH of the medium should be in the range from 5 to 8,5, preferably around 7.0. The pH for cultivation can be controlled during cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled by employing antifoams such as, for example, fatty acid polygiycol esters. To maintain the stability of vector it is possible to add to the medium suitable substances having a selective effect, for example antibiotics. Aerobic conditions are maintained by introducing oxygen or oxygen-containing gas mixtures such as, for example, ambient air into the culture. The temperature of the culture is normally 20°C to 45°C and preferably 25°C to 40°C. The culture is continued until formation of the desired product is at a maximum. This aim is normally achieved within 24 to 96 hours.

The resultant media ("broth") can then be processed further. The biofuei may, according to requirement, be removed completely or partially from the broth by separation methods such as, for example, centrifugation, filtration, decanting or a combination of these methods or be left completely in said broth. It may be advantageous to process the biofuei after its separation.

The methods of the invention may include culturing the cells in conditions that promote direct product (i.e. aikane) secretion for easy recovery without the need to extract biomass. Preferably, the alkanes are secreted directly into the culture medium. Preferably, the secreted products are easily recovered and can be used directly or used with minimal processing. Product recovery efficiency is an important determinant of the total production cost.

Techniques known in the art for the large scale culture of host ceils are disclosed in for example, Bailey and Ollis (1986) Biochemical Engineering Fundamentals, McGraw-Hill, Singapore; or Shuler (2001) Bioprocess Engineering: Basic Concepts, Prentice Hall. All such techniques are incorporated herein by reference.

Transformed or transfected host cells can be cultured in aerobic or anaerobic conditions. In aerobic conditions, preferably, oxygen is continuously removed from the culture medium, by for example, the addition of reductants or oxygen scavengers, or, by purging the reaction medium with neutral gases.

The host ceils of the invention can be cultured in a vessel, for example a bioreactor. Bioreactors, for example fermenters, are vessels that comprise cells or enzymes and typically are used for the production of molecules on an industrial scale. The molecules can be recombinant proteins or compounds that are produced by the cells contained in the vessel or via enzyme reactions that are completed in the reaction vessel. Typically, cell based bioreactors comprise the cells of interest and include ail the nutrients and/or co- factors necessary to carry out the reactions.

In yet another embodiment, the method comprises cuituring the host ceil in the presence of an antibiotic, where said antibiotic selects for the presence of a corresponding "selectable marker" on the expression vector of the invention in the host ceil.

Aspects of the invention are demonstrated by the following non-limiting examples. EXAMPLES

MATERIALS AND METHODS

Generation of luxCDE~fad constructs

All genetic manipulations were carried out in E.coli DH5a cells. Cells were made chemically competent and transformed using standard techniques (Sambrook et aL, 1989 Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY USA: Cold Spring Harbor Laboratory). PCR was carried out using Accuzyme 2x reaction mix (Bioline).

Using the primers indicated in Figure 7, the luxC, D and E genes were isolated by PCR from plasmid pSB4 7, which contains the luxCDABE genes from Photorhabdus

luminescens ATCC299 (Hb strain; Winson, M.K et ai. 1998 FEMS Microbiol Letts 163, 193- 202), and the fatty aldehyde decarbonyiase (fad) gene was isolated by PCR from

Synechococcus elongatus PCC 7942 genomic DNA. The luxCDE-fad gene cluster was built-up sequentially using the ne pot' Gibson method for cloning large DNA fragments as described previously (Gibson et ai. 2009 Nature Methods 6, 343-5). PCR primers were designed with 30-40 bp of sequence homologous to the preceding gene in the cluster upstream of the target gene and 30-40 bp of sequence homologous to the next gene in the cluster downstream of the target gene. The Gibson reaction mix contains T5 exonuclease to create homologous single stranded 3' overhangs allowing annealing and ligation of the target gene with the next gene in the cluster by Phusion polymerase and T4 ligase. Each forward primer for the LuxD, LuxE and fad genes contained a Synechocystis specific ribosome binding site upstream of the start codon. The luxC forward primer and reverse FAD primer contained sequence homologous to the expression vector pWH1274, which is a derivative of pBR322 containing the origin of replication of Acs netobacter sp. (Hunger, ., R. et ai. 990 Gene 87, 45-51). This enabled the luxCDE-fad gene cluster to be Gibson ligated into the EcoRV-Bam l site of pWH 1274 under the control of the

tetracycline resistance promoter; this new plasmid pWH1274: iuxCDE-fad was then transformed into Acinetobacter ADPWH_alkRM_/ux. This strain has been designed to act as a biosensor to indicate the presence of alkanes through expression of bioiuminescence (Zhang et al, Microbial Biotechnology 5, 87-97 (2012), doi: 10.1 11 1/j.1751- 7915.201 .00301.x).

The pB!uescript KS ^÷ (Strategene) derived plasmid pPD was generated to allow

transformation of gene sequences into Synechocystis by homologous recombination.

Using the primers shown in Figure 8 (SEQ ID NO:20 and SEQ ID NO:21) a 400bp fragment (f1) containing the upstream light-activated promoter region of the psbAII gene (encodes Synechocystis D1 photosystem I! reaction centre protein) and a 2 Kb fragment (f2) containing the kanamycin resistance gene and sequence downstream of the psbAii gene were isolated by PGR (using SEQ ID NO:22 and SEQ ID NO: 23) from plasmid pS2 (Lagarde, D. et al. 2000 Applied and Environmental Microbiology 88, 64-72)). The EcoRV- f1 -Nde\-EcoR\ fragment was inserted into pBluescript followed by the £coR1-Sg/il- f2 - EcoRI fragment creating plasmid pPD containing an Nde\-Bgi\\ cloning site between upstream and downstream psbAII sequence. Using the primers indicated in Figure 9, a new iuxCDE-fad fragment was generated from pWH1274: luxCDE-fad carrying 5' sequence homologous to the promoter region of psbAII and 3' sequence homologous to the kanamycin resistance gene. This fragment was Gibson ligated into pPD to create pPD:luxCDE-fad.

Synechocystis transformation

The construct pPD luxCDE-fad was used to transform Synechocystis sp. PCC8803 by homologous recombination allowing insertion of this construct into the Synechocystis genome instead of the psbAII gene. 300ng pPD.iuxCDE-fad was mixed with 1 ml of 2 day old ceils and left 24 hours on a BG-11 plate with no selection. Transformants of Synechocystis 8803 were selected on BG-11 plates with increasing concentrations of kanamycin (5 ml ^"1 ) to ensure complete segregation, which was checked for using PGR on single colonies with primers upstream and downstream (f1 for and f2 rev; see Figure 8; SEQ ID NQ:20 and SEQ ID NO: 23) of the psbAII gene.

Synechocystis growth conditions

Synechocystis strains were grown photoautotrophicaliy in liquid BG-11 medium (Rippka et al., 1979 J. Gen. Microbiol. 11 , 1-61) supplemented by 10 mM TES pH8.2 at 30°C with 30 μΓηο! photons s ^"1 m ^"2 on a rotary shaker unless indicated otherwise. Supplements of 5mM glucose or 10mM NaHC0 ₃ were added as indicated.

E.coH transformation and overexpression

The !uxCDE-fad gene cluster was chemically synthesized (GeneArt, Invitrogen) after being codon optimized for E.coli with the ribosome binding sequence AAG A AG G AG GTATAC AT , placed upstream of the ATG start codon of each gene in the cluster. The resulting DNA fragment was cloned into the Xba\-Bam \ and /Vdei-Sa/r?HI sites of the pET3a expression vector (Novagen). The pET3a: !uxCDE-fad construct was transformed using heat shock into BL21 (DE3) cells made chemically competent by standard method and transformanfs selected for on media containing 100 pg m ¹ ampicillin. Over-expression of the construct was performed by growing the ceils to log phase (OD ₆₀₀ = 0.6-0.7) at 30-37°C for 6-8 hrs with shaking at 200 rpm in either LB (Luria-Bertani) medium or 9 minimal media supplemented with glucose and then adding the inducer IPTG (final concentration 400μ ) followed by overnight growth at 20°C.

GC-!VIS (Gas chromatography-mass spectrometry) analysis

10g of each sample was weighed into clean glass 25ml volumetric flasks and spiked with deuterated PAH internal standard to 2 g Γ . 0,5ml of hexane was added to the flasks, which were then capped and manually shaken for 4 minutes. Ultra high quality water was added to bring the hexane layer up to the neck of the flasks. The hexane extract was dried with sodium sulphate and pipetted into a GC vial. Emulsions, when formed, were broken by centrifugation prior to the drying stage. 1 μΙ of the extract was analysed via GC/ S using cold splitiess injection and a temperature program from 50°C up to 270°C (eluting tetracosane around 29min). Compound identification was achieved by interpretation of mass spectra and correspondence of retention times to standards containing tridecane, tetradecane, pentadecane, heptadecane, 1~tetradecanol and 1-hexadecanol.

RESULTS

A novel biosynthetic pathway in the soil bacterium Acinetobacter baylyi ADP1, the cyanobacterium Synechocystis sp. PCC6803 and the enteric bacterium Escherichia coli is described that results in the secretion of significant amounts of alkanes of defined chain lengths in the range of C13-C17. The invention intercepts the native fatty acid pathway in bacteria and reroutes carbon flux to fatty acid aldehyde using Lux proteins. This aldehyde is then available for conversion to aikane either via cyanobacteriai aldehyde decarbonylase (not naturally present in A. baylyi ADP1 but naturally available in the cyanobacierium where increased amounts of this enzyme can also be engineered) or via other endogenous enzyme reactions.

The applicants have shown that this sequence of biosynthetic reactions, using genes from Photorhabdus luminescens, can be constructed in the bacterium A. baylyi ADP1 and in the cyanobacterium Synechocystis sp, PCC6803 and in the enteric bacterium Escherichia coli. A four-gene cluster of iuxCDE (from Photorhabdus luminescens ATCC299) and fad (from Synechococcus e!ongatus PCC 7942) was introduced into A. baylyi ADP1 and Synechocystis sp. PCC8803 and E. coli.

Figure 2 shows the results of PGR reactions that confirm the integration of the luxCDE-fad genes into the Synechocystis chromosome.

The GC-MS (gas chromatography-mass spectrometry) data in Figures 3 and 4 demonstrate that the applicants have engineered the first cyanobacterium to convert dissolved carbon dioxide (bicarbonate) to biodiesei of a defined chain length (C ₁₃ _C ₁₇ ).

Furthermore the biodiesei is excreted into the growth medium in significant amounts (Figure 3), eliminating the need to obtain ceil mass by centrifugation and subsequent extraction of the hydrocarbon using solvents.

A similar result is obtained with E. coli containing the luxCDE-fad genes on the expression p!asmid pET3a where significant amounts of pentadecane are also secreted into the growth medium (Figures 5 and 6).

The reader's attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent, or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.