Methods of developing terpene synthase variants
AssigneeAmyris, Inc.
InventorsLishan Zhao, Lan Xu, Patrick Westfall, Andrew Main
Life of this patent
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Abstract
The present disclosure relates to methods of developing terpene synthase variants through engineered host cells. Particularly, the disclosure provides methods of developing terpene synthase variants with improved in vivo performance that are useful in the commercial production of terpene products. Further encompassed in the present disclosure are superior terpene synthase variants and host cells comprising such terpene synthase variants.
Claims29
A method of testing a terpene synthase variant for improved in vivo performance, comprising the steps of:
- (a) dividing a population of host cells into a control population and a test population;
- (b) co-expressing in the control population a control terpene synthase (S1) and a comparison terpene synthase (S2), wherein the control terpene synthase can convert a polyprenyl diphosphate to a first terpene (T1) and the comparison terpene synthase can convert the same polyprenyl diphosphate to a different second terpene (T2) and wherein said control terpene synthase and comparison terpene synthase compete for the same pool of polyprenyl diphosphate;
- (c) co-expressing in the test population a test terpene synthase (S1′) and the comparison terpene synthase (S2), wherein the test terpene synthase is a variant of the control terpene synthase that can convert the same polyprenyl diphosphate to the same first terpene (T1) as the control terpene synthase, and wherein the comparison terpene synthase can convert the same polyprenyl diphosphate to a different second terpene (T2) and is expressed at similar levels in the test population and in the control population;
- and
- (d) measuring a ratio of the first terpene over the second terpene (T1/T2) in both the test population and in the control population, wherein the test population is identified as comprising a terpene synthase variant having improved in vivo performance compared to the control terpene synthase by having an increase in the ratio of the first terpene over the second terpene in the test population compared to that in the control population.
A composition comprising two cell subpopulations derived from a common population of host cells, wherein:
- (a) the first subpopulation comprises a heterologous control terpene synthase (S1) and a heterologous comparison terpene synthase (S2), wherein the control terpene synthase converts a polyprenyl diphosphate to a first terpene (T1), and wherein the comparison terpene synthase converts the same pool of polyprenyl diphosphate to a second, different terpene (T2);
- and
- (b) the second subpopulation comprises a heterologous test terpene synthase (S1′) which is a variant of the control terpene synthase (S1) of the first subpopulation, and the same comparison terpene synthase (S2) as in the first subpopulation,
- wherein the test terpene synthase (S1′) converts the polyprenyl diphosphate to the same first terpene (T1) in the first subpopulation and the comparison terpene synthase (S2) competes for the same of polyprenyl diphosphate as the test terpene synthase (S1′) and converts it to the same second terpene (T2) as in the first subpopulation;
- and wherein the comparison terpene synthase (S2) is expressed and present at similar levels in the first subpopulation and in the second subpopulation.
Dependencies are inferred from the claim text.