Nucleic acid molecule encoding triterpenoid synthase.
This application claims priority from prior foreign patent Application No. 2009904482, filed Sep. 15, 2009 in Australia, and foreign patent Application No. 2009905381, filed Nov. 4, 2009 in Australia, and foreign patent Application No. 2010900782, filed Feb. 24, 2010 in Australia, all hereby incorporated by reference. The present invention relates to nucleic acid molecules encoding a polypeptide capable of producing a triterpenoid hydrocarbon. Additionally, the invention relates to polypeptides encoded by such nucleic acid molecules and use of such nucleic acid molecules or their encoded polypeptides in triterpenoid hydrocarbon production. Fossil fuel is widely recognised as an unsustainable source of petroleum because of depleting supplies and the net contribution of these fuels to the carbon dioxide in the atmosphere. Renewable, carbon neutral fuels are necessary for environmental and economic sustainability. Biofuel derived from oil crops is a potential renewable and carbon neutral alternative to petroleum fuels. Currently, biofuels are produced mainly from soybeans, canola oil, animal fat, palm oil, corn oil and waste cooking oil. Biofuel from the above sources cannot realistically satisfy even a small fraction of the existing demand for fuels. While researchers are seeking alternative feedstock for biofuel, algae have emerged as one of the most promising sources for biofuel production for three main reasons: (1) the yields of oil from algae are orders of magnitude higher than those for traditional oilseeds; (2) algae can grow in places away from the farmlands and forests, thus minimising the damages caused to ecosystems and food chain systems; and (3) algae can be grown in sewers utilising sewage and next to power-plant smokestacks where they digest pollutants and produce oil. Algae are phototrophic cell factories, capable of deriving energy from sunlight and carbon from carbon dioxide. Algae convert carbon dioxide to potential biofuels, foods, feeds and high-value bioactives. Algae can provide several different types of renewable biofuels and valuable by-products such as antibiotics. Not all algae are satisfactory for producing biofuel because of their low oil content or slow growth. Some species of the genus The hydrocarbons produced by However, the production of photosynthetic fuel oils from Disclosed in the prior art, however, is a squalene synthase (SEQ ID NO: 10) and an alleged botryococcene synthase (SEQ ID NO: 42), each derived from the Race B Therefore, a need exists for synthetic or recombinant tools to facilitate triterpenoid hydrocarbon, particularly botryococcene, production. A first aspect provides an isolated nucleic acid molecule encoding a polypeptide capable of producing a triterpenoid hydrocarbon, wherein the polypeptide comprises at least 29 contiguous amino acid residues of any one of SEQ ID NOs: 1 to 5 or 20 to 29. The nucleic acid molecule provides a tool for triterpenoid hydrocarbon production. In one embodiment of the first aspect, the nucleic acid molecule comprises any one of SEQ ID NOs: 6 to 9 or 30. The nucleic acid molecule may be recombinant or synthetic. A second aspect provides an isolated polypeptide capable of producing a triterpenoid hydrocarbon, wherein the polypeptide comprises at least 29 contiguous amino acid residues of any one of SEQ ID NOs: 1 to 5 or 20 to 29. Particular examples of triterpenoid hydrocarbon molecules are botryococcene and squalene, which are isomers of each other and are produced from farnesyl pyrophosphate (FPP) substrate via presqualene pyrophosphate (PSPP) intermediate in The polypeptide of the second aspect may be used to synthesise a triterpenoid hydrocarbon in a cell-free system. Alternatively, the polypeptide of the second aspect may be expressed in a recombinant organism for production of a triterpenoid hydrocarbon or a botryococcene, respectively. In one embodiment of the second aspect, the polypeptide comprises any one of SEQ ID NOs: 1 to 5 or 20 to 29. The polypeptide may be recombinant or synthetic. The nucleic acid molecule encodes, or the polypeptide is, a botryococcene synthase, which is capable of converting FPP and/or PSPP to botryococcene. Alternatively, the nucleic acid molecule may encode, or the polypeptide may be, a squalene synthase, which is capable of converting FPP and/or PSPP to squalene. A third aspect provides a vector, comprising the nucleic acid molecule of the first aspect. A fourth aspect provides a recombinant non-human organism capable of producing a triterpenoid hydrocarbon, the organism comprising the nucleic acid molecule of the first aspect or the vector of the third aspect. In another embodiment of the fourth aspect, the organism is a microorganism. The microorganism may be an alga or a bacterium. In another embodiment, the microorganism is selected from the group consisting of: A fifth aspect provides use of the nucleic acid molecule of the first aspect, the polypeptide of the second aspect, the vector of the third aspect, or the recombinant organism of the fourth aspect, to produce a triterpenoid hydrocarbon. A sixth aspect provides a method for producing a triterpenoid hydrocarbon, comprising the step of growing the recombinant organism of the fourth aspect under conditions sufficient for the organism to produce a triterpenoid hydrocarbon. A seventh aspect provides a triterpenoid hydrocarbon produced by the method of the sixth aspect. In various embodiments, the triterpenoid hydrocarbon is squalene, botryococcene, dehydrosqualene or dehydrobotryococcene. An eighth aspect provides use of the nucleic acid molecule of the first aspect, the polypeptide of the second aspect, the vector of the third aspect, the recombinant organism of the fourth aspect, the triterpenoid hydrocarbon produced by the method of the sixth aspect, or the triterpenoid hydrocarbon of the seventh aspect, in the production of an intermediate of a biofuel, a bioplastic, a pharmaceutical, a food additive or an industrial chemical production. A ninth aspect provides a method for producing an intermediate of a biofuel, a bioplastic, a pharmaceutical, a food additive or an industrial chemical production, comprising the steps of growing the recombinant organism of the fourth aspect under conditions sufficient for the organism to produce a triterpenoid hydrocarbon, harvesting the triterpenoid hydrocarbon from the organism, and producing the intermediate from the triterpenoid hydrocarbon. A tenth aspect provides an intermediate produced by the method of the ninth aspect of a biofuel, a bioplastic, a pharmaceutical, a food additive or an industrial chemical, production. In an embodiment of any one of the first to ninth aspects, the triterpenoid hydrocarbon is produced from FPP and/or PSPP. In another embodiment of any one of the first to ninth aspects, the triterpenoid hydrocarbon is a botryococcene or a squalene. To overcome the slow growth rate of some organisms, such as FIGS. 1 to 5 and 20 to 28 depict the amino acid sequences encoded by exons of squalene synthase/botryococcene synthase homologues of the Ayamé 1 strain of FIGS. 6 to 9 depict the nucleic acid sequences of exons of squalene synthase/botryococcene synthase homologues of the Ayamé 1 strain of FIG. 10 depicts the amino acid sequence of squalene synthase of FIG. 11 depicts the nucleic acid sequence (mRNA, complete cds; accession identifications: gi|6636499; gb|AF205791.1; AF205791) encoding the amino acid sequence of FIG. 10 and corresponds with SEQ ID NO: 11. FIG. 12 depicts the partial amino acid sequence of squalene synthase of FIG. 13 depicts the nucleic acid sequence (partial cds; accession identifications: gi|7532838; gb|AH009227.1; SEG_AF205789S) encoding the amino acid sequence of FIG. 12 and corresponds with SEQ ID NO: 13. FIG. 14 depicts the amino acid sequence of FIG. 10 against which the consensus deduced amino acid sequences of FIGS. 1 to 5 have been positioned. An asterisk represents a stop codon. The consensus sequence corresponds with SEQ ID NO: 14. FIG. 15 illustrates the nucleic acid sequence (SEQ ID NO: 15) of the squalene synthase pSP124S construct transformed into FIGS. 16 and 17 depict the nucleic acid sequences (corresponding with SEQ ID NO: 16 and SEQ ID NO: 17, respectively) of primer pair 1 (PP1) used to detect the presence of the squalene synthase pSP124S construct in transformed FIGS. 18 and 19 depict the nucleic acid sequences (corresponding with SEQ ID NO: 18 and SEQ ID NO: 19, respectively) of primer pair 2 (PP2) used to detect the presence of the squalene synthase pSP124S construct in transformed FIG. 29 depicts the amino acid sequence of ATB1, a triterpenoid hydrocarbon synthetic polypeptide of FIG. 30 depicts the inferred cDNA nucleic acid sequence encoding the amino acid sequence of FIG. 29 and corresponds with SEQ ID NO: 30. The gene comprises 8 exons (labelled 1 to 7 and 9), of which exons 2, 4, 6 and 9 are underlined. The cDNA comprises exons that correspond with SEQ ID NOs: 6 to 9. FIG. 31 depicts a genomic nucleic acid sequence encoding the amino acid sequence of FIG. 29 and corresponds with SEQ ID NO: 31. FIG. 32 depicts a nucleic acid sequence used for cloning the ATB1 triterpenoid hydrocarbon synthetic nucleic acid sequence. The full sequence corresponds to SEQ NO: 32. The sequence, other than immediately flanking the start and stop codons, corresponds to the sequence of FIG. 30, i.e. SEQ ID NO: 30. The sequence was used to generate pUC57/ATB1, pET11a/ATB1, pET302/NT-His-ATB1, and CT-His-ATB1/pET302, referred to herein as constructs 1, 2, 3, and 4, respectively. FIG. 33 is a photograph of an agarose gel for molecular detection of the squalene synthase pSP124S construct in transformed FIG. 34 is a photograph of agarose gel electrophoresis separating restriction digests of pET11a/ATB1, pET302/NT-His-ATB1, and CT-His-ATB1/pET302 (constructs 2, 3 and 4, respectively) and confirming that the expected fragment sizes are present. FIG. 34A shows that construct 2 was cut with NdeI and BstXI. FIG. 34B shows that constructs 3 and 4 were each cut with NdeI and XhoI. FIGS. 34A and B 100 bp ladder fragment sizes: 1500; 1000; 900; 800; 700; 600; 500; 400; 300; 200; 100 bp. FIG. 34A 1 kb ladder fragment sizes: 10; 8; 6; 5; 4; 3; 2.5; 2; 1.5; 1; 0.75; 0.5; 0.25 kb. FIG. 34B 1 kb ladder fragment sizes: 10; 8; 6; 5; 4; 3; 2; 1.5; 1.0; 0.5. FIG. 35 is a photograph of an SDS-PAGE minigel of proteins of whole untransformed (U) FIG. 36 depicts the chemical conversion of two farnesyl pyrophosphate C15 units to the squalene C30 unit by the enzyme squalene synthase with NADPH cofactor (A and B) and the chemical conversion of two farnesyl pyrophosphate C15 units to the botryococcene C30 unit by the enzyme botryococcene synthase with NADPH cofactor (B). FIG. 37 is a gas chromatography-mass spectrometry (GC-MS) analysis of authentic squalene. FIG. 38 is a GC-MS analysis of authentic C30 botryococcene. FIG. 39 is a GC-MS analysis of heptane extracts of untransformed FIG. 40 is a GC-MS analysis of heptane extracts of FIG. 41 is a GC-MS analysis of heptane extracts of FIG. 42 is a GC-MS analysis of heptane extracts of lysates, derived from FIG. 43 depicts a nucleic acid sequence (SEQ ID NO: 33) of nucleotides inserted immediately upstream of the native start codon of the nucleotide sequence provided in FIG. 30. FIG. 44 depicts the nucleic acid sequence of the NsiI restriction site (SEQ ID NO: 34). FIG. 45 depicts the nucleic acid sequence of the NdeI restriction site (SEQ ID NO: 35). FIG. 46 depicts the amino acid sequence (SEQ ID NO: 36) of the ATB1-MH polypeptide encoded by pET11a/ATB1 (construct 2), i.e. the ATB1 polypeptide of FIG. 29 (SEQ ID NO: 29) augmented consecutively with one methionine and one histidine residue at the C-terminus of ATB1. The asterisk represents the stop codon. FIG. 47 depicts a nucleic acid sequence (SEQ ID NO: 37) of nucleotides, comprising XhoI and SmaI restriction sites, inserted immediately downstream of the native stop codon of the nucleotide sequence provided in FIG. 30. FIG. 48 depicts the amino acid sequence (SEQ ID NO: 38) of the MHHHHHHHHMH-ATB1-MH polypeptide encoded by pET302/NT-His-ATB1 (construct 3), i.e. the ATB1 polypeptide of FIG. 29 (SEQ ID NO: 29) augmented consecutively with one methionine, eight histidine, one methionine, and one histidine residues at the N-terminus of ATB1 and augmented consecutively with one methionine and one histidine residue at the C-terminus of ATB1 (FIG. 29, SEQ ID NO: 29). The asterisk represents the stop codon. FIG. 49 depicts the amino acid sequence (SEQ ID NO: 39) of the MH-ATB1-MH-HHHHHVNSLEIDDIRA polypeptide encoded by CT-His-ATB1/pET302 (construct 4), i.e. the ATB1 polypeptide of FIG. 29 (SEQ ID NO: 29) augmented consecutively with at its N-terminus with one histidine and one methionine residue and augmented consecutively at its N-terminus with five histidine residues and residues VNSLEIDDIRA at the C-terminus of ATB1 (FIG. 29, SEQ ID NO: 29). The asterisk represents the stop codon. FIG. 50 depicts the nucleic acid sequence of the BstXI restriction site (SEQ ID NO: 40). FIG. 51 depicts the nucleic acid sequence of the XhoI restriction site (SEQ ID NO: 41). FIG. 52 depicts the amino acid sequence (SEQ ID NO: 42) provided as SEQ ID NO: 2 of US20100041120. FIG. 53 illustrates a polypeptide sequence alignment of SEQ ID NO: 29 disclosed herein, and SEQ ID NOs: 10 and 42 disclosed previously. The boxed regions indicate Domains I, II, III, IV and V, which have been observed previously to be highly conserved amongst diverse squalene synthases. Domains III and IV have been correlated with conversion of FPP to PSPP, whereas Domains III and IV have been correlated with conversion of PSPP to squalene. SEQ ID NO: 10 comprises a hydrophobic C-terminus, which is not present in SEQ ID NOs: 29 or 42. SEQ ID NO: 29 is 87% identical to SEQ ID NO: 42 and 38% identical to SEQ ID NO: 10. SEQ ID NO: 42 is 39% identical to SEQ ID NO: 10. FIG. 54 depicts the nucleic acid sequence of plasmid for FIGS. 55 to 62 depict nucleic acid sequences of oligonucleotide primers used in Example 19. Recombinant DNA technology has provided the main impetus for the rise in biotechnology since the 1980s. Currently, almost every major industrial biotechnological process is based around the use of genetically modified organisms. Disclosed herein are synthetic and recombinant tools useful for triterpenoid hydrocarbon production. Such tools include nucleic acid molecules, polypeptides, vectors, recombinant organisms, uses and methods. Triterpenoid hydrocarbons can be used for production of biofuel, bioplastic, pharmaceuticals, food additives, industrial chemicals or specialty chemicals, for example. Examples of use of triterpenoid hydrocarbons for manufacture of intermediates and products such as biofuel, bioplastic, pharmaceuticals (e.g. vaccine adjuvant), food additives (e.g. for cancer prevention), industrial chemicals (e.g. lubricant), specialty chemicals or cosmetics (e.g. light protection) is disclosed in Ab Gapor et al. Exemplary triterpenoid hydrocarbon synthetic nucleic acid molecules encode a botryococcene synthase or a squalene synthase. Whilst not wishing to be bound to any particular hypothesis, a botryococcene synthase may be advantageous when compared with a squalene synthase, since squalene synthase is subject to biosynthetic feedback inhibition, whereas it is proposed that botryococcene synthase is not subject to biosynthetic feedback inhibition. Alternatively, it is proposed that botryococcene synthase is subject to minimal or reduced biosynthetic feedback inhibition relative to biosynthetic feedback inhibition exerted upon squalene synthase. This hypothesis may account for the unusually high level of triterpenoid hydrocarbons synthesised and accumulated by In one embodiment of the first aspect, a nucleic acid molecule encoding a botryococcene synthase polypeptide may be isolated from Race B Whilst not wishing to be bound by any particular hypothesis, strains Ayamé 1 and Paquemar were considered to be high hydrocarbon-producing strains, a consideration that appeared to be confirmed in respect of Ayamé 1 by photographs showing substantial accumulation of oil deposits. Qualitatively, Ayamé 1 has been confirmed by gas chromatography to contain greater amounts of hydrocarbon than other strains. Accordingly, Ayamé 1 was selected for isolation of triterpenoid hydrocarbon nucleic acid molecules. In another embodiment of the first aspect, a nucleic acid molecule encoding a squalene synthase polypeptide may be isolated from a Race A, a Race B or a Race L Alternatively, a nucleic acid molecule encoding a triterpenoid hydrocarbon synthetic polypeptide may be synthesised. The terms “nucleic acid molecule” and “polynucleotide” are used synonymously and refer to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. A nucleic acid of the present disclosure will generally contain phosphodiester bonds, although in some cases, nucleic acid analogues may be used that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphosphoroamidite linkages, and peptide nucleic acid backbones and linkages. Other analogue nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones. Thus, nucleic acid molecules or polynucleotides may also include modified nucleotides that permit correct read-through by a polymerase. “Nucleic acid sequence” or “polynucleotide sequence” includes both the sense and antisense strands of a nucleic acid molecule as either individual single strands or in a duplex. As will be appreciated by those skilled in the art, the depiction of a single strand also defines the sequence of the complementary strand; thus the sequences described herein also provide the complement of the sequence. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses complementary sequences as well as the sequence explicitly indicated. The nucleic acid molecule may be DNA, both genomic and cDNA, RNA or a hybrid. The phrase “a nucleic acid molecule encoding” refers to a nucleic acid molecule which contains sequence information for a structural RNA such as rRNA, a tRNA, or the primary amino acid sequence of a specific protein or polypeptide, or a binding site for a trans-acting regulatory agent. This phrase specifically encompasses degenerate codons (i.e., different codons which encode a single amino acid) of the native sequence or sequences that may be introduced to conform to codon preference in a specific host cell. Letters other than A, T, C, and G when present in a nucleic acid sequence disclosed herein represent ambiguity. That is, of all the molecules sampled, there is more than one kind of nucleotide at that position. The symbols (letters) are defined as follows: A=adenine; C=cytosine; G=guanine; T=thymine; R=G A (purine); Y=T C (pyrimidine); K=G T (keto); M=A C (amino); S=G C (strong bonds); W=A T (weak bonds); B=G T C (all but A); D=G A T (all but C); H=A C T (all but G); V=G C A (all but T). The term “gene” may be used interconvertibly with the term “nucleic acid molecule” or “nucleic acid sequence”. Nucleic acid molecules disclosed herein, e.g. encoding botryococcene synthase or squalene synthase can be expressed recombinantly in organisms, e.g., algae, cyanobacteria, photosynthetic or non-photosynthetic bacteria, fungi, including yeasts, or plant cells. As appreciated by one of skill in the art, expression constructs can be designed taking into account such properties as codon usage frequencies of the organism in which the nucleic acid molecule is to be expressed. Codon usage frequencies can be tabulated using known methods. Codon usage frequency tables, including those for algae and cyanobacteria, are also available in the art. The term “complementary” is used herein to mean that the sequence is complementary to all or a portion of a reference polynucleotide sequence. A “polypeptide” refers to a polymer formed from the linking, in a defined order, of amino acids by means of an amide bond. Usually, a polypeptide performs some specific function in an organism. As used herein, the term “polypeptide” includes a peptide. As used herein, “enzyme” refers to a polypeptide that catalyses the specific conversion of a molecular substrate to a molecular product. The term “enzyme” is used herein interconvertibly with the term “polypeptide”. The term “isolated”, when applied to a nucleic acid molecule or polypeptide, denotes that the nucleic acid or polypeptide is essentially free of other cellular components with which it is associated in the natural state. It is preferably in a homogeneous state and may be in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high pressure liquid chromatography. A polypeptide that is the predominant species present in a preparation is substantially purified. In particular, an isolated nucleic acid molecule is separated from open reading frames, which encode polypeptides other than the polypeptide of interest, that flank the gene from which the nucleic acid molecule is derived. As used herein, an “isolated” nucleic acid molecule or polypeptide may be purified to at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12.5%, 15%, 17.5%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more purity by weight. As used herein, “homologue”, “homology” and “homologous” refer to nucleotide sequence similarity and amino acid sequence similarity among nucleic acid molecules and polypeptides, respectively. “Homology” may also refer to functional similarity. Homology is an indicator of ancestry or evolution. An “orthologue” and a “paralogue” are two types of homologous sequences and are encompassed by the term “homologue”. “Orthology” describes nucleotide sequences or polypeptides in different species that derive from a common ancestor. “Orthologous” nucleic acid molecules or polypeptides may or may not have the same function. “Paralogy” describes homologous nucleotide sequences or polypeptides within a single species that diverged by gene duplication. As used herein, a “homologue” of a first nucleic acid molecule or sequence (e.g., any one of SEQ ID NOs: 6 to 9 or 30), or first polypeptide (e.g., any one of SEQ ID NOs: 1 to 5 or 20 to 29), is a second nucleic acid molecule or sequence, or second polypeptide, in the same organism type or in a different organism type, that is substantially similar to the first nucleic acid sequence or first polypeptide, in terms of its sequence. Otherwise, the “homologue” is substantially similar to the first nucleic acid molecule or first polypeptide in terms of function. Accordingly, a triterpenoid hydrocarbon synthetic nucleic acid molecule, or the triterpenoid hydrocarbon synthetic polypeptide, includes a homologue, an orthologue or a paralogue of a nucleic acid molecule or polypeptide disclosed herein. The homologue, orthologue or paralogue corresponds with a nucleic acid molecule or polypeptide disclosed herein. The homologue, orthologue or paralogue may be a homologue, orthologue or paralogue of SEQ ID NO: 29 or SEQ ID NO: 30, but not of SEQ ID NOs: 10 to 13 or 42. As used herein, a “triterpenoid hydrocarbon” is a compound that may be characterised by the presence of six 5-carbon isoprene (2-methyl-1,3-butadiene) units, or three 10-carbon terpene units. A “triterpenoid hydrocarbon” includes triterpenoids, methylated triterpenoids and partially cyclic triterpenoids. Particular examples of triterpenoid hydrocarbons include those synthesised by As used herein, “botryococcene” refers to a hydrocarbon compound that is produced by a living organism, and is sparingly soluble in water, but is readily soluble in organic solvents. More particularly, “botryococcene” refers to a hydrocarbon comprising isoprene units. More specifically, “botryococcene” refers to a triterpenoid hydrocarbon comprising the formula CnH2n-10, where n=30 to 37 (C30 to C37). Whilst not wishing to be bound to any particular hypothesis, triterpenoid synthesis, hence botryococcene synthesis, is dependent upon isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) precursors. IPP and DMAPP combine to produce geranyl pyrophosphate (GPP), which then combines with IPP to produce FPP. Two molecules of FPP combine to form the intermediate molecule PSPP within the active site of the botryococcene synthase enzyme, which then forms botryococcene. Botryococcene, in particular C30 botryococcene, which is the precursor of all higher botryococcenes (C31 to C37), is a triterpene resulting from head-to-head 1′-3 linkage of two FPP molecules (FIG. 36). Ultimately, botryococcene refers to any one of C30 to C37 hydrocarbons produced by the initial activity of a botryococcene synthase, including cyclic and acyclic botryococcenes and dehydrobotryococcenes. As used herein, “squalene” refers to a hydrocarbon compound that is produced by a living organism, and is sparingly soluble in water, but is readily soluble in organic solvents. More particularly, “squalene” refers to a hydrocarbon comprising isoprene units. More specifically, squalene refers to a triterpenoid hydrocarbon comprising 30 carbon atoms (C30). Triterpenoid synthesis, hence squalene synthesis, is dependent upon IPP and DMAPP precursors. IPP and DMAPP combine to produce GPP, which then combines with IPP to produce FPP. Two molecules of FPP combine to form the intermediate molecule PSPP within the active site of the squalene synthase enzyme, which then forms squalene. Squalene is a triterpene resulting from head-to-head 1′-1 linkage of two FPP molecules (FIG. 36). Ultimately, squalene refers to a C30 hydrocarbon produced by a squalene synthase. As used herein, a “substrate” is any compound that may be acted upon by a triterpenoid hydrocarbon synthetic polypeptide to produce a triterpenoid hydrocarbon. The substrate may itself be a terpenoid hydrocarbon. More particularly, the substrate may be FPP and/or PSPP or a precursor of FPP. The substrate, FPP and/or PSPP or precursor of FPP may be synthetic, recombinant or may be produced by an organism via one or more enzymes. The substrate, FPP and/or PSPP or precursor of FPP may be produced by a recombinant organism of the fourth aspect harbouring a nucleic acid molecule of the first aspect, a polypeptide of the second aspect, or a vector of the third aspect. The substrate, FPP and/or PSPP or precursor of FPP may be provided from an exogenous source to a polypeptide of the second aspect or a recombinant organism of the fourth aspect. As stated above, triterpenoid hydrocarbon synthetic polypeptide has an enzymatic activity of catalysing the condensation of two C15 molecules of FPP to a long-chain C30 triterpenoid hydrocarbon, e.g. squalene or botryococcene. In addition, a triterpenoid hydrocarbon synthetic polypeptide may catalyse the condensation of two C20 geranylgeranyl pyrophosphate (GGPP) molecules to a long-chain C40 lycopadiene hydrocarbon. A “triterpenoid hydrocarbon synthetic nucleic acid molecule” is a nucleic acid molecule encoding a polypeptide capable of converting a substrate to a triterpenoid hydrocarbon. In one embodiment, a “triterpenoid hydrocarbon synthetic nucleic acid molecule” comprises a nucleic acid molecule that encodes a polypeptide that comprises, or is derived from, any one of SEQ ID NOs: 1 to 5 or 20 to 29. In another embodiment, the triterpenoid hydrocarbon synthetic nucleic acid molecule comprises, or is derived from, any one of SEQ ID NOs: 6 to 9 or 30. A “triterpenoid hydrocarbon synthetic nucleic acid molecule” may: 1) comprise a region of about 100, 150, 200, 300, 400, 500, 1000, 1500, 2000, or 5000, or more nucleotides encoding a polypeptide comprising any one of SEQ ID NOs: 1 to 5 or 20 to 29; 2) comprise a region of about 100, 150, 200, 300, 400, 500, 1000, 1500, 2000, or 5000, or more nucleotides comprising any one of SEQ ID NOs: 6 to 9 or 30; 3) under stringent conditions hybridise to a nucleic acid molecule encoding a polypeptide comprising any one of SEQ ID NOs: 1 to 5 or 20 to 29, or the complement thereof; 4) under stringent conditions hybridise to a nucleic acid molecule comprising any one of SEQ ID NOs: 6 to 9 or 30, or the complement thereof; 5) encode a polypeptide comprising at least 15 contiguous amino acids, or at least 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, or 550, or more contiguous residues of a polypeptide comprising any one of SEQ ID NOs: 1 to 5 or 20 to 29; 6) be amplified by primers to a nucleic acid molecule encoding a polypeptide comprising any one of SEQ ID NOs: 1 to 5 or 20 to 29; 7) is amplified by primers to a nucleic acid molecule comprising any one of SEQ ID NOs: 6 to 9 or 30. Exons 2.1 (SEQ ID NO: 6, FIG. 6), 3.11 and 4.1 (SEQ ID NO: 7, FIG. 7), 5.3 and 6.1 (SEQ ID NO: 8, FIG. 8) and 9.2 (SEQ ID NO: 9, FIG. 9) correspond with SEQ ID NO: 30 (FIG. 30) and encode amino acids that correspond with SEQ ID NO: 29 (FIG. 29). Exons 2.1, 4.1, 5.3, 6.1 and 9.2 encode amino acids (SEQ ID NOs: 1 to 5, FIGS. 1 to 5) that correspond with SEQ ID NO: 29 (FIG. 29). As used herein, an “exon” refers to a nucleic acid sequence (either DNA or RNA) that is represented in the mature form of an RNA molecule after portions (introns) of a precursor RNA have been removed by splicing. The mature RNA molecule can be a messenger RNA or a functional form of a non-coding RNA such as rRNA or tRNA. The person skilled in the art will appreciate that the amino acid translation of a nucleotide sequence encoding an exon may not provide all amino acid residues due to the presence of incomplete codons arising from splice junctions. In all cases, a “triterpenoid hydrocarbon synthetic nucleic acid molecule” encodes a polypeptide capable of producing a triterpenoid hydrocarbon. The term “triterpenoid hydrocarbon synthetic nucleic acid molecule” refers to a double stranded or single stranded nucleic acid molecule. The triterpenoid hydrocarbon synthetic nucleic acid molecule encodes an active or functional triterpenoid hydrocarbon synthetic polypeptide that catalyses the conversion of a substrate to a hydrocarbon. A “triterpenoid hydrocarbon synthetic polypeptide” is an active or functional polypeptide that is capable of converting a substrate to a triterpenoid hydrocarbon. In one embodiment, a “triterpenoid hydrocarbon synthetic polypeptide” comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 5 or 20 to 29. Thus, a triterpenoid hydrocarbon synthetic polypeptide can: 1) comprise at least 15 contiguous amino acids, or at least 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, or more contiguous amino acids of any one of SEQ ID NOs: 1 to 5 or 20 to 29; or 2) bind to an antibody raised against an immunogen comprising a partial or full amino acid sequence of any one of SEQ ID NOs: 1 to 5 or 20 to 29. In all cases, a “triterpenoid hydrocarbon synthetic polypeptide” is a polypeptide capable of producing a triterpenoid hydrocarbon. A triterpenoid hydrocarbon synthetic polypeptide may be used to produce a triterpenoid hydrocarbon in a host organism. Alternatively, a triterpenoid hydrocarbon synthetic polypeptide may be used to synthesise a triterpenoid hydrocarbon, i.e. in the absence of a host cell. Any reference herein to “botryococcene synthase” or a “botryococcene synthase polypeptide” is to a polypeptide or amino acid sequence having activity sufficient to synthesise a botryococcene, including a dehydrobotryococcene. In a particular embodiment, botryococcene synthase is capable of converting FPP and/or PSPP to botryococcene. In another embodiment, botryococcene synthase is capable of converting FPP and/or PSPP to dehydrobotryococcene, by limiting, decreasing, minimising or omitting reducing equivalents such as NADPH or NADH from the reaction system. “Botryococcene synthase” refers to a protein that belongs to a family of prenyl transferases. Accordingly, specific motifs that are characteristic of prenyl transferases are also present in botryococcene synthase, such as, for example, an aspartate-rich region and/or a substrate-Mg2+ binding site. Methods for identifying amino acid residues necessary for the biochemical activity of a triterpenoid synthetic polypeptide polypeptide are described in Pandit et al., If the polypeptide of the second aspect is a botryococcene synthase and the polypeptide is able to produce a squalene and a botryococcene, more botryococcene will be produced than squalene. For example, the ratio of botryococcene to squalene will be at least greater than about 1, or at least greater than about 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10 000, 20 000, 50 000, 100 000, 200 000, 500 000, 1 000 000, 2 000 000, 5 000 000, 10 000 000 or more. As used herein, “botryococcene synthase gene” refers to a nucleic acid molecule that encodes botryococcene synthase polypeptide. Thus, such a gene is often a cDNA sequence that encodes botryococcene synthase. In other embodiments, a botryococcene synthase gene may include sequences, such as introns, that are not present in a cDNA. The term “botryococcene synthase coding region” refers to the region of the nucleic acid molecule that encodes a mature polypeptide. A “botryococcene synthase nucleic acid molecule” is a nucleic acid molecule encoding a polypeptide capable of converting FPP and/or PSPP to botryococcene. The term “botryococcene synthase nucleic acid molecule” refers to a double stranded or single stranded nucleic acid molecule. Appropriate primers for identifying a botryococcene synthase gene from an organism can be generated from comparisons of the sequences provided herein. The term “primers” refers to oligonucleotides of 10 to 100 nucleotides, which are used to amplify a gene by the polymerase chain reaction (PCR) as known by one skilled in the art. Any reference herein to “squalene synthase” or “squalene synthase polypeptide” is to an active or functional polypeptide having activity sufficient to synthesise a squalene, including a dehydrosqualene. In a particular embodiment, squalene synthase is capable of converting FPP and/or PSPP to squalene. In another embodiment, squalene synthase is capable of converting FPP and/or PSPP to a dehydrosqualene. A squalene synthase of the present disclosure does not encompass any one of SEQ ID NOs: 10, 12 or 42. If a squalene synthase polypeptide is able to produce a squalene and a botryococcene, more squalene will be produced than botryococcene. For example, the ratio of squalene to botryococcene will be at least greater than about 1, or at least greater than about 2, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10 000, 20 000, 50 000, 100 000, 200 000, 500 000, 1 000 000, 2 000 000, 5 000 000, 10 000 000 or more. As used herein, “squalene synthase gene” refers to a nucleic acid molecule that encodes a squalene synthase polypeptide. Thus, such a gene is often a cDNA sequence that encodes squalene synthase. In other embodiments, a squalene synthase gene may include sequences, such as introns that are not present in a cDNA. The term “squalene synthase coding region” refers to the region of the nucleic acid molecule that encodes the polypeptide. A “squalene synthase nucleic acid molecule” is a nucleic acid molecule encoding a polypeptide capable of converting FPP and/or PSPP to squalene. The term “squalene synthase nucleic acid molecule” refers to a double stranded or single stranded nucleic acid molecule. Appropriate primers for identifying and characterising a squalene synthase gene from an organism can be generated from the sequences provided herein. Because botryococcene synthase and squalene synthase are exemplary triterpenoid hydrocarbon synthetic polypeptides, a botryococcene synthase or squalene synthase nucleic acid molecule may have any of the features of a triterpenoid hydrocarbon synthetic nucleic acid molecule. Similarly, a botryococcene synthase or squalene synthase polypeptide may have any of the features of a triterpenoid hydrocarbon synthetic polypeptide. The person skilled in the art will understand that optimal alignment of sequences for comparison may be conducted by the local homology algorithm, by the homology alignment algorithm, by the search for similarity method, by computerized implementations of these algorithms (GAP, BESTFIT, BLAST, FASTA, TFASTA, and DASH), or by inspection. Two nucleic acid molecules or polypeptides are said to be “identical” if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is the same when aligned for maximum correspondence as described below. Optionally, the percentage of sequence identity between a reference sequence and a query sequence is only considered over the length of the query sequence. “Percentage of sequence identity” is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the nucleic acid or amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. A “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions, e.g., 15 or 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. The triterpenoid hydrocarbon synthetic nucleic acid molecule or polypeptide, e.g. botryococcene synthase or squalene synthase nucleic acid molecule or polypeptide, disclosed herein includes nucleic acid molecules and polypeptides identified and characterised by techniques such as hybridisation and/or sequence analysis using exemplary nucleic acid sequences including any one of SEQ ID NOs: 6 to 9 or 30, and exemplary polypeptide sequences including any one of SEQ ID NOs: 1 to 5 or 20 to 29. The phrase “stringent hybridisation conditions” refers to conditions under which a probe will hybridise to its target subsequence, typically in a complex mixture of nucleic acid molecules, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridise specifically at higher temperatures. Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid molecule concentration) at which 50% of the probes complementary to the target hybridise to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent hybridisation conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 M to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent hybridisation conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridisation, a positive signal is at least two times background, optionally 10 times background hybridisation. Exemplary stringent hybridisation conditions can be as following: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 55° C., 60° C., or 65° C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes. Nucleic acids that do not hybridise to each other under stringent hybridisation conditions are still substantially identical if the polypeptides that they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid molecule is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acid molecule typically hybridises under moderately stringent hybridisation conditions. For example, a botryococcene synthase or squalene synthase nucleic acid molecule can also be identified by its ability to hybridise under moderately stringent hybridisation conditions (e.g., Tm ˜40° C.) to nucleic acid probes having the sequence of any one of SEQ ID NOs: 6 to 9 or 30. Such a botryococcene synthase nucleic acid sequence can have, e.g., about 25-30% base pair mismatches or fewer relative to the selected nucleic acid probe. Any one of SEQ ID NOs: 6 to 9 or 30, comprises an exemplary botryococcene synthase or squalene synthase nucleic acid sequence. Exemplary “moderately stringent hybridisation conditions” include hybridisation in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 1×SSC at 45° C. Such washes can be performed for 5, 15, 30, 60, 120, or more minutes. A positive hybridisation is at least twice background. Those of ordinary skill in the art will readily recognise that alternative hybridisation and wash conditions can be utilized to provide conditions of similar stringency. Appropriate probes for identifying a triterpenoid hydrocarbon synthetic gene, such as a botryococcene synthase or squalene synthase gene, from an organism can be generated from comparisons of the sequences provided herein. Consequently, a “percentage of sequence identity” and/or hybridisation can aid identification of a homologue, an orthologue or a paralogue of a triterpenoid hydrocarbon synthetic gene or polypeptide disclosed herein. Isolation or generation of a homologous triterpenoid hydrocarbon synthetic nucleic acid molecule, such as a botryococcene synthase or squalene synthase nucleic acid molecule, can be accomplished by a number of techniques. Cloning and expression of such technique will be addressed in the context of botryococcene synthase or squalene synthase nucleic acid molecules. For instance, oligonucleotide probes based on the sequences disclosed here can be used to identify the desired nucleic acid molecule in a cDNA or genomic DNA library from a desired species. Such a cDNA or genomic library can then be screened using a probe based upon the sequence of a cloned botryococcene synthase or squalene synthase gene, e.g., any one of SEQ ID NOs: 6 to 9 or 30. Probes may be used to hybridise with genomic DNA or cDNA sequences to isolate homologous genes in the same or different species. Alternatively, the nucleic acid molecule of interest can be amplified from nucleic acid samples using amplification techniques. For instance, PCR may be used to amplify the sequences of the genes directly from mRNA, from cDNA, from genomic libraries or cDNA libraries. PCR and other in vitro amplification methods may also be useful, for example, to clone nucleic acid molecules that code for polypeptides to be expressed, to make nucleic acid molecules to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes. As used herein, “expression” of a triterpenoid hydrocarbon synthetic nucleic acid molecule, such as a botryococcene synthase or squalene synthase nucleic acid molecule, refers to introducing a triterpenoid hydrocarbon synthetic nucleic acid molecule, e.g. a botryococcene synthase or squalene synthase nucleic acid molecule, into a cell, e.g., algae, such as green algae, cyanobacteria, photosynthetic or non-photosynthetic bacteria, fungi, including yeasts, or a plant, in which it is not normally expressed. Accordingly, an “increase” in triterpenoid hydrocarbon synthetic activity or expression is generally determined relative to wild type cells, e.g., algae, cyanobacteria, photosynthetic or non-photosynthetic bacteria, fungi, including yeasts, or plants that have no, or low, triterpenoid hydrocarbon synthetic activity. “Increased” or “enhanced” activity or expression of a triterpenoid hydrocarbon synthetic nucleic acid molecule or polypeptide, e.g. a botryococcene synthase or squalene synthase nucleic acid molecule or polypeptide, refers to a change in triterpenoid hydrocarbon synthetic activity or expression, e.g. botryococcene synthase or squalene synthase activity or expression. Examples of such increased activity or expression include the following: botryococcene synthase expression or activity or squalene synthase expression or activity of a botryococcene synthase nucleic acid molecule or polypeptide or squalene synthase nucleic acid molecule or polypeptide is increased above the level of that in the wild-type, non-transgenic control organism (i.e., the quantity of botryococcene synthase or squalene synthase activity, or expression of botryococcene synthase or squalene synthase nucleic acid molecule, is increased); botryococcene synthase or squalene synthase activity, or expression of a botryococcene synthase or squalene synthase nucleic acid molecule, is in a cell where it is not easily detected in wild-type, non-transgenic cells (i.e., expression of the botryococcene synthase or squalene synthase protein is increased, as is its activity); botryococcene synthase or squalene synthase activity, or botryococcene synthase or squalene synthase nucleic acid molecule expression, is also increased when botryococcene synthase or squalene synthase activity or expression of the botryococcene synthase or squalene synthase nucleic acid molecule is present in a cell for a longer period than in a wild-type, non-recombinant control (i.e., duration of botryococcene synthase or squalene synthase activity, or expression of the botryococcene synthase or squalene synthase protein is increased). The activity or expression of a triterpenoid hydrocarbon synthetic nucleic acid molecule or polypeptide, or the production of a triterpenoid hydrocarbon, is considered to be “increased” or “enhanced” if the activity, expression or production is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12.5%, 15%, 17.5%, 20%, 25%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82.5%, 85%, 87.5%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or more, for example at least about 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 20-, 50-, 100-fold or more, compared with an organism lacking a triterpenoid hydrocarbon synthetic gene or polypeptide, or compared with an organism comprising a wild-type or endogenous triterpenoid hydrocarbon synthetic gene or polypeptide, or in a cell-free system compared to another putative or homologous triterpenoid hydrocarbon synthetic polypeptide. A “vector” as used herein refers to any vehicle used to transfer genetic material, particularly to transfer exogenous genetic material into a cell. An “expression vector” refers to a nucleic acid construct, which when introduced into a host cell, results in transcription and/or translation of a RNA or polypeptide, respectively. The introduction of polynucleotide sequences can be either temporary, e.g. by use of vectors, or permanent, e.g. by integration of the entire vector or a fragment thereof into either the nuclear genome, the plastid genome, or the mitochondrial genome of the host alga. Suitable vectors for increasing expression of the polynucleotide sequence that encodes the desired polypeptide sequence in an algal cell are known in the art, such as the expression vectors described in U.S. Pat. No. 7,232,679. Such vectors for increasing expression of the polynucleotide sequence are incorporated herein by reference. For example, suitable vectors include pBBR-K-mev-opl6-I, pBBR-K-mev-opl6-2, pDS-mvaA, pDS-idi, pDS-hcs, pDS-mvk, pDS-pmk, pDS-mvd, pDS-His-mvaA, pDS-His-idi, pDS-His-hcs, pDS-His-mvk, pDS-His-pmk, pDS-His-mvd, pBBR-K-Zea4, pBBR-K-Zea4-up, pBBR-K-Zea4-down, pBBR-K-PcrtE-crtE-3, pBBR-tK-PcrtE-mvaA, pBBR-tK-PcrtE-idi, pBBR-tK-PcrtE-hcs, pBBR-tK-PcrtE-mvk, pBBR-tK-PcrtE-pmk, pBBR-tK-PcrtE-mvd, pBBR-K-PcrtE-mvaA-crtE-3, pDS-His-phaA, pBBR-K-PcrtE-crtW, pBBR-K-PcrtE-crtWZ, pBBR-K-PcrtE-crtZW, and combinations thereof. A vector comprising a triterpenoid hydrocarbon synthetic nucleic acid molecule, e.g. botryococcene synthase nucleic acid molecule or squalene synthase nucleic acid molecule, may comprise a marker gene that confers a selectable phenotype on algae, plant or bacterial cells. For example, the marker may encode antibiotic resistance, such as resistance to kanamycin, G418, phleomycin (Zeocin™), bleomycin, hygromycin, and the like. In one embodiment, a selectable marker for use in Selectable markers for host organisms are well known in the art. A “regulatory element” includes a promoter, which may be either constitutive or inducible. In one embodiment, a promoter can be used to direct expression of botryococcene synthase nucleic acid molecules under the influence of changing environmental conditions. Examples of environmental conditions that may effect transcription via an inducible promoter include anaerobic conditions, elevated temperature, or the presence of light. Promoters that are inducible upon exposure to chemical reagents may also be used to express a triterpenoid hydrocarbon synthetic nucleic acid molecule, e.g. a botryococcene synthase or a squalene synthase nucleic acid molecule. Other useful inducible regulatory elements include copper-inducible regulatory elements, tetracycline and chlor-tetracycline-inducible regulatory elements, ecdysone inducible regulatory elements, heat shock inducible regulatory elements, and lac operon elements, which are used in combination with a constitutively expressed lac repressor to confer, for example, IPTG-inducible expression. An inducible regulatory element also can be, for example, a nitrate-inducible promoter, e.g., derived from the spinach nitrite reductase gene, or a light-inducible promoter, such as that associated with the small subunit of RuBP carboxylase or the LHCP gene families, or a light. The term “promoter” or “regulatory element” refers to a region or sequence determinants located upstream or downstream from the start of transcription that direct transcription. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal elements, which can be located as much as several thousand base pairs from the start site of transcription. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. An “inducible” promoter is a promoter that is active under environmental or developmental regulation. The term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter) and a second nucleic acid sequence, such as a botryococcene synthase or squalene synthase gene, wherein the expression control sequence directs transcription of the nucleic acid molecule corresponding to the second sequence. An “algae promoter”, “bacterial promoter”, “fungal promoter”, “yeast promoter” or “plant promoter” is a promoter capable of initiating transcription in algae, bacteria, fungi, including yeasts, and/or plant cells, respectively. Such a promoter is therefore active in algae, cyanobacteria, bacterial, or plant cells, but need not originate from that organism. It is understood that limited modifications can be made without destroying the biological function of a regulatory element and that such limited modifications can result in regulatory elements that have substantially equivalent or enhanced function as compared to a wild type regulatory element. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental such as through mutation in hosts harbouring the regulatory element. All such modified nucleotide sequences are included in the definition of a regulatory element as long as the ability to confer expression is substantially retained. In one example, a promoter sequence that is responsive to light may be used to drive expression of a botryococcene synthase nucleic acid construct that is introduced into In one embodiment, the promoter may be from a gene associated with photosynthesis in the species to be transformed or another species. For example such a promoter from one species may be used to direct expression of a protein in transformed algal cells or cells of another photosynthetic marine organism. Suitable promoters may be isolated from or synthesized based on known sequences from other photosynthetic organisms. Preferred promoters are those for genes from other photosynthetic species that are homologous to the photosynthetic genes of the algal host to be transformed. For example, a series of light harvesting promoters from the fucoxanthin chlorophyll binding protein have been identified in In one embodiment, a promoter used to drive expression of a heterologous triterpenoid hydrocarbon synthetic nucleic acid molecule, e.g. a botryococcene synthase or squalene synthase nucleic acid molecule, is a constitutive promoter. Examples of constitutive strong promoters for use in algae include, e.g., the promoters of the atpA, atpB, and rbcL genes. Various promoters that are active in cyanobacteria are also known. These include promoters such as the (constitutive) promoter of the psbA3 gene in cyanobacteria. Other promoters that are operative in plants, e.g., promoters derived from plant viruses, such as the CaMV35S promoters, can also be employed in algae. Promoter sequences for an algal cell are preferably isolated from an algal species or a closely related organism. Promoters that are functional in higher plants are less preferred except for groups of algae closely related to higher plants. For example, the 35S CaMV promoter, which is active in many plant species, is completely inactive in Examples of suitable promoters include hydrogenase promoters, Cytochrome C6 (Cyc6) promoter, Nia1 promoter, Cabll-1 promoter, Ca1 promoter, Ca2 promoter, coprogen oxidase promoter, algal ribulose bisphosphate carboxylase small subunit gene (SSU) promoter, and algal pyruvate kinase promoter. Additional suitable promoters include the arylsulfatase promoter, and the aminoglycoside 3′-phosphotransferase gene (aph VIII) promoter from the multicellular green alga Volvox, and RbcS2 promoter which has been widely used to drive gene expression in the nucleus of Another suitable promoter for use in In one embodiment, promoters are identified by analysing the 5′ sequences of a genomic clone corresponding to a triterpenoid hydrocarbon synthetic gene, e.g. a botryococcene synthase or squalene synthase gene. Sequences characteristic of promoter sequences can be used to identify the promoter. A promoter can be evaluated, e.g., by testing the ability of the promoter to drive expression in plant cells, e.g., green algae, in which it is desirable to introduce a triterpenoid hydrocarbon synthetic construct, such as a botryococcene synthase or squalene synthase expression construct. Examples of suitable regulatory elements, e.g. enhancer elements, include: EE-1 and EE-2 described in Kucho et al. ( Enhancer elements suitable for transgene expression in “ As used herein, “strain” refers to an organism belonging to the same species, but displaying at least one different characteristic compared with a second organism of the same species. The different characteristic can be caused by differences in the genomes of the strains under comparison. Thus, a recombinant organism is also a “strain” of the host organism. Specific types of strains are referred to herein as “races”. “Race A” refers to strains of As used herein, “organism” refers to an individual living system. A “host organism” refers to a first organism that comprises exogenous DNA or RNA, or a polypeptide, which is derived from a second organism other than the first organism. The host organism is a “non-human” organism, which excludes vertebrate organisms. The host organism may be a a single cell organism, a filamentous organism, a higher plant or a macroalgae such as a seaweed, for example Pacific giant kelp. Pacific giant kelp is a species of marine alga found on the Pacific coast of North and Central America. It begins life as a microscopic spore, but may grow up to 60 m with the upper fronds forming a dense canopy at the surface. Kelp fronds may grow at 300 mm to 600 mm per day which makes it ideal to produce large amounts of triterpenoid hydrocarbons in a short period of time. See, for example, Copping, et al., 2008, Techno-Economic Feasibility Analysis of Offshore Seaweed Farming for Bioenergy and Biobased products, Independent Research and Development Report, Battelle Pacific Northwest Division, incorporated herein by reference. In a particular embodiment, the host organism is a microorganism. As used herein, “microorganism” refers to microalgae, bacteria, fungi, including yeast, cyanobacteria, diatoms, photosynthetic bacteria or non-photosynthetic bacteria, or Euglenids. In one embodiment, an alga is used. “Alga”, “algae”, “microalga”, “microalgae” or the like, refer to plants belonging to the subphylum Algae of the phylum Thallophyta. The algae are unicellular, photosynthetic, oxygenic algae and are non-parasitic plants without roots, stems or leaves; they contain chlorophyll and have a great variety in size, from microscopic to large seaweeds. Green algae, which are single cell eukaryotic organisms of oxygenic photosynthesis, endowed with chlorophyll a and chlorophyll b, belonging to Eukaryota-Viridiplantae-Chlorophyta-Chlorophyceae, may be used. However, algae may also be blue-green, red, or brown, so long as the algae are able to host a nucleic acid molecule of the first aspect or a vector of the third aspect to produce a triterpenoid hydrocarbon such as botryococcene or squalene. In one embodiment, a green alga, cyanobacterium (blue-green algae), or a diatom is used. Examples of green algae include: Alternatively, the alga may be In another embodiment, a euglanid may be used, e.g. In yet another embodiment, a bacterium may be used. The bacterium may be photosynthetic, e.g. In an alternative embodiment, another photosynthetic organism may be used, e.g. a plant cell. Plant cells that may be suitable include those of In one embodiment, a fungus may be used. For example, a yeast may be used, e.g. As used herein, “recombinant organism” or “transgenic organism” refers to a second organism, or “host organism”, that harbours genetic material derived from a first, or donor, source. “Recombinant organism” refers to any organism, such as an alga, bacterium, fungus, including a yeast, or plant, in which an exogenous nucleic acid molecule is expressed. More specifically, the term refers to any transformed alga, bacterium, fungus, including a yeast, or plant that displays the desired characteristics of both the donor and host organisms. The desired characteristic of the donor organism is a triterpenoid hydrocarbon synthetic nucleic acid molecule, e.g. a botryococcene synthase or squalene synthase nucleic acid molecule, or the desired characteristic is a triterpenoid hydrocarbon synthetic polypeptide, e.g. a botryococcene synthase or a squalene synthase polypeptide, either of which contributes in the host organism to high triterpenoid hydrocarbon content similar to the donor organism. The desired characteristics of the host organism include stability and/or fast growth. Therefore, a “recombinant organism” will synthesise levels of triterpenoid hydrocarbon in excess of the host organism and more similar to those of the donor strain. A desired “recombinant organism” will also be more stable and/or grow faster than the donor organism. As used herein, “recombinant” describes a second, or host species of genetic material that harbours genetic material derived from a first, or donor, source. Thus, the host harbours exogenous genetic material, e.g. DNA or RNA. “Recombinant” is used interchangeably with “genetically manipulated” or “transgenic”. As used herein, “genetic manipulation” or “molecular biotechnology” refers to any human-exerted artificial process that is performed on DNA or RNA, including, but not limited to, disruption, isolation, replication, modification and introduction into a different organism. An organism that has undergone genetic manipulation is also referred to herein as “recombinant”. As used herein, “exogenous” refers to a molecule, e.g. DNA, RNA or compound, originating externally to an organism. With respect to DNA or RNA, the term can be used interchangeably with “heterologous”. Conversely, as used herein, “endogenous” refers to a molecule, e.g. DNA, RNA or compound, originating within an organism. The term “heterologous” refers to a host, e.g. a strain, species, or genus, not normally possessing a gene that is transformed into the host, or refers to a gene not normally present in a host, e.g. a strain, species, or genus, artificially endowed with the gene. The term “heterologous” is used interconvertibly with the term “exogenous”, in the case of an exogenous gene. A nucleic acid molecule is “heterologous to” a second nucleic acid molecule if it originates from a foreign species, or, if from the same species, is modified by human action from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is different from any naturally occurring allelic variants. This disclosure contemplates a recombinant organism or a host organism further transformed with a nucleic acid molecule or vector encoding, in addition to the triterpenoid hydrocarbon synthetic polypeptide, a polypeptide useful in synthesis of a triterpenoid hydrocarbon such as a botryococcene. For example, the recombinant organism may also comprise an FPP synthase or a methyltransferase such as a botryococcene methyltransferase or a squalene methyltransferase. As used herein, “transform”, and variations thereof, refers to the process of introducing exogenous DNA or RNA from a donor organism into a host organism. The term is not used in a limiting sense and includes any mechanism by which foreign DNA can be introduced into a host organism. Thus, “transform” includes, but is not limited to, electroporation, transfection, gene guns, viral methods, and glass beads methods for example. The term “transformed” essentially unites a gene and organism not previously containing that gene, and refers to the artificial conjunction of the organism and the gene by genetic modification of the organism by a human being, using processes other than sexual conjugation of organisms. After transformation, function of the gene may or may not occur in the new organism. In algae, the nuclear, mitochondrial, and chloroplast genomes are transformed through a variety of known methods, including by microparticle bombardment (e.g. DNA-coated gold particles), or using a glass bead method. In one embodiment, a triterpenoid hydrocarbon synthetic nucleic acid molecule, e.g. a botryococcene synthase or squalene synthase nucleic acid molecule, is introduced into and/or targeted to the chloroplast of an alga. In another embodiment, a triterpenoid hydrocarbon synthetic nucleic acid molecule, e.g. a botryococcene synthase or squalene synthase nucleic acid molecule, is introduced into and/or targeted to the nucleus. To use isolated sequences in the above techniques, synthetic or recombinant DNA vectors suitable for transformation of green algae, other eukaryotic algae, cyanobacteria, photosynthetic or non-photosynthetic bacteria, fungi, including yeasts, or plant cells are prepared. Techniques for transformation are well known in the art and described in the technical and scientific literature. For example, a DNA sequence encoding a triterpenoid hydrocarbon synthetic nucleic acid molecule, e.g. a botryococcene synthase or squalene synthase nucleic acid molecule, can be combined with transcriptional and other regulatory sequences which will direct the transcription of the nucleic acid molecule in the intended cells of the transformed alga, cyanobacterium, bacterium, fungus, including a yeast, or plant. In one embodiment, an expression vector that comprises the botryococcene synthase or squalene synthase nucleic acid molecule further comprises a promoter operably linked to the botryococcene synthase or squalene synthase nucleic acid molecule. In other embodiments, a promoter and/or other regulatory elements that direct transcription of the botryococcene synthase or squalene synthase nucleic acid molecule are endogenous to the organism and the vector comprising the botryococcene synthase or squalene synthase nucleic acid molecule is introduced, e.g., by homologous recombination, such that the heterologous botryococcene synthase or squalene synthase nucleic acid molecule is operably linked to an endogenous promoter and expression is driven by the endogenous promoter. The present disclosure employs various routine synthetic or recombinant nucleic acid techniques. Generally, the nomenclature and the laboratory procedures in synthetic or recombinant DNA technology described are those well known and commonly employed in the art. As used herein, “biofuel” refers to fuel derived from carbon sources that can be replenished and make reduced, minimal or no net contribution to atmospheric carbon dioxide levels. More specifically, biofuel refers to carbon sources derived from an organism that expresses a triterpenoid hydrocarbon synthetic gene, and the lipid or hydrocarbon can be used as fuel or to produce fuel. To produce biofuel, it may be necessary for hydrolytic cracking or hydrocracking of the triterpenoid hydrocarbon. Biofuel is distinct from traditional “fossil” fuels. A triterpenoid hydrocarbon produced according to this disclosure may be combusted directly as a fuel. However for performance in internal combustion engines, the triterpenoid hydrocarbon should be modified by processes such as pyrolysis and catalytic cracking. Crude triterpenoid hydrocarbons can be converted to petrol (gasoline), light cycle oil, heavy cycle oil, and coke by catalytic cracking as has been taught by Kitazato et al., Sekiyu Gakkaishi 32:28. The yields of petrol (gasoline) obtained by catalytic cracking of algal hydrocarbons are comparable to yields obtained from petroleum. Also the gasoline produced has sufficiently high octane numbers for direct use in transportation vehicles. Aviation fuel may also be obtained using processes known in the art. Similarly, “bioplastic” refers to plastic derived from carbon sources that can be replenished and make minimal or no net contribution to atmospheric carbon dioxide levels. More specifically, bioplastic refers to carbon sources derived from an organism that expresses a triterpenoid hydrocarbon synthetic gene, and the triterpenoid hydrocarbon can be used to produce plastic. The disclosure contemplates application to any product traditionally derived from petrochemical sources of fossil fuels to which the triterpenoid hydrocarbon is amenable. For example, in addition to biofuel and bioplastic, it is envisaged that the disclosure will be useful for, but not limited to, production of pharmaceuticals, food additives, industrial chemicals, specialty chemicals, detergents, fertilizers, medicines, paints, plastics, synthetic fibres, or synthetic rubber. In an embodiment of the method of the sixth aspect, the triterpenoid hydrocarbon is squalene or botryococcene and the conditions comprise supplementing the recombinant organism with NADPH or NADH. In another embodiment, the triterpenoid hydrocarbon is dehydrosqualene or dehydrobotryococcene and the conditions comprise not supplementing the recombinant organism with, or minimising the concentration of, NADPH or NADH. By limiting, decreasing, minimising or omitting reducing equivalents such as NADPH or NADH in the reaction system, the method provides for the production of a dehydrosqualene or a dehydrobotryococcene, which each lack at least two hydrogen atoms when compared with squalene or botryococcene, respectively. Dehydrosqualene and/or dehydrobotryococcene can be identified using GC-MS. A triterpenoid hydrocarbon synthetic nucleic acid molecule, e.g. botryococcene synthase or squalene synthase nucleic acid molecule, can be expressed in any number of algae, e.g., green algae, other eukaryotic algae, cyanobacteria, or photosynthetic or non-photosynthetic bacteria, fungi, including yeasts, or plant cells where it is desirable to produce a triterpenoid hydrocarbon, e.g. botryococcene or squalene. Transformed algae, cyanobacteria, bacteria (photosynthetic bacteria or non-photosynthetic bacteria), or fungi, including yeasts, that express a heterologous triterpenoid hydrocarbon synthetic nucleic acid molecule, e.g. botryococcene synthase or squalene synthase nucleic acid molecule, may be grown under mass culture conditions for the production of the triterpenoid hydrocarbon, e.g. botryococcene or squalene. The transformed or recombinant organisms are grown in bioreactors or fermentors that provide an enclosed environment to contain the triterpenoid hydrocarbon, e.g. botryococcene or squalene. In certain embodiments for mass culture, the algae, cyanobacteria, bacteria, or fungi, including yeasts, are grown in enclosed reactors in quantities of at least about 100 litres, 200 litres, 500 litres, at least about 1000 litres, 2000 litres, 5000 litres, 10,000 litres, 20,000 litres, 50,000 litres, 100,000 litres, 200,000 litres, 500,000 litres or greater, and in one embodiment, in quantities of about 1,000,000 litres or more, even 1,500,000 litres, or more. As used herein, “mass-culturing” refers to growing large quantities of algae, cyanobacteria, photosynthetic or non-photosynthetic bacteria, or fungi, including yeasts, that have been modified to express a triterpenoid hydrocarbon synthetic nucleic acid molecule, e.g. botryococcene synthase or squalene synthase. A “large quantity” is generally in the range of about 10 litres or more, 20 litres, 50 litres, or about 100 litres to about 1,500,000 litres, for example, 200 litres, 500 litres, 1000 litres, 2000 litres, 5000 litres, 10,000 litres, 20,000 litres, 50,000 litres, 100,000 litres, 200,000 litres, 500,000 litres, 1,000,000 litres, even 1,500,000 litres, or more. In one embodiment, the organism may be cultured in large quantities in modular bioreactors, each having a capacity of about 1,000 to about 1,000,000 litres, for example, 200 litres, 500 litres, 1000 litres, 2000 litres, 5000 litres, 10,000 litres, 20,000 litres, 50,000 litres, 100,000 litres, 200,000 litres, or 500,000 litres. In one embodiment, a recombinant plant may be amenable to mass culture. A “bioreactor” refers to any enclosed large-capacity vessel in which microalgae, cyanobacteria or photosynthetic or non-photosynthetic bacteria, or fungi, including yeasts, are grown. A “large-capacity vessel” can hold about 10 litres, or about 500 litres to about 1,000,000 litres, or more, for example, 20 litres, 50 litres, 100 litres, 200 litres, 500 litres, 1000 litres, 2000 litres, 5000 litres, 10,000 litres, 20,000 litres, 50,000 litres, 100,000 litres, 200,000 litres, or 500,000 litres, or more. Methods of mass-culturing algae are known. For example, algae can be grown in high density photobioreactors, bioreactors such as those for sewage and waste water treatments, mass-cultured for the elimination of heavy metals from contaminated water, mass-cultured for the production of β-carotene, hydrogen, and pharmaceutical compounds, as well as nutritional supplements for both humans and animals and for the production of other compounds of nutritional value. Conditions for growing triterpenoid hydrocarbon-expressing algae, cyanobacteria, photosynthetic or non-photosynthetic bacteria, or fungi, including yeasts, e.g. botryococcene synthase-expressing or squalene synthase-expressing algae or bacteria, are known in the art. For example, methods for growing Alternatively, a triterpenoid hydrocarbon may be produced in a cell-free system. Examples of cell-free systems are disclosed herein at Example 10 and Example 16, and in US 2010/0041120, Okada et al. Triterpenoid hydrocarbon, e.g. botryococcene or squalene, produced by the genetically modified organisms disclosed herein is not currently known in the art, but could be harvested using known techniques. As used herein, to “harvest” a triterpenoid hydrocarbon, e.g. botryococcene or squalene, refers to capturing and sequestering the triterpenoid hydrocarbon, e.g. botryococcene or squalene, in a closed or contained environment. Harvesting may include flocculating the microorganism, e.g. alga, by adjusting the pH of the medium in with the organism resides. Botryococcenes and squalene are not miscible in water and they may rise to and float at the surface of the growth medium if released by the organism. If so, they may be siphoned off from the surface and sequestered in suitable containers. Alternatively, destructive harvesting of the triterpenoid hydrocarbon, e.g. botryococcene or squalene, from the recombinant organism, such as algae (including seaweed) or bacteria, may be required, for example pressing, solvent extraction or supercritical fluid extraction. Under suitable conditions, 70% of hydrocarbons can be released by contact with hexane for 30 minutes. Hydrocarbons can be continuously extracted with proper agitation and recovery methodologies known in the art. Supercritical fluids such as carbon dioxide (CO2) allow rapid extraction of hydrocarbons, and are nontoxic, inexpensive, easily separated from the extracted hydrocarbons and are reusable. Also disclosed herein is a business method comprising providing a carbon credit to a party growing a genetically modified organism, comprising a nucleic acid molecule according to the first aspect, adapted to produce a biofuel, a pharmaceutical, a food additive, an industrial chemical, a specialty chemical, a detergent, a fertilizer, a medicine, a paint, a plastic, a synthetic fibre, or a synthetic rubber. As such, the methods and compositions described herein may be used in a business method in exchange for carbon credits. Carbon credits may be an allowance, permit, credit, or the like which are or have been allowed, authorised, or recognised by some relevant sovereign entity (such as, but not limited to, a city (including municipalities of all sizes and types including both incorporated and unincorporated municipalities), a county, a state or province, or a nation, as well as related governmental entities such regional, multi-national, or other international bodies such as the United Nations, the European Union, or the United Arab Emirates). The carbon credit may be substantially received directly from a regulatory agency or administrative entity. In other instances, they may be received indirectly, for example, an entity using the methods or compositions herein may receive the carbon credits directly from a regulatory agency, and may then transfer the carbon credits to another entity. Transfer of the carbon credit may be in association with a given process or product using the genetically modified organism. For example, a first entity may be identified that provides a consumable product that is distributed for consumption in an end-user mobile platform, wherein the consumption and/or production of the consumable product includes a corresponding resultant emission. For example, combustion of diesel fuel often results in the environmental release of corresponding nitrogen oxides and combustion of gasoline often results in the environmental release of corresponding sulfur oxide. The first party may adopt a method of producing its product using the genetically modified organism, or use the product generated by the genetically modified organisms in their compositions, resulting in less harmful effects on the environment than conventional methods of generating, for example, diesel fuel, thus off-setting the environmental effects of the end product. The first party may then receive a carbon, or emission, credit as a result of a reduction of the total emission. The carbon credit may be received from a regulatory or administrative agency, or may be transferred to the first party from a second party, wherein the second party may have sold the genetically modified organism or the product of the genetically modified organism to the first party. The carbon credit may be exchanged for a substantially liquid monetary instrument. For example, the carbon credit may be exchanged for a cash equivalent, such as cash, cheque, and the like. The carbon credit may also be exchanged for a legal grant regarding an intellectual property right, for example, but not limited to, an assignment or a license. The carbon credit may also be exchanged for a government tax subsidy or access to purchasers of a given market. The carbon credit may also be exchanged for use of another carbon emission process, such as one not comprising growing the organism. For example, a party may have a limited number of emissions it may release in a time period, for example, a month or a year, and going over the limit may incur fines and penalties. However, with carbon credits, the party going over the limit may exchange carbon credits to offset the fines or penalties or may be taken into account when determining the amount of emissions generated by the party. The business method can also involve the production of a product other than a biofuel, for example a pharmaceutical, a food additive, an industrial chemical, a specialty chemical, a detergent, a fertilizer, a medicine, a paint, a plastic, a synthetic fibre, or a synthetic rubber. A business method associated with a biofuel, including those involving the use of carbon credits, are also relevant to the production of other types of useful products and materials. It will be understood to persons skilled in the art of the invention that many modifications may be made without departing from the spirit and scope of the invention. In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country. Pursuant to the Budapest Treaty, plasmids pUC57/ATB1 (construct 1, Example 12), pET11/ATB1 (construct 2, Example 12), pSP124S (Example 11), and pBC1 were deposited on 14 Sep. 2010 with the National Measurement Institute (NMI), Australia in its capacity as an International Depository Authority. Deposited plasmid pUC57/ATB1 was assigned the accession number V10/022706. Deposited plasmid pET11/ATB1 was assigned the accession number V10/022707. Deposited plasmid pSP124S was assigned the accession number V10/022708. Deposited plasmid pBC1 was assigned the accession number V10/022709. The DNA was extracted from the Ayamé 1 strain of Example 1 as follows. First, approximately 2×200 milligrams of cell pellet was obtained by flocculation of cells in 50% ethanol (final concentration). Each cell pellet was resuspended in 2 mL of high salt DNA extraction buffer (200 mM Tris.HCl pH 7.5, 100 mM EDTA pH 8.0, 0.5 M NaCl) and subjected to 10 rounds of freeze-thaw using liquid nitrogen and a 45° C. water bath. Proteinase K was then added to a final concentration of 2 mg/mL and the mix was incubated at 55° C. for one hour, then 60° C. overnight. The resulting lysate was used whole in a Qiagen DNeasy Plant Maxi Kit (cat. no. 68163), to extract the DNA, according to the manufacturer's instructions. The genome of Twenty micrograms of the genomic DNA from Another twenty micrograms of the genomic DNA from Preparatory assembly of DNA contigs by merging of overlapping raw sequence data (Example 3) was performed at the AGRF. Subsequently, data from the Illumina platform was integrated at Flinders University using a variety of algorithms such as EULER, VELVET, CLC Genomics Workbench, DASH, to extend contigs and establish additional contigs. Using BLAST or DASH algorithms, putative genes were identified and the genome will be assembled. Exons encoded by amino acid sequences provided as SEQ ID NOs: 1 to 5 or 20 to 28were identified according to this example. The full polypeptide sequence (ATB1) provided as SEQ ID NO: 29 was identified according to this example. Cells of A. Algae The disclosure is not limited to algae, but in one embodiment the host cell is an alga. The desirable criteria for host algae include ease and rapidity of growth, and flagellate morphology, because flagellate algae have positive phototaxis and promote ease of harvest. Given these criteria, the non-limiting example of species listed below may be gene recipients. Alternatively, the alga may be a seaweed, for example Pacific giant kelp. 1. Algae i. ii. iii. iv. Other green algal species that may be used according to the disclosure include v. vi. Any alga of the dinophyceae, haptophyceae, stramenopiles, alveolates, cryptophyceae, euglenophyceae, or rhodophyceae may be used as hosts. vii. 2. Cyanobacteria i. ii. Other cyanobacteria that may be used according to the disclosure include B. Euglenids C. Bacteria 1. Photosynthetic Bacteria 2. Non-Photosynthetic Bacteria D. Other Photosynthetic Organisms E. Yeast Fungi may also be a suitable host cell, for example a yeast such as A nucleic acid construct suitable for use in an expression vector for expressing botryococcene synthase or squalene synthase in A second nucleic acid construct suitable for use in an expression vector for expressing botryococcene synthase in The expression construct may be ligated into a vector, for example a plasmid. Nucleic acid sequences may be manipulated using computer software such as Sequencher (GeneCodes USA) or Bioedit. Electroporation and the glass beads method may be used for the transformation of algal species. The transformed cells may be incubated for two weeks in 24 hour light at 25° C. Transformed cells may be subjected to antibiotic selection (phleomycin (Zeocin™) resistance). Colonies of resistant A botryococcene synthase gene or squalene synthase gene may be functionally tested in Identification and quantification of botryococcene or squalene production in the transgenic organism may be conducted by lysing the transgenic host cells, then detecting botryococcene or squalene directly. Botryococcene is not present at all in Squalene is present in Alternatively, the activity of the transgenic botryococcene synthase or squalene synthase enzyme in the transgenic host may be assayed by lysing the transgenic host cells, then detecting the botryococcene or squalene using a biochemical assay for conversion of FPP to botryococcene or squalene, for example incorporation of tritium from tritium-labelled FPP into tritium-labelled botryococcene or tritium-labelled squalene. The botryococcene or squalene can be detected by chromatography, for example gas chromatography (GC), liquid chromatography (LC), or thin layer chromatography (TLC). The TLC procedure is useful for detecting tritium-labelled botryococcene or tritium-labelled squalene using photographic film or other detectors. In representative experiments, a host organism such as Botryococcene synthase or squalene synthase enzymes expressed in Botryococcene or squalene production may be achieved by purification of transgenic botryococcene synthase or squalene synthase enzyme from the in vivo host, until it exists in a high state of purity substantially free of other enzymes of that host. Identification and quantification of botryococcene or squalene may be conducted using a biochemical assay for conversion of FPP to botryococcene or squalene. The botryococcene or squalene thus produced can be detected by chromatography, e.g. GC, LC or TLC. The TLC procedure may rely on incorporation of tritium-labelled FPP into tritium-labelled botryococcene or tritium-labelled squalene, and may be detected using photographic film or other detectors to detect tritium labelled botryococcene or squalene. The host strain of Squalene Synthase Construct To insert the Insertion into The squalene synthase construct was cut with the restriction enzyme SacI (Promega, WI, USA) to linearise it, and was then transformed into Bioinformatic Methods The bleomycin resistance plasmid was obtained from Dr Britta Forster (Australian National University) and Dr Saul Purton (University College London). The gene construct was designed by downloading from Genbank the genomic DNA sequence, and RNA sequence for Detection of Transgenic Primer pair 1 (5′-CCTTGATGGGGTATTTGAGCAC-3′, SEQ ID NO: 16 and 5′-GGAATTCGGTTAGGCGCTGAGTGTGGGTCTAGG-3′, SEQ ID NO: 17) and primer pair 2 (5′-TGAGCACTTGCAACCCTTATCCG-3′, SEQ ID NO: 18 and 5′-GGAATTCGGTTAGGCGCTGAGTGTGGGTCTAGG-3′, SEQ ID NO: 19) were used to amplify the Results Transformants After the electroporation and glass bead protocols had been carried out using the squalene synthase pSP124S construct, and the transformed cells incubated for two weeks at 25° C. in 24 hour light on Tris Acetate Phosphate (TAP) agar with Zeocin™, colonies of Zeocin™ resistant Detection of Recombinant Each transformed cell grew to a colony on the plate. Each colony represents a transformed cell carrying the bleomycin resistance gene. Thus, the Other CC400 colonies that were bleomycin resistant but were not PCR-positive for the artificial gene carried only the antibiotic resistance parts of the squalene synthase pSP124S construct that was transformed into the cells, but did not also carry a full squalene synthase cassette. In such colonies the other parts of the DNA (i.e. some section of the squalene synthase gene, or its entirety) had been excluded by the organism during transformation and recombination. Test of Function Colonies that were both bleomycin resistant and positive for the squalene synthase pSP124S construct (i.e. using primer pairs 1 and 2) were assayed for squalene content. The transformed cells did not contain significantly more squalene than untransformed control cells. Gene ATB1 was assembled from the Similarly, the nucleotides ATGCAT (SEQ ID NO: 34) comprising an NsiI restriction site were inserted immediately upstream of the native stop codon of the nucleotide sequence of the ATB1 gene provided in FIG. 30 (SEQ ID NO: 30). In all constructs this resulted in addition to the polypeptide sequence provided in FIG. 29 (SEQ ID NO: 29), of one methionine and one histidine residue, upstream of the native stop codon (giving ATB1-MH: FIG. 46, SEQ ID NO: 36). Furthermore, the nucleotides TCACTCGAGCCCGGG (FIG. 47, SEQ ID NO: 37) comprising XhoI and SmaI restriction sites were inserted immediately downstream of the native stop codon to facilitate cloning. The resulting ATB1 sequence with these 33 additional nucleotides was synthesised (by Genscript, NJ USA). Initially, ATB1 was cloned into standard vector pUC57 (construct 1) for verification by sequencing. Subsequently, ATB1 was subcloned into vector pET-11a for overexpression of ATB1 protein including one methionine and one histidine residue at the C-terminus of ATB1 (ATB1-MH; SEQ ID NO: 36) via the inducible T7-lac promoter in that vector. The synthesised ATB1 gene was cut with restriction enzymes NdeI and BclI and cloned into the NdeI and BamHI sites of pET-11a to produce construct pET11a/ATB1 (construct 2). ATB1 was also subcloned into vectors that supply an inducible T7-lac promoter and either a N-terminal or C-terminal poly-histidine tag that can be used for affinity purification of the ATB1 protein. The synthesised ATB1 gene (SEQ ID NO: 32) was cut with restriction enzyme XhoI and cloned into the PmlI and XhoI sites of pET-302, to produce pET302/NT-His-ATB1 (construct 3) that encodes protein ATB1-MH augmented consecutively with one methionine, eight histidine, one methionine, and one histidine residues at its N-terminus (to produce MHHHHHHHHMH-ATB1-MH; FIG. 48, SEQ ID NO: 38). Alternatively, the synthesised ATB1 gene was cut with restriction enzyme NsiI and cloned directionally into the NsiI site of pET-302 (such that the T7-lac promoter is upstream) to produce CT-His-ATB1/pET302 (construct 4) that encodes protein ATB1-MH augmented consecutively at its N-terminus with one histidine and one methionine residue and augmented consecutively at its N-terminus with five histidine residues and residues VNSLEIDDIRA (to produce MH-ATB1-MH-HHHHHVNSLEIDDIRA; FIG. 49, SEQ ID NO: 39). Transformation The inducible promoter in ATB1 constructs 2, 3, and 4 is the viral T7 promoter. Constructs 2, 3, and 4 were transformed into Three vials of Glycerol stocks of Restriction Digests Plasmid DNA from the transformants was verified by extraction and restriction digestion. To prepare for DNA extraction, a small aliquot of each transformant was grown overnight at 37° C. in rich LB growth medium. DNA was extracted from transformed All constructs were cut with restriction enzyme NdeI, which cuts at the specific DNA sequence CATATG at the start of the synthesised ATB1 gene. Each construct was also cut with a second restriction enzyme as follows: construct 2—BstXI (cuts at sequence CCANNNNNNTGG; FIG. 50, SEQ ID NO: 40); constructs 3 and 4—XhoI (cuts at sequence CTCGAG; FIG. 51, SEQ ID NO: 41). This was achieved by combining 0.5 μl of NdeI and 0.5 μl of the appropriate second restriction enzyme, together with 2 μl of 10× “Promega restriction buffer 4”, 0.5 μl of bovine serum albumin (stock 10 mg/ml, final concentration 250 ng per μl), and 480 ng of DNA (about 6 to 12 μl), making a total reaction volume of 20 μl per reaction (by adding nuclease free water), and then incubating at 37° C. for 1 hour. Samples were electrophoresed on a 1% agarose gel in TAE buffer (40 mM Tris acetate, 1 mM EDTA) at 120 volts for 30 minutes, and DNA bands were visualised with SYBR-Safe fluorescent dye, on a UV illuminator, and digitally captured to disc using an 8 second exposure, with camera aperture fully open. The pattern observed when construct 2 was cut with NdeI and BstXI included a band of 1070 base pairs (FIG. 34A) as predicted, confirming the presence of ATB1. The pattern observed when constructs 3 and 4 were cut with NdeI and XhoI included a band of 1230 base pairs (FIG. 34B) as predicted, confirming the presence of ATB1. Untransformed The inducing agent isopropylthiogalactoside (IPTG) was added to each culture at a final concentration of 1 mM. At 0, 2 and 4 hours post IPTG induction, 1 ml of culture was removed and centrifuged. The resulting pellet was boiled for 10 minutes in 200 μl of protein assay buffer (50 mM Tris-HCl pH 6.8, 10 mM Dithiothreitol, 1% sodium dodecyl sulfate), and then protein concentration was measured on a Nanodrop spectrophotometer set at a wavelength of 280 nm. A sample containing 60 μg of protein was added to 3× strength Laemmli buffer (6% SDS, 30% glycerol, 15% □-mercaptoethanol, 0.006% bromophenol blue, 0.1875 M Tris-HCl) and boiled for 2 min. Also at 4 hours post IPTG induction, 5 ml of culture of The samples were loaded on a SDS-PAGE minigel comprising a stacking gel (4% acrylamide) and a resolving gel (10% acrylamide). The minigel was run at 170 volts. All lanes of the minigel contained 1× Laemmli buffer final concentration. The proteins on the minigel were visualised using Coomassie dye (FIG. 35). ATB1 protein was not present in untransformed A few microlitres of each of untransformed Each 200 ml culture was centrifuged and the pellet resuspended in 10 ml of LB medium, then added to 990 ml of fresh LB medium containing the inducer IPTG (final concentration 1 mM), and grown for 1 hour at 37° C. to allow cells to recover from lag phase and begin to divide. The 1 litre cultures were then incubated overnight at 28° C. to 33° C. with shaking. The temperature 28° C. to 33° C. may increase the yield of correctly folded ATB1 protein relative to the yield at 37° C. Following the overnight incubation, the cultures were centrifuged and the pellets resuspended in 10 ml of lysis buffer A (50 mM MOPS (4-Morpholinepropanesulfonic acid) buffer pH 7.3; 25 mM □-mercaptoethanol; 20 mM MgCl2; 0.5 mM PMSF (Phenylmethylsulfonyl fluoride)). First the sample of untransformed Heptane was separated from aqueous material and cell debris by centrifugation in PTFE/Teflon® tubes. The heptane fraction was removed using a single-use heptane-rinsed glass pipette. Samples were evaporated in the order untransformed Heptane-soluble polar compounds, consisting of mostly fatty acids, their esters and lipids, were removed by: 1) Adding sufficient heptane to the sample to increase its volume to 10 ml; 2) Passing the entire 10 ml of heptane through a column (LCTech (Germany), Elufix Column, Part number 9370) consisting of 2 g of anhydrous sodium sulfate and 2 g of synthetic magnesia-silica gel (Florisil®) and collecting the eluent; 3) Reducing the volume of heptane eluent collected to approximately 0.8 ml under a stream of nitrogen. A few microlitres of each of Untransformed Each of the samples to be induced at 37° C. was resuspended in 75 ml of LB (without glucose), grown for 2 hours at 37° C. to approximately attain log phase, and then induced with 0.5 mM IPTG. These were then further incubated for 2 hours at 37° C., to induce transgene expression. Each of the samples to be induced at 18° C. were passaged directly from the pellets obtained after removing glucose from overnight cultures. The pellets were resuspended in 4 ml of overnight culture, then transferred to 500 ml of LB containing 0.5 mM IPTG (final concentration) and incubated overnight at 18° C. After induction, cultures were centrifuged. The pellet was resuspended in 5 ml of lysis buffer A. Lysis was achieved by sonication for 1 minute, on ice (ice-water slurry) in a glass beaker. Samples were clarified by aliquotting into microfuge tubes, and centrifuging at 13000 rpm, at 4° C. for 15 minutes, then combining aliquots of identical samples to make 3.6 ml in total of each sample. To each clarified lysate, 35 μl of FPP stock (stock 10 mM in ethanol) and 15 mg of NADPH powder was added and the mix was incubated with gentle shaking at 37° C. for 30 minutes (FIG. 36). After incubation with FPP and NADPH, 10 ml of heptane was added to each lysate, and each was vigorously shaken by hand for about 20 seconds. Then the lysates were allowed to separate into hydrophobic and hydrophilic fractions overnight at room temperature with gentle shaking. Next day, the samples were centrifuged in PTFE/Teflon® tubes then evaporated as described in Example 14. Samples derived from Examples 15 and 16 were analysed by GC-MS. GC-MS was performed on an Agilent Technologies 5975C mass spectrometer equipped with an Agilent technologies 7890A gas chromatograph and a 7683B autosampler. The capillary column was an Alltech EC-5 (15 m×0.25 mm i.d.). Splitless injection was used with a split time of 60 seconds. Injection port temperature was 300° C. The carrier gas was helium in constant flow mode at 1.8 mL/min. Injection volume was 1 μL. The GC temperature program was: initial temperature of 40° C. for 4 min, ramp rate of 20° C./min, final temperature of 350° C. for 4 min. The GC/MS interface temperature was set to 280° C. The FID temperature was 325° C. For full scan mass spectra, the mass spectrometer was scanned from m/z 35 to m/z 550. Electron ionisation (EI) was used with the electron energy set to 70 eV. The source temperature was 230° C. The MS quadruple was set to 150° C. Authentic squalene and authentic C30 botryococcene were analysed by GC-MS (FIGS. 37 and 38, respectively). Squalene (elution time 16.5 min, mass 410) and botryococcene (elution time 15.8 min, mass 410) were undetectable in untransformed When supplemented with FPP and NADPH, a cell lysate of The mass spectrum of Compound X after electron cracking (FIG. 39) is similar to that of both squalene (FIG. 37) and botryococcene (FIG. 38), except that a majority of peaks are smaller by two mass units than those of squalene and botryococcene (Table 1). The absence of two hydrogens, demonstrated both by the overall molecular weight and the electron cracked mass spectrum, indicates that the triterpenoid Compound X is a “dehydro” triterpenoid Indeed, the mass spectrum is consistent with a dehydrotriterpenoid, such as 12,13-dehydrosqualene, also known as 4,4′-diapophytoene. On the basis of GC-MS, Compound X and Compound Y are identified as triterpenoid hydrocarbons. Specifically, Compound X is dehydrosqualene and Compound Y is squalene. Algae, for example Alternatively, algae is grown in TAP medium, which is described in Gorman and Levine, Algae are cultured in raceway ponds made of opaque plastic with dimensions 1.52 m wide, 2.44 m long and 0.61 m deep with a capacity of 2000 L. The raceway ponds are powered by a paddle wheel and operate at 20-30 cm depth. The paddle wheel rotation is adjusted to generate a flow rate of 21±3 cm s−1. Alage are also cutured in vertical tank reactors (VTR) with dimensions 0.45 m diameter and 1.52 m height with 100 L working volume and made of transparent acrylic sheets. A roll of low density polyethylene (LDPE-Uline-6-Mil heavy duty polytubing with 50.8 cm circumference) material is used to fabricate hanging polybags with dimensions 95 cm deep, 15 cm diameter, 20 L working volume. All the reactors are attached with delivery tubings and air stones for bubbling 5-6% CO2 and air mixture. Supplemental CO2 is derived from a liquid CO2 storage tank blended with air using a Concoa BlendMaster Model 1000 mixer and passed through a Whatman HEPA-Vent filter at 5-6% CO2 concentration in air. Cole Parmer rotameters are used to regulate air flow rates among the raceways, VTR and polybags. Raceways are stirred continuously with paddle wheels powered by Dayton Model 4Z129B motors (90 V, 1.27 Amps). For the VTR, mixing is accomplished by bubbling CO2 and air mixture through rectangular air stones (15×4×4 cm), whereas for the polybags, the mixture is simply bubbled into a port disk (0.72 cm opening) placed at the bottom of the polybag. To keep the VTR and polybags stirred after terminating the supply of supplemental CO2, each evening ambient air is pumped into these cultures at the same flow rate as that of the supplemental CO2 gas mix during the day. Culture temperature and pH for the raceways, VTR and polybags are measured daily. A MasterFlex Model 77250-62 peristaltic pump is used to fill polybags at a flow rate of 3-4 L min−1. Inoculum is drained from the bottom of VTR through a gate valve fitted with a 1.3 cm internal diameter garden hose that is either directly sent into the raceways or into pre-autoclaved glass carboys/Erlenmeyer flasks for subsequent delivery to the intended culture system. Algae are grown batch-wise in a green house. The raceways, VTR and polybags are arranged in a row parallel to the east-west direction. Biomass is harvested with a Lavin process centrifuge (Model: 12-413 V) operated at 2250 g, manually removed from the interior drum with a spoon and dried at 40° C. in a hot air oven for 72 h. It is stored at 4-5° C. Raceway ponds are filled with 800 L of growth medium. These are inoculated with 50 L each of exponentially growing cultures of algae. After inoculation, the raceways operate at 30 cm depth. Alternatively, raceways with 496 L growth medium are inoculated with 18 L of algal culture and operate at 20 cm depth with a total volume of 550 L. VTR have 88 L growth medium and 4 L of algal culture is added as inoculum. VTR operate with 100 L working volume and the depth of water column is maintained at 61 cm. Alternatively, algae are grown in raceways (working volume: 500 L; Depth: 18 cm), VTR (working volume: 100 L; diameter: 45 cm; depth: 61 cm) or polybags (working volume: 20 L; diameter: 16 cm; depth: 95 cm). To each of the raceways, VTR and polybags, 450, 90 and 18 L of growth medium is filled and 25, 5 and 1 L of the inoculum is added, respectively. Final volumes are, 500 L (18 cm deep) in raceways, 100 L (61 cm deep) in VTR and 20 L (95 cm deep) in polybags. Disclosed is a method for generating an expression vector pCE2 (plasmid for An interim vector pCE1 is first constructed. The Stepwise, firstly the RbcS2 terminator is amplified from pSP124S using forward primer RbcS2-termforw (5′-GAGTACGCGGCCGCCCCGCTCCGTGTAAAT-3′; FIG. 55, SEQ ID NO: 44), which contains a NotI restriction site, and reverse primer RbcS2-termrev (5′-GCACTCGAATTCGCTTCAAATACGCCCAGC-3′; FIG. 56, SEQ ID NO: 45), which contains an EcoRI restriction site. The polymerase used is Promega (Madison, Wis., USA) GoTaq. All conditions used of GoTaq enzyme activity are within the manufacturer's instructions (dNTPs concentration 0.1 mM, MgCl2 concentration 1 mM, primer concentration 0.4 pmol/μl, template concentration 1 pg/μl, 1× manufacturer's buffer) and the PCR cycle conditions used are 94° C. for 3 min, then 40 cycles of: 94° C. for 30 s, 55° C. for 30 s, 72° C. for 1 min, and a final extension of 72° C. for 7 min. The amplicon generated is 260 bp long and is purified in a Promega Wizard PCR purification column, then cut with NotI and EcoRI for 1 hr at 37° C., and subsequently heat treated for 10 minutes at 65° C. to denature enzymes, before purifying again through a Wizard PCR purification column. The amplified and restricted terminator is then ligated into vector pSP124S that has been prepared as follows: the vector is cut with NotI and EcoRI by incubating with these enzymes for 1 hr at 37° C., and the vector is then purified through a Wizard PCR purification column; the cut and purified vector is treated with Promega Calf Intestinal Alkaline Phosphatase according to the manufacturer's instructions, in order to prevent self ligation; the phosphatase-treated vector is purified through a Wizard PCR purification column. Ligation of the restriction-cut terminator amplicon and phosphatase treated pSP124S is accomplished with Promega T4 ligase according to the manufacturer's instructions. The ligation mix is transformed into DH10α electrocompetent Transformants are screened by plating the transformation mix onto Oxoid (Basingstoke, Hampsire, UK) nutrient agar (1 g/L Lab-Lemco powder, 2 g/L yeast extract, 5 g/L peptone, 5 g/L NaCl, 15 g/L agar) plates, containing 100 μg/ml (final concentration in agar) of sterile-filtered ampicillin. Colonies are picked and grown overnight in LB medium (nutrient broth), and their plasmid DNA extracted the following day. These plasmids are screened by PCR using forward primer pSP124S-preSacI (5′-CACTAAAGGGAACAAAAGCTG-3′; FIG. 57, SEQ ID NO: 46) and reverse primer RbcS2-termrev (5′-GCACTCGAATTCGCTTCAAATACGCCCAGC-3′; FIG. 56, SEQ ID NO: 45), and those plasmids which yield a PCR amplicon of 291 bp are considered positive. Positive plasmids are sequenced by the Australian Genomic Research Facility (AGRF) using a T3 primer (5′-GCAATTAACCCTCACTAAAGGGA-3′; FIG. 58, SEQ ID NO: 47) and dye-terminator chemistry to confirm the RbcS2 terminator sequence. This interim vector that contains a Similarly, in making pCE2, the RbcS2 promoter is first amplified from pSP124S using forward primer RbcS2-promforw (5′-GAGTGCGAGCTCTAAATGCCAGAAGGAGCG-3′; FIG. 59, SEQ ID NO: 48), which contains a SacI restriction site, and reverse primer RbcS2-promrev (5′-GTCACTGCGGCCGCTTTAAGATGTTGA-3′; FIG. 60, SEQ ID NO: 49), which contains a NotI restriction site. The polymerase used is Promega GoTaq. All conditions used of GoTaq enzyme activity are within the manufacturer's instructions (dNTPs concentration 0.1 mM, MgCl2 concentration 1 mM, primer concentration 0.4 pmol/μl, template concentration 1 pg/μl, 1× manufacturer's buffer) and the PCR cycle conditions used are 94° C. for 3 min, then 40 cycles of: 94° C. for 30 s, 52° C. for 30 s, 72° C. for 1 min, and a final extension of 72° C. for 7 min. The amplicon generated is 239 bp long and is purified in a Wizard PCR purification column, then cut with SacI and NotI for 1 hr at 37° C., and subsequently heat treated for 10 minutes at 65° C. to denature enzymes, before purifying again through a Wizard PCR purification column. The amplified and restricted promoter is then ligated into vector pCE1 that has been prepared as follows: the vector is cut with SacI and NotI, by incubating with these enzymes for 1 hr at 37° C., and the vector is then purified through a Wizard PCR purification column. The cut and purified vector is treated with Promega Calf Intestinal Alkaline Phosphatase according to the manufacturer's instructions in order to prevent self ligation. The phosphatase-treated vector is purified through a Wizard PCR purification column. Ligation of the restriction-cut promoter amplicon and phosphatase treated pCE1 is accomplished with Promega T4 ligase according to the manufacturer's instructions. The ligation mix is transformed into DH10α electrocompetent This novel expression vector that contains a As a further example, the ATB1 gene is ligated into vector pCE2 at the NotI site between the RbcS2 promoter and terminator, to create an expression construct pCE3-ATB1. Specifically, in generating the expression construct pCE3-ATB1, the ATB1 gene is amplified from construct pUC57/ATB1 that was manufactured at Genscript (Piscataway, N.J., USA). Construct pUC57/ATB1 contains the inferred cDNA of the The amplified and restricted ATB1 gene is then ligated into vector pCE2 that has been prepared as follows: the vector is cut with NotI, by incubating with these enzymes for 1 hr at 37° C., and the vector is then purified through a Wizard PCR purification column; the cut and purified vector is treated with Promega Calf Intestinal Alkaline Phosphatase according to the manufacturer's instructions to prevent self ligation; the phosphatase-treated vector is purified through a Wizard PCR purification column. Ligation of the restriction-cut ATB1 gene and phosphatase treated pCE2 is accomplished with Promega T4 ligase according to the manufacturer's instructions. The ligation mix is transformed into DH10α electrocompetent Construct pCE3-ATB1 is transformed into Zeocin resistant colonies of Functional units (motifs) in triterpenoid synthetic polypeptides have been identified. Prosite accession number PDO000802 discloses two squalene and phytoene synthase signatures (PS01044 signature 1 and PS01045 signature 2). PS01044 signature 1 provides the consensus pattern: Y-[CSAM]-x(2)-[VSG]-A-[GSA]-[LIVAT]-[IV]-G-x(2)-[LMSC]-x(2)-[LIV]. PS01045 signature 2 provides the consensus pattern: [LIVM]-G-x(3)-Q-x(2,3)-[ND]-[IFL]-x-[RE]-D-[LIVMFY]-x(2)-[DE]-x(4,7)-R-x-[FY]-x-P. These consensus signatures are used to find homologous signatures in the triterpenoid hydrocarbon synthetic polypeptide, thereby identifying one or more amino acids, and the corresponding codon(s), to be mutated. Alternatively, the consensus sequence of FIG. 53 is used to identify one or more amino acids, and the corresponding codon(s), to be mutated. Oligonucleotide primers for site directed mutagenesis are designed to mutate the one or more codons selected for mutation in a plasmid encoding ATB1. The mutated triterpenoid hydrocarbon synthetic polypeptide is then expressed and its function assayed according to Examples 15 to 17 or Examples 20 and 21 for its triterpenoid hydrocarbon synthetic activity. The function of the mutated polypeptide is then correlated with the structure of the mutated polypeptide to ascertain amino acid residues that contribute to triterpenoid hydrocarbon synthetic activity. The invention relates to an isolated nucleic acid molecule encoding a polypeptide capable of producing a triterpenoid hydrocarbon. The invention also relates to the encoded polypeptide, a vector comprising the nucleic acid molecule, a recombinant non-human organism comprising the nucleic acid molecule, and to methods of producing a triterpenoid hydrocarbon or an intermediate of biofuel using the nucleic acid molecule, polypeptide or recombinant organism. 1. An isolated nucleic acid molecule encoding a polypeptide capable of producing a triterpenoid hydrocarbon, the polypeptide comprising SEQ ID NO: 29. 2. The nucleic acid molecule of 3-5. (canceled) 6. A recombinant non-human organism capable of producing a triterpenoid hydrocarbon, the organism comprising an isolated nucleic acid molecule encoding a polypeptide capable of producing a triterpenoid hydrocarbon, the polypeptide comprising SEQ ID NO: 29. 7. The recombinant organism of 8. The recombinant organism of 9. The recombinant organism of 10. A method for producing a triterpenoid hydrocarbon, comprising expressing an isolated nucleic acid molecule encoding a polypeptide capable of producing a triterpenoid hydrocarbon, the polypeptide comprising SEQ ID NO: 29. 11-12. (canceled) 13. The method of 14. The method of 15-19. (canceled) 20. The nucleic acid molecule of 21. The nucleic acid molecule or 22. (canceled) 23. The recombinant organism of 24. The recombinant organism of 25. The recombinant organism of 26. The recombinant organism of 27. The method of 28. The method of 29. The nucleic acid molecule of CROSS REFERENCE TO RELATED APPLICATIONS
FIELD
BACKGROUND
SUMMARY
BRIEF DESCRIPTION OF THE FIGURES
DETAILED DESCRIPTION
EXAMPLES
Example 1
Culture Conditions
Example 2
Genomic DNA Preparation
Example 3
DNA Sequencing
Example 4
Genome Assembly and Gene Identification
Example 5
RNA Preparation
Example 6
Host Cell Species (Gene Recipient)
Example 7
Expression Vector
Example 8
Transformation
Example 9
Functional Tests of Botryococcene Synthase or Squalene Synthase In Vivo
Example 10
Functional Tests of Botryococcene Synthase or Squalene Synthase In Vitro
Example 11
Transformation of
Example 12
Cloning the ATB1 Triterpenoid Hydrocarbon Synthetic Nucleic Acid Sequence
Example 13
Transformation of
Example 14
Expression and Folding of ATB1 by Transformed
Example 15
Generation of a Triterpenoid Hydrocarbon by Transformed
Example 16
Generation of a Triterpenoid Hydrocarbon by Lysate of Transformed
Example 17
Detection of a Triterpenoid Hydrocarbon by Gas Chromatography—Mass Spectrometry (GC-MS)
Mass spectra of triterpenoids squalene, botryococcene and Compound X after electron cracking. Major peaks (companion peaks in brackets) are reported. Compound X Squalene C30 Botryococcene 408 410 410 393 395 395 378 (377) 365 367 367 356 (352) 354 339 (340) 341 341 327 (326) 325 325 (327, 328) 311 313 297 299 299 (297) 285 (282) 285 (287) 285 271 273 271 (273) 257 (255, 258) 257 (259) 257 (259) 243 (241) 245 (244) 245 (243) 229 231 231 (229) 215 217 (218) 217 (215) 203 (201) 203 203 187 (189) 191 (189, 192) 191 (189) 173 (175) 175 (177) 175 (177) 159 (161) 161 (163) 161 (163) 147 (145) 149 149 (147) 133 137 (136) 135 119 (121) 121 (123) 121 (123) 109 (107, 105) 109 (107) 109 (107) 93 (95, 91) 95 (93) 95 (93) 81 (79) 81 81 69 69 69 55 55 55 41 41 41 Example 18
Algae Culture
Example 19
Generating an ATB1 Construct Containing
Example 20
Transforming pCE3-ATB1 into
Example 21
Assaying Triterpenoids in Transgenic Alga CC503
Example 22
Structure-Function Analysis