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A Fungal P450 Enzyme from Thanatephorus cucumeris with Steroid Hydroxylation Capabilities.

ABSTRACT: In this study, we identified a P450 enzyme (STH10) and an oxidoreductase (POR) from Thanatephorus cucumeris NBRC 6298 by a combination of transcriptome sequencing and heterologous expression in Pichia pastoris The biotransformation of 11-deoxycortisol was performed by using Pichia pastoris whole cells coexpressing sth10 and por, and the product analysis indicated that the STH10 enzyme possessed steroidal 19- and 11?-hydroxylase activities. This is a novel fungal P450 enzyme with 19-hydroxylase activity, which is different from the known steroidal aromatase cytochrome P450 19 (CYP19) and CYP11B families of enzymes.IMPORTANCE Hydroxylation is one of the most important reactions in steroid functionalization; in particular, C-19 hydroxylation produces a key intermediate for the synthesis of 19-nor-steroid drugs without a C-19 angular methyl group in three chemoenzymatic steps, in contrast to the current industrial process, which uses 10 chemical reactions. However, hydroxylation of the C-19 angular methyl group remains a very challenging task due to the high level of steric resistance to the C-19 methyl group between the A and B rings. The present report describes a novel fungal P450 enzyme with 19-hydroxylase activity. This opens a new venue for searching effective biocatalysts for the useful process of steroidal C-19 hydroxylation, although further studies for better understanding of the structural basis of the regioselectivity and substrate specificity of this fungal steroidal 19-hydroxylase are warranted to facilitate the engineering of this enzyme for industrial applications.

SUBMITTER: Lu W

PROVIDER: S-EPMC6007115 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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A Fungal P450 Enzyme from Thanatephorus cucumeris with Steroid Hydroxylation Capabilities.

Lu Wei W Chen Xi X Feng Jinhui J Bao Yun-Juan YJ Wang Yu Y Wu Qiaqing Q Zhu Dunming D

Applied and environmental microbiology 20180618 13

In this study, we identified a P450 enzyme (STH10) and an oxidoreductase (POR) from Thanatephorus cucumeris NBRC 6298 by a combination of transcriptome sequencing and heterologous expression in Pichia pastoris The biotransformation of 11-deoxycortisol was performed by using Pichia pastoris whole cells coexpressing sth10 and por, and the product analysis indicated that the STH10 enzyme possessed steroidal 19- and 11β-hydroxylase activities. This is a novel funga ...[more]

PMID: 29728383

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Project description:Purpose: In order to identify genes for steroid 14α-hydroxylase P450lun and its associated redox partner CPRlun in filamentous fungus Cochliobolus lunatus ATCC#12017 Methods: Strand-specific RNA-seq libraries were constructed for two samples, including (I) Mycelia of C. lunatus treated with mock N, N-dimethylformamide (DMF); (II) Mycelia of C. lunatus treated with 170 mg/L 11-deoxycortisol 21-acetate (RSA) dissolved in N,N-dimethylformamide. For preparation of RNA samples, germinated young mycelia of C. lunatus after 22-24 pre-culture in liquid potato dextrose broth medium were harvested and transferred into a new 30 ml potassium phosphate buffer supplement with either 170 mg/L RSA dissolved in DMF, or an equal volume of DMF as mock. Additional two-hours cultivation was performed to induction of the P-450lun gene expression. Total RNA for samples were isolated using TRIzol reagent (Invitrogen, Grand Island, USA), then used for high-throughput RNA sequencing. The two 150-nt strand-specific paired-end RNA-seq libraries were generated commercially at Novogene Biotechnology Co. Ltd (Tianjin, China) by using Illumina’s Hiseq X Ten platform (Illumina, San Diego, USA). The clean reads were used to create a de novo transcriptome assembly using the Trinity pipeline (v2.4.0). The abundance for each of the resulted transcripts was calculated by RSEM (v1.2.0) using the Fragments Per Kilobase per Million (FPKM) method. Results: A total of 7.1 and 7.9 million 150-bp paired-end clean reads were separately generated from the DMF mock and RSA induced samples,and 23957 transcripts with length more than 600nt were assembled from the pooled reads of both samples using Trinity program. The translated sequences of these transcripts were predicted using TransDecoder, and then used as queries to search against the SWISS-PROT and the Pfam databases for functional annotation. Transcripts DN8493 and DN2353 were separately identified as C.lunatus P-450lun and CPRlun genes. Conclusions: The 14β-hydroxylase system of C.lunatus was deciphered by transcriptome analysis.

Dataset Information

A Fungal P450 Enzyme from Thanatephorus cucumeris with Steroid Hydroxylation Capabilities.

Publications

A Fungal P450 Enzyme from Thanatephorus cucumeris with Steroid Hydroxylation Capabilities.

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