Asp305Gly mutation improved the activity and stability of the styrene monooxygenase for efficient epoxide production in Pseudomonas putida KT2440

Abstract

BackgroundStyrene monooxygenase (SMO) catalyzes the first step of aromatic alkene degradation yielding the corresponding epoxides. Because of its broad spectrum of substrates, the enzyme harbors a great potential for an application in medicine and chemical industries.ResultsIn this study, we achieved higher enzymatic activity and better stability towards styrene by enlarging the ligand entrance tunnel and improving the hydrophobicity through error-prone PCR and site-saturation mutagenesis. It was found that Asp305 (D305) hindered the entrance of the FAD cofactor according to the model analysis. Therefore, substitution with amino acids possessing shorter side chains, like glycine, opened the entrance tunnel and resulted in up to 2.7 times higher activity compared to the wild-type enzyme. The half-lives of thermal inactivation for the variant D305G at 60 °C was 28.9 h compared to only 3.2 h of the wild type SMO. Moreover, overexpression of SMO in Pseudomonas putida KT2440 with NADH regeneration was carried out in order to improve biotransformation efficiency for epoxide production. A hexadecane/buffer (v/v) biphasic system was applied in order to minimize the inactivation effect of high substrate concentrations on the SMO enzyme. Finally, SMO activities of 190 U/g CDW were measured and a total amount of 20.5 mM (S)-styrene oxide were obtained after 8 h.ConclusionsThis study offers an alternative strategy for improved SMO expression and provides an efficient biocatalytic system for epoxide production via engineering the entrance tunnel of the enzyme’s active site.Electronic supplementary materialThe online version of this article (10.1186/s12934-019-1065-5) contains supplementary material, which is available to authorized users.

Accessing Enantiopure Epoxides and Sulfoxides: Related Flavin‐Dependent Monooxygenases Provide Reversed Enantioselectivity

“…If assuming a similar position of the isoalloxazine ring of PHBH from P. aeruginosa in the Pp StyA S12, the C4a‐hydroperoxide then would be about 4 to 5 Å closer to the substrate and in a range that is reasonable for direct oxygen transfer. In accordance with that, mutations within loop 1 and loop 2 showed already an impact on the enzyme activity . It should be mentioned that the loops that interact with the isoalloxazine are composed differently in SMOs and IMOs, which might also result in a different FAD‐protein interaction.…”

“…For instance, Tan et al . just recently identified a residue that sits on top of the substrate cavity and affects activity by influencing FAD access to the active site . They were able to show by saturation mutagenesis that glycine at this position, which is conserved in over 96 % of GEMs, preserves highest activity and stability …”

“…Artificial fusion proteins were generated and several mutations were identified that changed and/or improved biocatalytic properties. These resulted in GEMs with, inter alia , up to 12‐fold higher activity and improved stability, change in substrate preference towards bulkier substrates, reversed enantioselectivity and higher catalytic efficiency . However, up to now it was not possible to predict activity or even enantioselectivity of these enzymes.…”

Production of Enantiopure Chiral Epoxides with E. coli Expressing Styrene Monooxygenase

“…Initially, wild-type SMOs of Pseudomonas were widely employed for biotransformation of styrene, but, to date, many other bacterial species that share styrene degradation pathways have been described [ 1 ]. Such discoveries, together with the expansion of recombinant technologies, metagenomics, and protein engineering, have led to the generation of SMOs with higher stability, selectivity, and activity towards a broad substrate spectrum, usually expressed as recombinant enzymes in Escherichia coli [ 2 , 4 , 5 , 6 , 7 ]. Significant progress was achieved by Panke et al in previous studies [ 8 , 9 , 10 , 11 ].…”

Substantially improving the enantioconvergence of PvEH1, a Phaseolus vulgaris epoxide hydrolase, towards m-chlorostyrene oxide by laboratory evolution

“…Optically pure epoxides and vicinal diols, the highly value-added and versatile building blocks, were widely applied in pharmaceutical, fine chemical and agrochemical industries [1–3]. For example, ( R )- p CPED was used for the synthesis of ( R )-Eliprodil, a neuroprotective agent for the treatment of ischemic stroke, while ( R )- m CPED for β3-adrenergic receptor agonists, such as SR 58611 and AJ-9677 [4].…”