GSE122890
Identification of Absidia orchidis steroid 11ß-hydroxylation system and its application in engineering Saccharomyces cerevisiae for one-step biotransformation to produce hydrocortisone
Purpose: In order to identify genes for 11ß-hydroxylase CYP5311B2 and associated redox partners CPR and cytochrome b5 in filamentous fungus Absidia orchidis AS3.65 Methods: Strand-specific RNA-seq libraries were constructed for two samples, including (I) Mycelia of A. orchidis treated with mock N, N-dimethylformamide (DMF); (II) Mycelia of A. orchidis treated with 170 mg/L 11-deoxycortisol 21-acetate (RSA) dissolved in N, N-dimethylformamide. For preparation of RNA samples, germinated young mycelia of A. orchidis 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 11ß-hydroxylase 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 10.4 and 10.5 million 150-bp paired-end clean reads were separately generated from the DMF mock and RSA induced samples,and 57549 unique transcripts were assembled from the pooled reads of the two samples using Trinity.  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.A group of 87 translated sequences were found have a typical CYP domain (PF00067), among which,the expression levels of DN10804 and DN10492 separately increased 306- and 48-fold upon RSA induction and identifed as A. orchidis 11a- and 11ß- hydroxylase genes. In addition, DN17017 and DN7580 were identifed as the transcripts for the cytochrome P450 reductase and cytochrome b5,respectively. Conclusions: The 11ß-hydroxylase system of A. orchidis was deciphered by transcriptome analysis. Overall design: mRNA profiles of A. orchidis treat with DMF mock or steroid RSA were generated by deep sequencing, using Illumina's Hiseq X Ten platform