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Series GSE122890 Query DataSets for GSE122890
Status Public on Nov 06, 2019
Title Identification of Absidia orchidis steroid 11β-hydroxylation system and its application in engineering Saccharomyces cerevisiae for one-step biotransformation to produce hydrocortisone
Organism Absidia caerulea
Experiment type Expression profiling by high throughput sequencing
Summary Purpose: In order to identify genes for 11β-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 11β-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 11α- and 11β- hydroxylase genes. In addition, DN17017 and DN7580 were identifed as the transcripts for the cytochrome P450 reductase and cytochrome b5,respectively.
Conclusions: The 11β-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
 
Contributor(s) Chen J, Fan F, Qu G, Xi Y, Sun Z, Bi C, Zhang X
Citation(s) 31669370
Submission date Nov 25, 2018
Last update date Nov 09, 2019
Contact name feiyu fan
E-mail(s) fan_fy@tib.cas.cn
Phone 0086022-84861948
Organization name Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences.
Department Key Laboratory of Systems Microbial Biotechnology
Lab Tian Chaoguang Lab
Street address 32 West 7th Avenue, Tianjin Airport Economic Area
City Tianjin
State/province Tianjin
ZIP/Postal code 300308
Country China
 
Platforms (1)
GPL25844 HiSeq X Ten (Absidia caerulea)
Samples (2)
GSM3487681 AC_Mock
GSM3487682 AC_RSA
Relations
BioProject PRJNA506826
SRA SRP170615

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Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE122890_RAW.tar 1.5 Mb (http)(custom) TAR (of TXT)
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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