Genetic fusion of P450 BM3 and formate dehydrogenase towards self-sufficient biocatalysts with enhanced activity

Arsenij Kokorin,Pavel D Parshin,Patrick J Bakkes,Vladimir I Tishkov,Anastasia A Pometun,Vlada B Urlacher

doi:10.1038/s41598-021-00957-5

Arsenij Kokorin, Pavel D Parshin + Show 4 more

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https://doi.org/10.1038/s41598-021-00957-5

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Abstract

Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling. In this study, the genes coding for cytochrome P450 BM3 from B. megaterium and formate dehydrogenase from Pseudomonas sp. were fused to enable both substrate oxidation catalyzed by P450 BM3 and continuous cofactor regeneration by formate dehydrogenase within one construct. The order of the genes in the fusion as well as the linkers that bridge the enzymes were varied. The resulting constructs were compared to individual enzymes regarding substrate conversion, stability and kinetic parameters to examine whether fusion led to any substantial improvements of enzymatic properties. Most noticeably, an activity increase of up to threefold was observed for the fusion constructs with various substrates which were partly attributed to the increased diflavin reductase activity of the P450 BM3. We suggest that P450 BM3 undergoes conformational changes upon fusion which resulted in altered properties, however, no NADPH channeling was detected for the fusion constructs.

Highlights

Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling
While the native formate dehydrogenases (FDHs) from Pseudomonas sp. 101 prefers NAD+, site-directed mutagenesis resulted in variants with a shifted nicotinamide cofactor specificity towards N ADP+ that were successfully applied in cofactor regeneration experiments with P450 BM3 b efore[31,34,35]
With the process of enzyme fusion, substrate oxidation by BM3 4m and NADPH regeneration by FDH2 would be combined in one construct (Fig. 1a)

Summary

Introduction

Fusion of multiple enzymes to multifunctional constructs has been recognized as a viable strategy to improve enzymatic properties at various levels such as stability, activity and handling. The resulting constructs were compared to individual enzymes regarding substrate conversion, stability and kinetic parameters to examine whether fusion led to any substantial improvements of enzymatic properties. A positive effect of enzyme proximity has been reported in in vitro a pplications[5], substrate channeling in these cases remains highly d ebated[19,20,21] Under consideration of these criteria, enzymes from various classes have been merged to bifunctional units that exhibit improved properties compared to their individual enzyme parts. Dependency on the cofactor NADPH makes large-scale applications of P450 BM3 expensive when the cofactor is used in stoichiometric amounts To circumvent this issue, additional enzymes such as glucose dehydrogenase (GDH)28, PTDH29 as well as F DH30 have been employed for in situ cofactor regeneration by turnover of cheap sacrificial substrates. Thermal and solvent stability were determined for the fusions and compared to the individual enzymes, and probability for substrate channeling in fusions was investigated

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