Determination of steroid hydroxylation specificity of an industrial strain Aspergillus ochraceus TCCC41060 by cytochrome P450 gene CYP68J5

Xue Wang,Peng Jin,Xi Jia,Xingwei Yang,Zhengxiang Wang,Xiaoguang Liu,Fuping Lu

doi:10.1186/s13213-020-01577-6

Xue Wang, Peng Jin + Show 5 more

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https://doi.org/10.1186/s13213-020-01577-6

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Abstract

PurposeThe use of Aspergillus ochraceus TCCC41060 for synthesis of 11α-OH-ethylgonendione, an important intermediate for synthesis of desogestrel-a major ingredient of the “third-generation” oral contraceptives, is hampered by its low regioselectivity of hydroxylation. In the present study, we sought to characterize gene(s) involved in steroid hydroxylation specificity in strain TCCC41060.MethodsTaking advantage of the fact that expression of the 11α-hydroxylase, a member of the cytochrome P450 family, is highly induced by steroid substrates, we combined RNA-seq, qRT-PCR, and yeast functional expression to search for responsible steroid hydroxylase gene(s).ResultsTwo highly inducible P450 genes (CYP68L8 and CYP68J5) were isolated and recombinant yeast cells expressing CYP68J5 were capable of 11α-hydroxylating both 16,17α-epoxyprogesterone and D-ethylgonendione. Disruption of CYP68J5 in strain TCCC41060 resulted in complete loss of hydroxylation activities towards D-ethylgonendione, indicating that CYP68J5 was solely responsible for hydroxylation activity on D-ethylgonendione in TCCC41060.ConclusionThe above results demonstrated that low hydroxylation specificity of CYP68J5 on D-ethylgonendione fully accounted for high by-product contents in TCCC41060, thus pointing to a strategy to engineer 11α-hydroxylase variants with higher hydroxylation specificity.

Highlights

Steroid drugs are a very important class of clinical drugs widely prescribed for the treatment of inflammation, cancer, rheumatic arthritis, allergy as well as contraception (Hogg 1992; Mahato et al 1989; Tong and Dong 2009)
The fungal steroid hydroxylation system consists of a cytochrome P450 monooxygenase (CYP) and a cytochrome P450 reductase (CPR) that mediate the transfer of electrons from NADPH to the P450 monooxygenases (Tong and Dong 2009; Wachenfeldt and Johnson 1995)
To gain insights into the mechanistic aspects of steroid hydroxylation in A. ochraceus, we isolated two highly inducible P450 genes, CYP68L8 and CYP68J5, by transcriptome sequencing and qRT-PCR, and we provide definitive genetic evidence that in A. ochraceus TCCC41060 CYP68J5 is solely responsible for 11α-hydroxylation activity on 16,17α-epoxyprogesterone and D-ethylgonendione

Summary

Introduction

Steroid drugs are a very important class of clinical drugs widely prescribed for the treatment of inflammation, cancer, rheumatic arthritis, allergy as well as contraception (Hogg 1992; Mahato et al 1989; Tong and Dong 2009). C11α, β-hydroxylations of steroids, one of the most important steroid microbial transformations for the production of key intermediates of corticosteroids, are performed by employing various. It has been well documented that in filamentous fungi, the hydroxylases (monooxygenases) involved in steroid hydroxylation are members of the cytochrome P450 superfamily (Črešnar and Petrič 2011; Den Besten et al 1990). It is still a challenge to assign a specific steroid hydroxylation ability to individual genes, given the existence of dozens or even hundreds of CYP genes in many filamentous fungi (Moktalil et al 2012; Petrič et al 2010) and the low homology between cytochrome P450s catalyzing steroid hydroxylation reaction even at the same position among closely related species (Nebert and Gonzalez 1987; Nebert et al 1989). The molecular basis of steroid hydroxylation in many filamentous fungi is poorly understood

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