In Vivo Evolution of Butane Oxidation by Terminal Alkane Hydroxylases AlkB and CYP153A6

Abstract

Enzymes of the AlkB and CYP153 families catalyze the first step in the catabolism of medium-chain-length alkanes, selective oxidation of the alkane to the 1-alkanol, and enable their host organisms to utilize alkanes as carbon sources. Small, gaseous alkanes, however, are converted to alkanols by evolutionarily unrelated methane monooxygenases. Propane and butane can be oxidized by CYP enzymes engineered in the laboratory, but these produce predominantly the 2-alkanols. Here we report the in vivo-directed evolution of two mediumchain-length terminal alkane hydroxylases, the integral membrane di-iron enzyme AlkB from Pseudomonas putida GPo1 and the class II-type soluble CYP153A6 from Mycobacterium sp. strain HXN-1500, to enhance their activity on small alkanes. We established a P. putida evolution system that enables selection for terminal alkane hydroxylase activity and used it to select propane-and butane-oxidizing enzymes based on enhanced growth complementation of an adapted P. putida GPo12(pGEc47⌬B) strain. The resulting enzymes exhibited higher rates of 1-butanol production from butane and maintained their preference for terminal hydroxylation. This in vivo evolution system could be useful for directed evolution of enzymes that function efficiently to hydroxylate small alkanes in engineered hosts.Microbial utilization and degradation of alkanes was discovered almost a century ago (27). Since then, several enzyme families capable of hydroxylating alkanes to alkanols, the first step in alkane degradation, have been identified and categorized based on their preferred substrates (30). The soluble and particulate methane monooxygenases (sMMO and pMMO) and the related propane monooxygenase and butane monooxygenase (BMO) are specialized on gaseous small-chain alkanes (C 1 to C 4 ), while medium-chain (C 5 to C 16 ) alkane hydroxylation seems to be the domain of the CYP153 and AlkB enzyme families.Conversion of C 1 to C 4 alkanes to alkanols is of particular interest for producing liquid fuels or chemical precursors from natural gas. The MMO-like enzymes that catalyze this reaction in nature, however, exhibit limited stability or poor heterologous expression (30) and have not been suitable for use in a recombinant host that can be engineered to optimize substrate or cofactor delivery. Alkane monooxygenases often cometabolize a wider range of alkanes than those which support growth (12). We wished to determine whether it is possible to engineer a medium-chain alkane monooxygenase to hydroxylate small alkanes, thereby circumventing difficulties associated with en-

Biofiltration of gasoline and diesel aliphatic hydrocarbons

“…The authors explained this trend by the fact that most of the known alkane hydroxylases are variants of either cytochrome P-450 (Van Beilen and Funhoff, 2007) or integral membrane di-iron proteins (Van Beilen et al, 2006), which are designed for hydrophobic substrates. As was shown by Koch et al (2009), these enzymes can transform, through mutations of the relevant genes, into those designed for shorter chain hydrocarbons. However, such an evolution does not seem to occur in nature despite the abundance of virtually obligate alkane degrading microbial species (Yakimov et al, 2004).…”

Heme-thiolate ferryl of aromatic peroxygenase is basic and reactive

“…Copper-containing methane monooxygenases (MMO) (3), nonheme, diiron hydroxylases such as soluble MMO (4,5), and the alkane ω-hydroxylases, AlkB (6,7), allow microorganisms to grow on petroleum and natural gas as their sole sources of carbon. The widely distributed heme-thiolate monooxygenases, such as cytochrome P450 (CYP), serve similar roles in catalyzing C−H hydroxylation reaction.…”

A System for the Rapid Determination of the Mutation Spectrum in Escherichia coli

“…[43][44][45][46] In addition to the high and controllable mutation rate, it is important to have a well-balanced mutation spectrum to generate high-quality libraries in protein engineering. 45 To optimally navigate the sequence space with minimal bias, it is important to quickly survey the mutator spectrum of mutator strains under various conditions.…”