MOLECULE PRODUCTION BY PHOTOSYNTHETIC ORGANISMS
The present invention provides compositions and methods for producing products by photosynthetic organisms. The photosynthetic organisms are genetically modified to effect production, secretion, or both, of products. The methods and compositions are particularly useful in the petrochemical industry.
1 . An isolated vector comprising: (a) a nucleic acid encoding an enzyme that produces an isoprenoid with two phosphates; and (b) a promoter configured for expression of said nucleic acid in a chloroplast of a non-vascular, photosynthetic organism, wherein the vector does not comprise the entire genome of a chloroplast.
2 . The vector of claim 1 , wherein said isoprenoid with two phosphates is GPP, IPP, FPP, GGPP or DMAPP.
3 - 4 . (canceled)
5 . The vector of claim 1 , further comprising a nucleic acid encoding a second enzyme which modifies the isoprenoid with two phosphates.
6 . The vector of claim 5 , wherein said second enzyme is botyrococcene synthase, limonene synthase, cineole synthase, pinene synthase, camphene synthase, sabinene synthase, myrcene synthase, abietadiene synthase, taxadiene synthase, bisabolene synthase, diapophytoene desaturase, diapophytoene synthase, monoterpene synthase, terpinolene synthase, zingiberene synthase, ocimene synthase, sesquiterpene synthase, curcumene synthase, farnesene synthase, geranylgeranyl reductase, chlorophyllidohydrolase, β-caryophyllene synthase, germacrene A synthase, 8-epicedrol synthase, valencene synthase, (+)-δ-cadinene synthase, germacrene C synthase, (E)-β-farnesene synthase, casbene synthase, vetispiradiene synthase, 5-epi-aristolochene synthase, aristolchene synthase, α-humulene, (E,E)-α-farnesene synthase, (−)-β-pinene synthase, γ-terpinene synthase, limonene cyclase, linalool synthase, (+)-bornyl diphosphate synthase, levopimaradiene synthase, isopimaradiene synthase, (E)-γ-bisabolene synthase, copalyl pyrophosphate synthase, kaurene synthase, longifolene synthase, γ-humulene synthase, δ-selinene synthase, β-phellandrene synthase, terpinolene synthase, (+)-3-carene synthase, syn-copalyl diphosphate synthase, α-terpineol synthase, syn-pimara-7,15-diene synthase, ent-sandaaracopimaradiene synthase, sterner-13-ene synthase, E-β-ocimene, S-linalool synthase, geraniol synthase, gamma-terpinene synthase, linalool synthase, E-β-ocimene synthase, epi-cedrol synthase, α-zingiberene synthase, guaiadiene synthase, cascarilladiene synthase, cis-muuroladiene synthase, aphidicolan-16b-ol synthase, elizabethatriene synthase, sandalol synthase, patchoulol synthase, zinzanol synthase, cedrol synthase, scareol synthase, copalol synthase, or manool synthase.
7 - 8 . (canceled)
9 . The vector of claim 1 , wherein said chloroplast is from a microalga.
10 . The vector of claim 9 , wherein said microalga is C. reinhardtii, D. salina, H. pluvalis, S. dimorphus, D. viridis , or D. tertiolecta.
11 . The vector of claim 1 , comprising any of the sequences in Table 5 or a sequence having at least 70% identity thereto.
12 - 31 . (canceled)
32 . A genetically modified chloroplast comprising the vector of claim 1 .
33 . A non-vascular, photosynthetic organism comprising the chloroplast of claim 32 .
34 . A method of producing an isoprenoid comprising: (a) transforming a chloroplast of a non-vascular, photosynthetic organism with a nucleic acid encoding an enzyme that produces an isoprenoid with two phosphates, and; (b) collecting at least one isoprenoid produced by said transformed organism.
35 . The method of claim 34 , further comprising growing said organism in an aqueous environment, wherein CO 2 is supplied to said organism.
36 . The method of claim 35 , wherein said CO 2 is at least partially derived from a burned fossil fuel.
37 . (canceled)
38 . The method of claim 34 , wherein said isoprenoid with two phosphates is GPP, IPP, FPP, GGPP or DMAPP.
39 . The method of claim 34 , wherein said collecting step comprises one or more of the following steps: (a) harvesting said transformed organism; (b) harvesting said isoprenoid from a cell medium; (c) mechanically disrupting said organism; or (d) chemically disrupting said organism.
40 - 41 . (canceled)
42 . A method for producing an isoprenoid comprising: (a) transforming the chloroplast of a non-vascular, photosynthetic organism to produce said isoprenoid, wherein said organism is not transformed with isoprene synthase or a methyl-butenol synthase; and (b) collecting said isoprenoid.
43 . The method of claim 42 , further comprising growing said organism in an aqueous environment, wherein CO 2 is supplied to said organism.
44 . The method of claim 43 , wherein said CO 2 is at least partially derived from a burned fossil fuel.
45 . (canceled)
46 . The method of claim 42 , wherein isoprenoid is GPP, IPP, FPP, GGPP or DMAPP.
47 . The method of claim 42 , wherein said chloroplast is transformed with a nucleic acid encoding botyrococcene synthase, limonene synthase, cineole synthase, pinene synthase, camphene synthase, sabinene synthase, myrcene synthase, abietadiene synthase, taxadiene synthase, bisabolene synthase, diapophytoene desaturase, diapophytoene synthase, monoterpene synthase, terpinolene synthase, zingiberene synthase, ocimene synthase, sesquiterpene synthase, curcumene synthase, farnesene synthase, geranylgeranyl reductase, chlorophyllidohydrolase, β-caryophyllene synthase, germacrene A synthase, 8-epicedrol synthase, valencene synthase, (+)-δ-cadinene synthase, germacrene C synthase, (E)-β-farnesene synthase, casbene synthase, vetispiradiene synthase, 5-epi-aristolochene synthase, aristolchene synthase, α-humulene, (E,E)-α-farnesene synthase, (−)-β-pinene synthase, γ-terpinene synthase, limonene cyclase, linalool synthase, (+)-bornyl diphosphate synthase, levopimaradiene synthase, isopimaradiene synthase, (E)-γ-bisabolene synthase, copalyl pyrophosphate synthase, kaurene synthase, longifolene synthase, γ-humulene synthase, δ-selinenesynthase, β-phellandrene synthase, terpinolene synthase, (+)-3-carene synthase, syn-copalyl diphosphate synthase, α-terpineol synthase, syn-pimara-7,15-diene synthase, ent-sandaaracopimaradiene synthase, sterner-13-ene synthase, E-β-ocimene, S-linalool synthase, geraniol synthase, gamma-terpinene synthase, linalool synthase, E-β-ocimene synthase, epi-cedrol synthase, α-zingiberene synthase, guaiadiene synthase, cascarilladiene synthase, cis-muuroladiene synthase, aphidicolan-16b-ol synthase, elizabethatriene synthase, sandalol synthase, patchoulol synthase, zinzanol synthase, cedrol synthase, scareol synthase, copalol synthase, or manool synthase.
48 . The method of claim 42 , wherein said collecting step comprises one or more of the following steps: (a) harvesting said transformed organism; (b) harvesting said isoprenoid from a cell medium; (d) mechanically disrupting said organism; or (e) chemically disrupting said organism.
49 - 66 . (canceled)
67 . The organism of claim 33 , wherein said organism comprises: a first nucleic acid encoding a botryococcene synthase, and a second nucleic acid encoding an FPP synthase.
68 . The host cell of claim 67 , wherein said first and second nucleic acids are integrated into a chloroplast genome.
69 - 127 . (canceled)
128 . The method of claim 34 , wherein phytol production in said non-vascular photosynthetic organism is increased above a level produced by said organism not containing said nucleic acid.
129 . The method of claim 128 , wherein said nucleic acid encodes an enzyme selected from the group consisting of a GPP synthase, a FPP synthase, a geranylgeranyl reductase, a chlorophyllidohydrolase, and a pyrophosphatase.
130 . (canceled)
131 . The method of claim 129 , wherein said enzyme is endogenous to said organism or is homologous to an endogenous enzyme of said organism.
132 . The method of claim 131 wherein said enzyme is overexpressed.
133 . The method of claim 129 , wherein said enzyme is exogenous to said organism.
134 . (canceled)
135 . The method of claim 128 , further comprising transformation of said organism with a nucleic acid which results in production of dimethylallyl alcohol, isopentyl alcohol, geraniol, farnesol or geranylgeraniol.
136 - 137 . (canceled)
138 . The method of claim 128 , wherein said nucleic acid comprises a sequence from Table 7 or a sequence with 70% homology thereto.
139 . A host cell comprising an introduced nucleic acid, wherein said nucleic acid results in an increase in production of phytol by said host cell above a level produced by a host cell not containing said nucleic acid, and wherein said host cell is a non-vascular photosynthetic organism.
140 - 142 . (canceled)
143 . The host cell of claim 139 , wherein said nucleic acid is present in a chloroplast.
144 . The host cell of claim 139 , wherein said nucleic acid encodes an enzyme selected from the group consisting of a GPP synthase, a FPP synthase, a geranylgeranyl reductase, a chlorophyllidohydrolase, and a pyrophosphatase.
145 . The host cell of claim 139 , further comprising a nucleic acid which results in production of dimethylallyl alcohol, isopentyl alcohol, geraniol, farnesol or geranylgeraniol.
146 - 147 . (canceled)
148 . The method of claim 128 , further comprising:
collecting said phytol from said organism.
149 . The method of claim 148 , wherein said nucleic acid encodes an enzyme selected from the group consisting of a GPP synthase, a FPP synthase, a geranylgeranyl reductase, a chlorophyllidohydrolase, and a pyrophosphatase.
150 . (canceled)
151 . The method of claim 148 , wherein said nucleic acid encodes an enzyme endogenous to said organism or an enzyme homologous to an endogenous enzyme of said organism.
152 - 154 . (canceled)
155 . The method of claim 148 , further comprising transformation of said organism with a nucleic acid which results in production of dimethylallyl alcohol, isopentyl alcohol, geraniol, farnesol or geranylgeraniol.
156 - 163 . (canceled)