Regioselective alkane hydroxylation with a mutant CYP153A6 enzyme
AssigneeU.S. Department of Energy
InventorsDaniel J. Koch, Frances H. Arnold
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Abstract
Cytochrome P450 CYP153A6 from Myobacterium sp. strain HXN1500 was engineered using in-vivo directed evolution to hydroxylate small-chain alkanes regioselectively. Mutant CYP153A6-BMO1 selectively hydroxylates butane and pentane at the terminal carbon to form 1-butanol and 1-pentanol, respectively, at rates greater than wild-type CYP153A6 enzymes. This biocatalyst is highly active for small-chain alkane substrates and the regioselectivity is retained in whole-cell biotransformations.
Claims11
An isolated mutant enzyme comprising the amino acid sequence of SEQ ID NO:
- 2, wherein the mutation consists of the A97V point mutation and the mutant enzyme hydroxylates small-chain alkanes at rates greater than those of the wild-type enzyme.
An isolated mutant enzyme comprising the amino acid sequence of SEQ ID NO:
- 3, wherein the mutation consists of the A94V point mutation and wherein the enzyme hydroxylates small-chain alkanes at rates greater than those of the corresponding wild-type enzyme.
An isolated mutant enzyme having an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:2, wherein the mutation consists of the point mutation of A97V, and wherein the mutant enzyme can hydroxylate an alkane at the terminal position.
An isolated mutant enzyme having an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:3, wherein the mutation consists of the point mutation of A94V, and wherein the mutant enzyme can hydroxylate a small-chain alkane at the terminal position.
A method of making a mutant CYP153A6 enzyme having an altered ability to hydroxylate a small-chain alkane substrate at the terminal position, comprising:
- (a) creating a CYP153A6 enzyme mutant library, wherein the CYP153A6 enzyme that is mutated has the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3;
- (b) providing a host microbial cell culture capable of growing on a 1-alkanol;
- (c) transforming said host cell culture with said mutant library;
- (d) growing said host cell culture on an alkane substrate;
- and,
- (e) selecting and isolating the mutant enzymes based on the ability to hydroxylate a small-chain alkane at the terminal position.
Dependencies are inferred from the claim text.