Engineered sesquiterpene synthases
AssigneeGinkgo Bioworks, Inc.
InventorsJeffrey Ian Boucher, Dylan Alexander Carlin, Cinzia Formighieri, Bryan Greenhagen, Jaide Jensen, Scott Marr
Life of this patent
Estimating the term…
Abstract
Provided in this disclosure are engineered sesquiterpene synthases, host cells expressing engineered sesquiterpene synthases and methods for making alpha-guaiene.
Claims113
A host cell that comprises a heterologous polynucleotide encoding a sesquiterpene synthase, wherein the sequence of the sesquiterpene synthase comprises one or more amino acid substitutions relative to SEQ ID NO:
- 1, and wherein at least one of the amino acid substitutions is at a position corresponding to position 122, 274, 275, 293, 295, 301, 368, 398, 404, 407, 507, 515, 525, and/or 533 in SEQ ID NO:
- 1.
A host cell that comprises a heterologous polynucleotide encoding a sesquiterpene synthase, wherein the sequence of the sesquiterpene synthase comprises two or more amino acid substitutions relative to SEQ ID NO:
- 1, wherein:
- (i) at least one of the amino acid substitutions is at a position corresponding to position 122, 274, 275, 293, 295, 301, 368, 398, 404, 407, 507, 515, 525, and/or 533 in SEQ ID NO:
- 1;
- and
- (ii) at least one of the amino acid substitutions is at a position corresponding to position 72, 124, 153, 191, 201, 205, 289, 290, 291, 292, 346, 406, 442, 480, 494, 509, 512, and/or 526 in SEQ ID NO:
- 1.
A host cell that comprises a heterologous polynucleotide encoding a sesquiterpene synthase, wherein the sesquiterpene synthase comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:
- 1, wherein the sequence of the sesquiterpene synthase comprises two or more amino acid substitutions relative to SEQ ID NO:
- 1, wherein the two or more amino acid substitutions are at positions corresponding to position 72, 122, 124, 153, 191, 201, 205, 274, 275, 289, 290, 291, 292, 293, 295, 301, 346, 368, 398, 404, 406, 407, 442, 480, 494, 507, 509, 512, 526 and/or 533 in SEQ ID NO:
- 1, wherein the host cell is capable of producing sesquiterpene products, and wherein at least 50% of the total sesquiterpene products produced by the host cell is alpha-guaiene.
A non-naturally occurring sesquiterpene synthase, wherein the sequence of the sesquiterpene synthase comprises one or more amino acid substitutions relative to SEQ ID NO:
- 1, wherein at least one of the amino acid substitutions is at a position corresponding to position 122, 274, 275, 293, 295, 301, 368, 398, 404, 407, 507, 515, 525, and/or 533 in SEQ ID NO:
- 1.
A non-naturally occurring sesquiterpene synthase, wherein the sequence of the sesquiterpene synthase comprises two or more amino acid substitutions relative to SEQ ID NO:
- 1, wherein:
- (i) at least one amino acid substitution is at a position corresponding to position 122, 274, 275, 293, 295, 301, 368, 398, 404, 407, 507, 515, 525, and/or 533 in SEQ ID NO:
- 1;
- and
- (ii) at least one amino acid substitution is at a position corresponding to position 72, 124, 153, 191, 201, 205, 289, 290, 291, 292, 346, 406, 442, 480, 494, 509, 512, and/or 526 in SEQ ID NO:
- 1.
A non-naturally occurring sesquiterpene synthase, wherein the sesquiterpene synthase comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:
- 1, wherein the sesquiterpene synthase comprises an amino acid sequence having two or more amino acid substitutions relative to SEQ ID NO:
- 1, and wherein the two or more amino acid substitutions are at positions corresponding to position 72, 122, 124, 153, 191, 201, 205, 274, 275, 289, 290, 291, 292, 293, 295, 301, 346, 368, 398, 404, 406, 407, 442, 480, 494, 507, 509, 512, 526 and/or 533 in SEQ ID NO:
- 1, wherein the sesquiterpene synthase is capable of producing sesquiterpene products, and wherein at least 50% of sesquiterpene products produced by the sesquiterpene synthase is alpha-guaiene.
A non-naturally occurring nucleic acid encoding a sesquiterpene synthase, wherein the non-naturally occurring nucleic acid comprises a sequence that is at least 90% identical to SEQ ID NOs:
- 40-76 or 93-109.
A composition comprising (a) a sesquiterpene, wherein at least 50% of the sesquiterpene is alpha-guaiene, and (b) one or more additional components, wherein the one or more additional components include fermentation medium, cell culture supernatant and/or a hydrophobic overlay.
A host cell that comprises a heterologous polynucleotide encoding a sesquiterpene synthase, wherein the sequence of the sesquiterpene synthase comprises one or more amino acid substitutions relative to SEQ ID NO:
- 1, and wherein at least one of the amino acid substitutions is at a position corresponding to position 44, 212, 217, 293, 295, 404, 515, and/or 542 in SEQ ID NO:
- 1.
A host cell that comprises a heterologous polynucleotide encoding a sesquiterpene synthase, wherein the sequence of the sesquiterpene synthase comprises two or more amino acid substitutions relative to SEQ ID NO:
- 1, wherein:
- (i) at least one of the amino acid substitutions is at a position corresponding to position 44, 212, 217, 293, 295, 404, 515, and/or 542 in SEQ ID NO:
- 1;
- and
- (ii) at least one of the amino acid substitutions is at a position corresponding to position 23, 72, 86, 111, 118, 134, 147, 188, 201, 224, 252, 255, 289, 290, 291, 292, 346, 381, 390, 406, 419, 433, 442, 443, 444, 448, 458, 467, 494, 499, 512, 516, and/or 519 in SEQ ID NO:
- 1.
A host cell that comprises a heterologous polynucleotide encoding a sesquiterpene synthase, wherein the sesquiterpene synthase comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:
- 1, wherein the sequence of the sesquiterpene synthase comprises two or more amino acid substitutions relative to SEQ ID NO:
- 1, wherein the two or more amino acid substitutions are at positions corresponding to position 23, 44, 72, 86, 111, 118, 134, 147, 188, 201, 212, 217, 224, 252, 255, 289, 290, 291, 292, 293, 295, 346, 381, 390, 404, 406, 419, 433, 442, 443, 444, 448, 458, 467, 494, 499, 512, 515, 516, 519, or 542 in SEQ ID NO:
- 1, wherein the host cell is capable of producing sesquiterpene products, and wherein at least 50% of the total sesquiterpene products produced by the host cell is alpha-guaiene.
A non-naturally occurring sesquiterpene synthase, wherein the sequence of the sesquiterpene synthase comprises one or more amino acid substitutions relative to SEQ ID NO:
- 1, wherein at least one of the amino acid substitutions is at a position corresponding to position 44, 212, 217, 293, 295, 404, 515, and/or 542 in SEQ ID NO:
- 1.
A non-naturally occurring sesquiterpene synthase, wherein the sequence of the sesquiterpene synthase comprises two or more amino acid substitutions relative to SEQ ID NO:
- 1, wherein:
- (i) at least one amino acid substitution is at a position corresponding to position 44, 212, 217, 293, 295, 404, 515, and/or 542 in SEQ ID NO:
- 1;
- and
- (ii) at least one amino acid substitution is at a position corresponding to position 23, 72, 86, 111, 118, 134, 147, 188, 201, 224, 252, 255, 289, 290, 291, 292, 346, 381, 390, 406, 419, 433, 442, 443, 444, 448, 458, 467, 494, 499, 512, 516, and/or 519 in SEQ ID NO:
- 1.
A non-naturally occurring sesquiterpene synthase, wherein the sesquiterpene synthase comprises an amino acid sequence that is at least 90% identical to the sequence of SEQ ID NO:
- 1, wherein the sesquiterpene synthase comprises an amino acid sequence having two or more amino acid substitutions relative to SEQ ID NO:
- 1, and wherein the two or more amino acid substitutions are at positions corresponding to position 23, 44, 72, 86, 111, 118, 134, 147, 188, 201, 212, 217, 224, 252, 255, 289, 290, 291, 292, 293, 295, 346, 381, 390, 404, 406, 419, 433, 442, 443, 444, 448, 458, 467, 494, 499, 512, 515, 516, 519, or 542 in SEQ ID NO:
- 1, wherein the sesquiterpene synthase is capable of producing sesquiterpene products, and wherein at least 50% of sesquiterpene products produced by the sesquiterpene synthase is alpha-guaiene.
A non-naturally occurring nucleic acid encoding a sesquiterpene synthase, wherein the non-naturally occurring nucleic acid comprises a sequence that is at least 90% identical to SEQ ID NOs:
- 133-154.
A composition comprising (a) a sesquiterpene, wherein at least 50% of the sesquiterpene is alpha-guaiene, and (b) one or more additional components, wherein the one or more additional components include fermentation medium, cell culture supernatant and/or a hydrophobic overlay.
Dependencies are inferred from the claim text.