Dataset Information

Genome-Wide Transcriptome Profiling Provides Insight on Cholesterol and Lithocholate Degradation Mechanisms in Nocardioides simplex VKM Ac-2033D.

ABSTRACT: Steroid microbial degradation plays a significant ecological role for biomass decomposition and removal/detoxification of steroid pollutants. In this study, the initial steps of cholesterol degradation and lithocholate bioconversion by a strain with enhanced 3-ketosteroid dehydrogenase (3-KSD) activity, Nocardioides simplex VKM Ac-2033D, were studied. Biochemical, transcriptomic, and bioinformatic approaches were used. Among the intermediates of sterol sidechain oxidation cholest-5-en-26-oic acid and 3-oxo-cholesta-1,4-dien-26-oic acid were identified as those that have not been earlier reported for N. simplex and related species. The transcriptomic approach revealed candidate genes of cholesterol and lithocholic acid (LCA) catabolism by the strain. A separate set of genes combined in cluster and additional 3-ketosteroid ?1-dehydrogenase and 3-ketosteroid 9?-hydroxylases that might be involved in LCA catabolism were predicted. Bioinformatic calculations based on transcriptomic data showed the existence of a previously unknown transcription factor, which regulates cholate catabolism gene orthologs. The results contribute to the knowledge on diversity of steroid catabolism regulation in actinobacteria and might be used at the engineering of microbial catalysts for ecological and industrial biotechnology.

SUBMITTER: Shtratnikova VY

PROVIDER: S-EPMC7593942 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Genome-Wide Transcriptome Profiling Provides Insight on Cholesterol and Lithocholate Degradation Mechanisms in <i>Nocardioides simplex</i> VKM Ac-2033D.

Shtratnikova Victoria Y VY Schelkunov Mikhail I MI Fokina Victoria V VV Bragin Eugeny Y EY Lobastova Tatyana G TG Shutov Andrey A AA Kazantsev Alexey V AV Donova Marina V MV

Genes 20201020 10

Steroid microbial degradation plays a significant ecological role for biomass decomposition and removal/detoxification of steroid pollutants. In this study, the initial steps of cholesterol degradation and lithocholate bioconversion by a strain with enhanced 3-ketosteroid dehydrogenase (3-KSD) activity, <i>Nocardioides simplex</i> VKM Ac-2033D, were studied. Biochemical, transcriptomic, and bioinformatic approaches were used. Among the intermediates of sterol sidechain oxidation cholest-5-en-26- ...[more]

PMID: 33092158

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Project description:BackgroundBacterial degradation/transformation of steroids is widely investigated to create biotechnologically relevant strains for industrial application. The strain of Nocardioides simplex VKM Ac-2033D is well known mainly for its superior 3-ketosteroid Δ1-dehydrogenase activity towards various 3-oxosteroids and other important reactions of sterol degradation. However, its biocatalytic capacities and the molecular fundamentals of its activity towards natural sterols and synthetic steroids were not fully understood. In this study, a comparative investigation of the genome-wide transcriptome profiling of the N. simplex VKM Ac-2033D grown on phytosterol, or in the presence of cortisone 21-acetate was performed with RNA-seq.ResultsAlthough the gene patterns induced by phytosterol generally resemble the gene sets involved in phytosterol degradation pathways in mycolic acid rich actinobacteria such as Mycolicibacterium, Mycobacterium and Rhodococcus species, the differences in gene organization and previously unreported genes with high expression level were revealed. Transcription of the genes related to KstR- and KstR2-regulons was mainly enhanced in response to phytosterol, and the role in steroid catabolism is predicted for some dozens of the genes in N. simplex. New transcription factors binding motifs and new candidate transcription regulators of steroid catabolism were predicted in N. simplex. Unlike phytosterol, cortisone 21-acetate does not provide induction of the genes with predicted KstR and KstR2 sites. Superior 3-ketosteroid-Δ1-dehydrogenase activity of N. simplex VKM Ac-2033D is due to the kstDs redundancy in the genome, with the highest expression level of the gene KR76_27125 orthologous to kstD2, in response to cortisone 21-acetate. The substrate spectrum of N. simplex 3-ketosteroid-Δ1-dehydrogenase was expanded in this study with progesterone and its 17α-hydroxylated and 11α,17α-dihydroxylated derivatives, that effectively were 1(2)-dehydrogenated in vivo by the whole cells of the N. simplex VKM Ac-2033D.ConclusionThe results contribute to the knowledge of biocatalytic features and diversity of steroid modification capabilities of actinobacteria, defining targets for further bioengineering manipulations with the purpose of expansion of their biotechnological applications.

Project description:BackgroundThe modification of glucose import capacity is an engineering strategy that has been shown to improve the characteristics of Escherichia coli as a microbial factory. A reduction in glucose import capacity can have a positive effect on production strain performance, however, this is not always the case. In this study, E. coli W3110 and a group of four isogenic derivative strains, harboring single or multiple deletions of genes encoding phosphoenolpyruvate:sugar phosphotransferase system (PTS)-dependent transporters as well as non-PTS transporters were characterized by determining their transcriptomic response to reduced glucose import capacity.ResultsThese strains were grown in bioreactors with M9 mineral salts medium containing 20 g/L of glucose, where they displayed specific growth rates ranging from 0.67 to 0.27 h-1, and specific glucose consumption rates (qs) ranging from 1.78 to 0.37 g/g h. RNA-seq analysis revealed a transcriptional response consistent with carbon source limitation among all the mutant strains, involving functions related to transport and metabolism of alternate carbon sources and characterized by a decrease in genes encoding glycolytic enzymes and an increase in gluconeogenic functions. A total of 107 and 185 genes displayed positive and negative correlations with qs, respectively. Functions displaying positive correlation included energy generation, amino acid biosynthesis, and sugar import.ConclusionChanges in gene expression of E. coli strains with impaired glucose import capacity could be correlated with qs values and this allowed an inference of the physiological state of each mutant. In strains with lower qs values, a gene expression pattern is consistent with energy limitation and entry into the stationary phase. This physiological state could explain why these strains display a lower capacity to produce recombinant protein, even when they show very low rates of acetate production. The comparison of the transcriptomes of the engineered strains employed as microbial factories is an effective approach for identifying favorable phenotypes with the potential to improve the synthesis of biotechnological products.

Project description:Water deficit limits plant growth and productivity in wheat. The effect of water deficit varies considerably in the contrasting genotypes. This study attempted comparative transcriptome profiling of the tolerant (Abura) and susceptible (AUS12671) genotypes under PEG-simulated water stress via genome-wide RNA-seq technology to understand the dynamics of tolerance mechanism. Morphological and physiological analyses indicated that the tolerant genotype Abura had a higher root growth and net photosynthesis, which accounted for its higher root biomass than AUS12671 under stress. Transcriptomic analysis revealed a total of 924 differentially expressed genes (DEGs) that were unique in the contrasting genotypes under stress across time points. The susceptible genotype AUS12671 had slightly more abundant DEGs (505) than the tolerant genotype Abura (419). Gene ontology enrichment and pathway analyses of these DEGs suggested that the two genotypes differed significantly in terms of adaptive mechanism. Predominant upregulation of genes involved in various metabolic pathways was the key adaptive feature of the susceptive genotype AUS12671 indicating its energy-consuming approach in adaptation to water deficit. In contrast, downregulation the expression of genes of key pathways, such as global and overview maps, carbohydrate metabolism, and genetic information processing was the main strategy for the tolerant genotype Abura. Besides, significantly higher number of genes encoding transcription factors (TF) families like MYB and NAC, which were reported to be associated with stress defense, were differentially expressed in the tolerant genotype Abura. Gene encoding transcription factors TIFY were only differentially expressed between stressed and non-stressed conditions in the sensitive genotype. The identified DEGs and the suggested differential adaptive strategies of the contrasting genotypes provided an insight for improving water deficit tolerance in wheat.

Dataset Information

Genome-Wide Transcriptome Profiling Provides Insight on Cholesterol and Lithocholate Degradation Mechanisms in Nocardioides simplex VKM Ac-2033D.

Publications

Genome-Wide Transcriptome Profiling Provides Insight on Cholesterol and Lithocholate Degradation Mechanisms in <i>Nocardioides simplex</i> VKM Ac-2033D.

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