Heterologous expression of cytochrome P450 monooxygenases in different ascomycetous yeasts
Theron, Chrispian William
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Cytochrome P450 monooxygenases (P450) are a diverse, ubiquitous family of important enzymes which catalyze a variety of activities. Their outstanding abilities to hydroxylate non-activated hydrocarbons make them attractive enzymes for applications in the fields of chemical synthesis and bioremediation. Large scale applications of P450s are however largely limited by: (i) their requirement for expensive cofactors; (ii) dependence on co-proteins; and (iii) limited enzyme stability. These problems are largely circumvented by using whole-cell biocatalysis. Therefore the identification of appropriate hosts for heterologous expression is important. E. coli has several limitations in its ability to express eukaryotic P450s, therefore yeasts are attractive alternatives, but have scarcely been used for whole-cell applications (Zollner et al, 2010). The aim of this study was therefore to investigate several ascomycetous yeasts for their potential as P450-expressing whole-cell biocatalysts. To perform parallel, unbiased comparisons, the vector, cultivation conditions and assay conditions needed to be consistent between the different yeasts. This was facilitated by a broad-range vector system designed in our group, which allowed genomic chromosomal integration of foreign DNA at the 18S rDNA regions, and expression using a constitutive TEF promoter. Cultures of different yeasts were grown for the same duration at the same temperature in a medium common among strains, prior to activity assays. Using CYP53B1 as a reporter enzyme we demonstrated whole-cell activity for all of the yeasts tested, except for H. polymorpha. The native reductase systems allowed detectable activities of CYP53B1 in all of the other yeasts, with the highest activity (2.3 μmol.h-1 .gDCW -1) found in A. adeninivorans. Coexpression of cytochrome P450 reductases (CPR) from Yarrowia lipolytica (YlCPR), Rhodotorula minuta (RmCPR) and Ustilago maydis (UmCPR) all led to improvements of the CYP53B1 activities, with the highest activity (11.5 μmol.h-1.gDCW -1) obtained with CYP53B1 and UmCPR coexpressed in A. adeninivorans. The effects of the cloned UmCPR also differed between the hosts, with the biggest improvements observed in A. adeninivorans and Y. lipolytica. RmCPR and UmCPR improved the CYP53B1 activity more dramatically than YlCPR, possibly because CYP53B1, RmCPR and UmCPR are all of basidiomycetous origin. The CYP53B1 activity achieved in this study was considerably better than results obtained previously in our group. In the previous study, a Y. lipolytica strain with multiple copies of CYP53B1 and an additional copy of YlCPR both under the regulation of strong inducible promoters was used, under optimised and constant induction conditions. (Shiningavamwe et al, 2006). The activity obtained in this study on the other hand was achieved using an A. adeninivorans strain carrying presumably only one or two copies of CYP53B1 and UmCPR, expressed under the control of a constitutive promoter, and under non-optimised conditions. Activity of the self-sufficient P450s CYP102A1 and CYP505A1 was assayed using hexylbenzoic acid (HBA) as a non-natural substrate, since wild-type β-oxidation pathways would not permit the use of fatty acids as substrates. HBA is hydroxylated at the ω-1 and ω-2 positions of the alkyl chain by both CYP102A1 and CYP505A1 during this study. Better expression of the bacterial CYP102A1 was obtained using K. marxianus and S. cerevisiae, than in A. adeninivorans; whereas expression of the eukaryotic CYP505A1 was better in Y. lipolytica and especially A. adeninivorans. The best activity observed with a self-sufficient P450 was obtained once more with A. adeninivorans expressing CYP505A1 (33 μmol.h-1 gDCW -1). Overall, the vector system allowed successful expression of P450s and CPRs from bacterial, ascomycetous and basidiomycetous fungal origin, in multiple ascomycetous yeast hosts. The differential effects of different CPRs on a class II P450 were demonstrated, and the UmCPR was in this case found to be an excellent P450 reductase. We report heterologous P450 expression in A. adeninivorans for the first time, and it proved to be, of the yeasts tested, the host with the highest potential for efficient P450 expression and whole cell biocatalysis. The findings of this study provide insight for the improvement of the field of eukaryotic P450 research and particularly whole-cell biocatalysis, and could potentially assist in enhancing the applications of these promising enzymes.