RNA-Seq analysis uncovers non-coding small RNA system of Mycobacterium neoaurum in the metabolism of sterols to accumulate steroid intermediates

Abstract

BackgroundUnderstanding the metabolic mechanism of sterols to produce valuable steroid intermediates in mycobacterium by a noncoding small RNA (sRNA) view is still limited. In the work, RNA-seq was implemented to investigate the noncoding transcriptome of Mycobacterium neoaurum (Mn) in the transformation process of sterols to valuable steroid intermediates, including 9α-hydroxy-4-androstene-3,17-dione (9OHAD), 1,4-androstadiene-3,17-dione (ADD), and 22-hydroxy-23, 24-bisnorchola-1,4-dien-3-one (1,4-BNA).ResultsA total of 263 sRNA candidates were predicted from the intergenic regions in Mn. Differential expression of sRNA candidates was explored in the wide type Mn with vs without sterol addition, and the steroid intermediate producing Mn strains vs wide type Mn with sterol addition, respectively. Generally, sRNA candidates were differentially expressed in various strains, but there were still some shared candidates with outstandingly upregulated or downregulated expression in these steroid producing strains. Accordingly, four regulatory networks were constructed to reveal the direct and/or indirect interactions between sRNA candidates and their target genes in four groups, including wide type Mn with vs without sterol addition, 9OHAD, ADD, and BNA producing strains vs wide type Mn with sterol addition, respectively. Based on these constructed networks, several highly focused sRNA candidates were discovered to be prevalent in the networks, which showed comprehensive regulatory roles in various cellular processes, including lipid transport and metabolism, amino acid transport and metabolism, signal transduction, cell envelope biosynthesis and ATP synthesis. To explore the functional role of sRNA candidates in Mn cells, we manipulated the overexpression of candidates 131 and 138 in strain Mn-9OHAD, which led to enhanced production of 9OHAD from 1.5- to 2.3-fold during 6 d’ fermentation and a slight effect on growth rate.ConclusionsThis study revealed the complex and important regulatory roles of noncoding small RNAs in the metabolism of sterols to produce steroid intermediates in Mn, further analysis of which will promote the better understanding about the molecular metabolism of these sRNA candidates and open a broad range of opportunities in the field.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0462-2) contains supplementary material, which is available to authorized users.

New Insights on Steroid Biotechnology

“…For example, a deep understanding of the enzymatic steps involved into the sterol side-chain degradation could be useful for the design of new AD/ADD-producing strains that do not accumulate the intermediates 4-HBC/1,4-HBC. In order to further understand the regulatory mechanisms of sterol catabolism and steroid production, several transcriptomic and proteomic studies have been also done in recent years, increasingly focused on higher-producer strains (Liu et al, 2016a , b ; Xiong et al, 2017a , b ).…”

Application of Transcriptomics to Enhance Early Diagnostics of Mycobacterial Infections, with an Emphasis on Mycobacterium avium ssp. paratuberculosis

“…sRNA mapping and function annotation facilitates RNA-seq data analysis. Several studies have concentrated on mapping the sRNA landscape of M. tuberculosis and other mycobacterial species, but the non-coding transcriptome of MAP has until now not been explored [81,82,83,84,85]. In MB, 34 novel sRNAs were detected that were also conserved in closely related M. tuberculosis and M. smegmatis strains [86].…”

Synthetic Biology of Small RNAs and Riboswitches

“…Recent examples of this method of sRNA-based metabolic engineering by overexpression of native sRNAs include increasing butanol tolerance in Clostridium acetobutylicum (127), ethanol and butanol tolerance in cyanobacterium Synechocystis sp. PCC 6803 (128) and Zymomonas mobilis (126), and steroid intermediate production in Mycobacterium neoaurum (129). Deletion of sRNAs that interfere with the desired pathway, or produce undesired phenotypes or growth rates, has also become a useful engineering method to ensure high production of final products (such as subtilisin in Bacillus licheniformis (130)).…”