A Chimeric Styrene Monooxygenase with Increased Efficiency in Asymmetric Biocatalytic Epoxidation

Abstract

The styrene monooxygenase (SMO) system from Pseudomonas sp. consists of two enzymes (StyA and StyB). StyB catalyses the reduction of FAD at the expense of NADH. After the transfer of FADH2 from StyB to StyA, reaction with O2 generates FAD‐OOH, which is the epoxidising agent. The wastage of redox equivalents due to partial diffusive transfer of FADH2, the insolubility of recombinant StyB and the impossibility of expressing StyA and StyB in a 1:1 molar ratio reduce the catalytic efficiency of the natural system. Herein we present a chimeric SMO (Fus‐SMO) that was obtained by genetic fusion of StyA and StyB through a flexible linker. Thanks to a combination of: 1) balanced and improved expression levels of reductase and epoxidase units, and 2) intrinsically higher specific epoxidation activity of Fus‐SMO in some cases, Escherichia coli cells expressing Fus‐SMO possess about 50 % higher activity for the epoxidation of styrene derivatives than E. coli cells coexpressing StyA and StyB as discrete enzymes. The epoxidation activity of purified Fus‐SMO was up to three times higher than that of the two‐component StyA/StyB (1:1, molar ratio) system and up to 110 times higher than that of the natural fused SMO. Determination of coupling efficiency and study of the influence of O2 pressure were also performed. Finally, Fus‐SMO and formate dehydrogenase were coexpressed in E. coli and applied as a self‐sufficient biocatalytic system for epoxidation on greater than 500 mg scale.

Enzyme Fusions in Biocatalysis: Coupling Reactions by Pairing Enzymes

“…However, the epoxidation activity was significantly decreased compared to that of the separate enzymes. In contrast, another group that studied the same enzymes as a fusion found it had a similar activity to the combination of separate StyA/StyB in a 1:1 ratio . This activity was higher than that of naturally fused StyA2B and the fusion enzymes addressed above .…”

“…Another enzyme (not shown) facilitates regeneration of NAD + to NADH (such as formate dehydrogenase). A) Fusion of SMO (StyA) with FR (StyB) for efficient coupling of a reduced flavin for styrene oxidation . B) Flavin halogenase can also be fused with a FR to couple the two reactions …”

“…For the two linkers that did show soluble expression (StyAL1B, StyAL2B, Table ), a decrease in activity compared to the separate enzymes was observed . However, when another group fused the same enzymes (Fus–SMO, Table ) with a 30‐resdue glycine‐rich linker, the level of activity was unaffected, and the expression level increased (40 mg L −1 compared to 12–15 mg L −1 for the StyAL1B and StyAL2B) . In a recent study of a natural FR–SMO fusion from Variovorax paradoxus EPS, the original linker (AREAV) was replaced with six distinct linkers .…”

“…By combining enzymes in this fashion, a single multifunctional biocatalyst can be produced that can catalyze a cascade of reactions. Remarkably, in a number of studies, evidence was found that the covalently tethered enzymes outperform the classic combination of the separate enzymes in some features such as enhanced expression, higher conversions of a cascade reaction, improved stability,, and improved catalytic activity . However, these promising observations are not consistent across studies, and generic methods to predictably design an effective bifunctional biocatalyst are still lacking.…”

Multicore Artificial Metalloenzymes Derived from Acylated Proteins as Catalysts for the Enantioselective Dihydroxylation and Epoxidation of Styrene Derivatives

“…For example the usage of a fused StyA/StyB system from pseudomonas sp. leads to very high specific epoxidation activities for styrene of 95 min −1 (99 % ee ( S )) within the first 5 min (TON=475) . Hollmann et al.…”