Terpene synthases for biofuel production and methods thereof
The present invention relates to terpene synthases capable of degrading precursors into biofuel compounds, such as terpenoid compounds. In one instance, a transformed organism can include such terpene synthases, as well as vectors encoding such synthases. Methods of employing such synthases and organisms are also described herein.
1. An isolated, genetically engineered organism comprising:
an exogenous terpenoid precursor, an exogenous enzyme configured to synthesize a terpenoid precursor, or a nucleic acid encoding the exogenous enzyme; and
an exogenous endophytic fungal terpene synthase or a nucleic acid encoding the exogenous endophytic fungal terpene synthase, wherein the exogenous endophytic fungal terpene synthase is selected from the group consisting of a pinene synthase, a guaiene synthase, a pinene and guaiene synthase, a caryophyllene synthase, a chamigrene synthase, a chamigrene and pinene synthase, a gurjunene synthase, a gurjunene and pinene synthase, a gumunene synthase, a selinene synthase, and an isoledene synthase, or a bifunctional synthase of any of these,
wherein the exogenous endophytic fungal terpene synthase comprises a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs or wherein the nucleic acid encoding the exogenous endophytic fungal terpene synthase comprises a nucleic acid sequence encoding a polypeptide sequence having at least 90% sequence identity to any one of the following SEQ ID NOs:
SEQ ID NO: 10, in which X at each position of SEQ ID NO:10 is an amino acid present at a position in one of SEQ ID NOs:11-14 when optimally aligned with SEQ ID NO:10;
SEQ ID NO:20, in which X at each position of SEQ ID NO:20 is an amino acid present at a position in one of SEQ ID NOs:21-25 when optimally aligned with SEQ ID NO:20;
SEQ ID NO:30, in which X at each position of SEQ ID NO:30 is an amino acid present at a position in one of SEQ ID NOs:31-34 when optimally aligned with SEQ ID NO:30;
SEQ ID NO:40, in which X at each position of SEQ ID NO:40 is an amino acid present at a position in one of SEQ ID NOs:41-46 when optimally aligned with SEQ ID NO:40;
SEQ ID NO:50, in which X at each position of SEQ ID NO:50 is an amino acid present at a position in one of SEQ ID NOs:51-54 when optimally aligned with SEQ ID NO:50; or
SEQ ID NO:60, in which X at each position of SEQ ID NO:60 is an amino acid present at a position in one of SEQ ID NOs:61-64 when optimally aligned with SEQ ID NO:60.
2. The organism of claim 1 , wherein the exogenous terpenoid precursor is selected from the group consisting of mevalonate, dimethylallyl pyrophosphate, isopentenyl pyrophosphate, farnesyl pyrophosphate, geranyl pyrophosphate, and geranylgeranyl pyrophosphate, or a salt thereof.
3. The organism of claim 1 , thereby configured to produce one or more terpenoid compounds selected from the group consisting of a monoterpene, a sesquiterpene, and a diterpene.
4. The organism of claim 1 , wherein the nucleic acid encoding the exogenous enzyme and/or the nucleic acid encoding the exogenous endophytic fungal terpene synthase is provided as a plasmid vector.
5. The organism of claim 1 , wherein the exogenous enzyme is selected from the group consisting of acetyl-CoA acetyltransferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, isoprenyl diphosphate isomerase, farnesyl pyrophosphate synthase, and geranyl pyrophosphate synthase.
6. The organism of claim 5 , wherein the nucleic acid encoding the exogenous enzyme comprises a nucleic acid sequence encoding the exogenous enzyme selected from the group consisting of acetyl-CoA acetyltransferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, isoprenyl diphosphate isomerase, farnesyl pyrophosphate synthase, and geranyl pyrophosphate synthase.
7. An isolated, genetically engineered organism comprising:
an exogenous terpenoid precursor, an exogenous enzyme configured to synthesize a terpenoid precursor, or a nucleic acid encoding the exogenous enzyme; and
an exogenous endophytic fungal terpene synthase or a nucleic acid encoding the exogenous endophytic fungal terpene synthase, wherein the exogenous endophytic fungal terpene synthase is selected from the group consisting of a pinene synthase, a guaiene synthase, a pinene and guaiene synthase, a caryophyllene synthase, a chamigrene synthase, a chamigrene and pinene synthase, a gurjunene synthase, a gurjunene and pinene synthase, a gumunene synthase, a selinene synthase, and an isoledene synthase, or a bifunctional synthase of any of these,
wherein the exogenous endophytic fungal terpene synthase comprises a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs:11-14, 21-25, 31-34, 41-46, 51-54, and 61-64; or wherein the nucleic acid encoding the exogenous endophytic fungal terpene synthase comprises a nucleic acid sequence encoding a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs:11-14, 21-25, 31-34, 41-46, 51-54, and 61-64.
8. The organism of claim 7 , wherein the exogenous endophytic fungal terpene synthase comprises a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs:23, 24, 25, 31, 32, 34, 41, 42, 44, 45, 54, 62, and 64; or wherein the nucleic acid encoding the exogenous endophytic fungal terpene synthase comprises a nucleic acid sequence encoding a polypeptide sequence having at least 90% sequence identity to any one of SEQ ID NOs:23, 24, 25, 31, 32, 34, 41, 42, 44, 45, 54, 62, and 64.
9. The organism of claim 7 , wherein the exogenous terpenoid precursor is selected from the group consisting of mevalonate, dimethylallyl pyrophosphate, isopentenyl pyrophosphate, farnesyl pyrophosphate, geranyl pyrophosphate, and geranylgeranyl pyrophosphate, or a salt thereof.
10. The organism of claim 7 , wherein the exogenous enzyme is selected from the group consisting of acetyl-CoA acetyltransferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, isoprenyl diphosphate isomerase, farnesyl pyrophosphate synthase, and geranyl pyrophosphate synthase.
11. An isolated, genetically engineered organism comprising:
an exogenous terpenoid precursor, an exogenous enzyme configured to synthesize a terpenoid precursor, or a nucleic acid encoding the exogenous enzyme; and
an exogenous endophytic fungal terpene synthase or a nucleic acid encoding the exogenous endophytic fungal terpene synthase, wherein the exogenous endophytic fungal terpene synthase is selected from the group consisting of a pinene synthase, a guaiene synthase, a pinene and guaiene synthase, a caryophyllene synthase, a chamigrene synthase, a chamigrene and pinene synthase, a gurjunene synthase, a gurjunene and pinene synthase, a gumunene synthase, a selinene synthase, and an isoledene synthase, or a bifunctional synthase of any of these,
wherein the exogenous endophytic fungal terpene synthase comprises a polypeptide sequence having at least 90% sequence identity to XXZZXXZX (SEQ ID NO:71); wherein X is any amino acid; and wherein Z is selected from the group consisting of Asp, Glu, and His.
12. The organism of claim 11 , wherein the exogenous endophytic fungal terpene synthase comprises a polypeptide sequence having at least 90% sequence identity to XXZDXXZX (SEQ ID NO:73); wherein X is selected from the group consisting of Ala, Ser, Thr, Val, Leu, Ile, Phe, Tyr, Trp, Glu, Asn, Gln, His, and Pro; and
wherein Z is selected from the group consisting of Asp and Glu.
13. The organism of claim 11 , wherein the exogenous terpenoid precursor is selected from the group consisting of mevalonate, dimethylallyl pyrophosphate, isopentenyl pyrophosphate, farnesyl pyrophosphate, geranyl pyrophosphate, and geranylgeranyl pyrophosphate, or a salt thereof.
14. The organism of claim 11 , thereby configured to produce one or more terpenoid compounds selected from the group consisting of a monoterpene, a sesquiterpene, and a diterpene.
15. The organism of claim 11 , wherein the nucleic acid encoding the exogenous enzyme and/or the nucleic acid encoding the exogenous endophytic fungal terpene synthase is provided as a plasmid vector.
16. The organism of claim 11 , wherein the exogenous enzyme is selected from the group consisting of acetyl-CoA acetyltransferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, isoprenyl diphosphate isomerase, farnesyl pyrophosphate synthase, and geranyl pyrophosphate synthase.
17. An isolated, genetically engineered organism comprising:
an exogenous terpenoid precursor, an exogenous enzyme configured to synthesize a terpenoid precursor, or a nucleic acid encoding the exogenous enzyme; and
an exogenous endophytic fungal terpene synthase or a nucleic acid encoding the exogenous endophytic fungal terpene synthase, wherein the exogenous endophytic fungal terpene synthase is selected from the group consisting of a pinene synthase, a guaiene synthase, a pinene and guaiene synthase, a caryophyllene synthase, a chamigrene synthase, a chamigrene and pinene synthase, a gurjunene synthase, a gurjunene and pinene synthase, a gumunene synthase, a selinene synthase, and an isoledene synthase, or a bifunctional synthase of any of these,
wherein the exogenous endophytic fungal terpene synthase comprises a polypeptide sequence having at least 90% sequence identity to XZZXXXSXXZ ZXX (SEQ ID NO:75); wherein X is any amino acid; and wherein Z is selected from the group consisting of Cys, Asp, Glu, Asn, Gln, Lys, Arg, and absent.
18. The organism of claim 17 , wherein the exogenous endophytic fungal terpene synthase comprises a polypeptide sequence having at least 90% sequence identity to XZDXXXSXXZZXX (SEQ ID NO:77); wherein X is selected from the group consisting of Gly, Ala, Thr, Val, Leu, Ile, Phe, Tyr, Trp, Asp, Glu, Gln, Lys, Arg, and absent; and wherein Z is selected from the group consisting of Cys, Asp, Glu, Asn, Gln, Lys, and Arg.
19. The organism of claim 17 , wherein the exogenous terpenoid precursor is selected from the group consisting of mevalonate, dimethylallyl pyrophosphate, isopentenyl pyrophosphate, farnesyl pyrophosphate, geranyl pyrophosphate, and geranylgeranyl pyrophosphate, or a salt thereof.
20. The organism of claim 17 , thereby configured to produce one or more terpenoid compounds selected from the group consisting of a monoterpene, a sesquiterpene, and a diterpene.
21. The organism of claim 17 , wherein the nucleic acid encoding the exogenous enzyme and/or the nucleic acid encoding the exogenous endophytic fungal terpene synthase is provided as a plasmid vector.
22. The organism of claim 17 , wherein the exogenous enzyme is selected from the group consisting of acetyl-CoA acetyltransferase, HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, mevalonate diphosphate decarboxylase, isoprenyl diphosphate isomerase, farnesyl pyrophosphate synthase, and geranyl pyrophosphate synthase.