Laboratory evolution of a soluble, self-sufficient, highly active alkane hydroxylase

Abstract

We have converted cytochrome P450 BM-3 from Bacillus megaterium (P450 BM-3), a medium-chain (C12-C18) fatty acid monooxygenase, into a highly efficient catalyst for the conversion of alkanes to alcohols. The evolved P450 BM-3 exhibits higher turnover rates than any reported biocatalyst for the selective oxidation of hydrocarbons of small to medium chain length (C3-C8). Unlike naturally occurring alkane hydroxylases, the best known of which are the large complexes of methane monooxygenase (MMO) and membrane-associated non-heme iron alkane monooxygenase (AlkB), the evolved enzyme is monomeric, soluble, and requires no additional proteins for catalysis. The evolved alkane hydroxylase was found to be even more active on fatty acids than wild-type BM-3, which was already one of the most efficient fatty acid monooxgenases known. A broad range of substrates including the gaseous alkane propane induces the low to high spin shift that activates the enzyme. This catalyst for alkane hydroxylation at room temperature opens new opportunities for clean, selective hydrocarbon activation for chemical synthesis and bioremediation.

Increased TCA cycle activity and reduced oxygen consumption during cytochrome P450‐dependent biotransformation in fission yeast

“…In addition, P450s have been the target of extensive protein engineering studies, including sophisticated methods of both rational design and molecular evolution (e.g. Bottner et al, 1996;Glieder et al, 2002;Lindberg and Negishi, 1989;Otey et al, 2004Otey et al, , 2006.…”

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

“…By applying a colorimetric screen using p-nitrophenoxy-derivatives (pNCA) as substrates, BM3 was evolved for increased activity on alkanes in five rounds. The obtained variant also proved to have a two times higher activity for C12:0 and C16:0 [113]. Rational site-directed mutagenesis was applied in order to establish BM3 variants to hydroxylate shorter chain carboxylic acids [114].…”

Catalytic Hydroxylation in Biphasic Systems using CYP102A1 Mutants

“…[13,14] Recently cloning and characterisation of two further fusion enzymes from Bacillus subtilis, which exhibit high homology to CYP102A1, was reported. [15][16][17] While a large number of publications describing novel evolved CYP102A1 mutants with altered substrate specificity exist, [18][19][20][21][22][23][24][25][26] there are only very few reports dealing with the use of these biocatalysts in preparative organic synthesis. [7,27] Beside strong doubts concerning the operational stability of the enzyme class, the high cost of the nicotinamide cofactor NADPH which has to be added in stochiometric or even higher amount (if uncoupling is taken into account) seems to make their in vitro application impractical.…”