Terpene synthases for biofuel production and methods thereof

Patent metadata

Field	Value
Publication number	US-10947563-B2
Application number	US-201916512072-A
Country	US
Kind code	B2
Filing date	Jul 15, 2019
Priority date	Mar 12, 2015
Publication date	Mar 16, 2021
Grant date	Mar 16, 2021

Abstract

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.

First claim

The invention claimed is:

1. A method of treating a biomass, the method comprising:
exposing the biomass to one or more organisms of an isolated, genetically engineered organism, wherein the organism comprises:
an exogenous terpenoid precursor, an exogenous enzyme configured to synthesize a terpenoid precursor, or a nucleic acid encoding the exogenous enzyme; and
an exogenous terpene synthase or a nucleic acid encoding the exogenous terpene synthase, wherein the exogenous 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; and

isolating one or more terpenoid compounds.

2. The method 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 method of claim 1 , wherein the organism is configured to produce one or more terpenoid compounds selected from the group consisting of a monoterpene, a sesquiterpene, and a diterpene.

4. The method of claim 1 , wherein the nucleic acid encoding the exogenous enzyme and/or the nucleic acid encoding the exogenous terpene synthase is provided as a plasmid vector.

5. The method 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, and geranyl pyrophosphate synthase.

6. The method 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, and geranyl pyrophosphate synthase.

7. The method of claim 1 , wherein the exogenous terpene synthase is a chamigrene synthase; or wherein the nucleic acid encoding the exogenous terpene synthase comprises a nucleic acid sequence encoding the chamigrene synthase.

8. The method of claim 1 , wherein the exogenous 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.

9. The method of claim 1 , wherein the nucleic acid encoding the exogenous 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 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.

10. The method of claim 1 , wherein the exogenous 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 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.

11. The method of claim 10 , wherein the exogenous 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 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.

12. The method of claim 1 , wherein the exogenous 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.

13. The method of claim 12 , wherein the exogenous 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.

14. The method of claim 1 , wherein the exogenous 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.

15. The method of claim 14 , wherein the exogenous 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.

16. The method of claim 1 , further comprising, prior to the exposing step:
pre-treating the biomass with one or more acids and/or enzymes.

17. The method of claim 1 , wherein the biomass comprises an alga, an amino a

Assignees

• Nat Tech & Eng Solutions Sandia Llc

Inventors

• Wu Weihua
• Gladden John Michael
• Wu Benjamin Chiau-Pin
• Davis Ryan Wesley

Classifications

• C12Y402/03057

  Beta-caryophyllene synthase (4.2.3.57) · CPC title

• C12Y402/03078

  Beta-chamigrene synthase (4.2.3.78) · CPC title

• C12Y402/03076

  (+)-Delta-selinene synthase (4.2.3.76) · CPC title

• C12Y402/03072

  Alpha-gurjunene synthase (4.2.3.72) · CPC title

• C12Y402/03014

  Pinene synthase (4.2.3.14) (C12Y402/03119 and C12Y402/0312 take precedence) · CPC title