Amorpha-4,11-diene 12-monooxygenase variants and uses thereof
AssigneeAmyris, Inc.
InventorsChristopher J. PADDON, Hanxiao JIANG, Stephanie H. KUNG
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Abstract
Provided herein are genetically modified host cells, compositions, and methods for improved production of artemisinic acid. The host cells are genetically modified to contain a heterologous nucleic acid that expresses novel and optimized variants of amorpha-4,11-diene 12-monooxygenase. Also provided herein are methods for screening for variants of cytochrome p450 enzymes that have increased enzymatic activity relative to a parental control enzyme.
Claims25
A variant amorpha-4,11-diene 12-monooxygenase polypeptide comprising the amino acid sequence of SEQ ID NO:1, wherein the sequence further comprises one or more amino acid substitutions.
A nucleic acid encoding the polypeptide of any one of the preceding claims.
A method of generating a variant of a target p450 enzyme comprising:
- obtaining a library of nucleic acids encoding variants of the target p 450;
- transforming a population of host cells with the library such that each library nucleic acid is operably linked to a weak promoter;
- plating individual transformed host cells into individual wells of a multi-well plate;
- culturing the host cells under conditions that produce a test compound;
- measuring the level of test compound produced by the transformed host cells;
- and
- selecting variants that increase the level of the test compound relative to a control.
The method of any of the preceding claims, wherein the target p450 enzyme is amorpha-4,11-diene 12-monooxygenase.
The method of any one of the preceding claims, further comprising:
- creating a second library of nucleic acids encoding variants of the target p450, wherein the nucleic acids comprise combinations of the selected variants;
- transforming a population of host cells with the second library such that each second library nucleic acid is operably linked to a second weak promoter;
- plating individual transformed host cells into individual wells of a multi-well plate;
- culturing the host cells under conditions that produce the test compound;
- measuring the level of test compound produced in each well of the multi-well plate;
- and
- selecting second order variants that increase the level of the test compound relative to a second control.
Dependencies are inferred from the claim text.