Abstract
SummaryCytochrome P450 monooxygenases (P450) are enzymes with high potential as biocatalysts for industrial applications. Their large‐scale applications are, however, limited by instability and requirement for coproteins and/or expensive cofactors. These problems are largely overcome when whole cells are used as biocatalysts. We previously screened various yeast species heterologously expressing self‐sufficient P450s for their potential as whole‐cell biocatalysts. Most P450s are, however, not self‐sufficient and consist of two or three protein component systems. Therefore, in the present study, we screened different yeast species for coexpression of P450 and P450‐reductase (CPR) partners, using CYP53B1 from Rhodotorula minuta as an exemplary P450. The abilities of three different coexpressed CPR partners to support P450 activity were investigated, two from basidiomycetous origin and one from an ascomycete. The various P450‐CPR combinations were cloned into strains of Saccharomyces cerevisiae, Kluyveromyces marxianus, Hansenula polymorpha, Yarrowia lipolytica and Arxula adeninivorans, using a broad‐range yeast expression vector. The results obtained supported the previous finding that recombinant A. adeninivorans strains perform excellently as whole‐cell biocatalysts. This study also demonstrated for the first time the P450 reductase activity of the CPRs from R. minuta and U. maydis. A very interesting observation was the variation in the supportive activity provided by the different reductase partners tested and demonstrated better P450 activity enhancement by a heterologous CPR compared to its natural partner CPR. This study highlights reductase selection as a critical variable for consideration in the pursuit of optimal P450‐based catalytic systems. The usefulness of A. adeninivorans as both a host for recombinant P450s and whole‐cell biocatalyst was emphasized, supporting earlier findings.
Highlights
The cytochrome P450 monooxygenases (P450s) are a promising group of enzymes for industrial applications, as they are capable of catalysing the stereo- and regiospecific hydroxylation of non-activated C–H bonds
Total RNA was extracted from R. minuta CBS2177 and reverse-transcribed to cDNA, which served as a template for the specific PCR amplification of the RmCPR, based on the deposited sequence (GenBank: AB055119)
The intronless U. maydis coexpression of P450 and P450-reductase (CPR) (UmCPR) ORF was PCR amplified directly from genomic DNA extracted from the DSM3121 strain, using primers based on the deposited sequence annotated as a putative NADPH-cytochrome P450 reductase
Summary
The cytochrome P450 monooxygenases (P450s) are a promising group of enzymes for industrial applications, as they are capable of catalysing the stereo- and regiospecific hydroxylation of non-activated C–H bonds As such, they are of interest in diverse research fields, most notably research on drug metabolism and design, fine chemical synthesis and bioremediation (Kumar, 2010; O’Reilly et al, 2011; Schewe et al, 2011). They are of interest in diverse research fields, most notably research on drug metabolism and design, fine chemical synthesis and bioremediation (Kumar, 2010; O’Reilly et al, 2011; Schewe et al, 2011) Largescale applications of these enzymes have, been limited by their requirement for expensive cofactors, and in most cases, coproteins are required for catalysis to occur, while poor enzyme stability is a further limitation (Gillam, 2007; O’Reilly et al, 2011; Urlacher and Girhard, 2012). While bacterial P450s tend to have higher activities and improved stability over eukaryotic P450s, the latter are of interest due to interest in their roles in drug design and metabolism, making them important tools in human medical research
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