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Transcriptome analysis using RNA-Seq reveals complex acid responsive mechanisms in 3-hydroxypropionic aicd producing strains of E. coli

ABSTRACT: RNA-Seq analysis was conducted at different growth phases of the cell cycle as well as various fermentation conditions. The genes encoding RNA polymerase were down-regulated in SH0012 compared to its mother strain, suggesting reprogramming of cells for survival in new environments. The micro-aerobic fermentation caused down-regulation of transporter proteins and 3-HP non-producing fermentation caused up-regulation of phage shock proteins and lipoproteins. Additionally, the clustering analyses revealed that there is a mode of temporal regulation of biological pathways during organic acid production.

ORGANISM(S): Escherichia coli

PROVIDER: GSE70507 | GEO | 2016/07/01

SECONDARY ACCESSION(S): PRJNA288904

REPOSITORIES: GEO

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Project description:In the present study, we have used a genomic approach to understand how the classical mutate-and-screen method actually generated an improved rifamycin B producer. Compared with the reference strains Amycolatopsis mediterranei S699 (rifamycin B producer) and U32 (rifamycin SV producer), a total of 250 variations affecting a total of 227 coding sequences (CDS) were found in rifamycin B overproducing strain HP-130. One hundred nine CDS variations were specific to HP-130 as they were absent in both S699 and U32. A lot of variations affected genes coding for fatty acid (an lipid) metabolism, which is tightly interconnected with rifamycin biosynthesis by common metabolic precursors (malonyl-CoA, methylmalonyl-CoA). Interestingly, a nonsense mutation was mapped within mutB coding for methylmalonyl-CoA mutase suggesting the importance of metabolic re-direction of carbon flow toward rifamycin biosynthesis in the over-producing phenotype of HP-130. Other key mutations were: i.) a missense mutation affecting ppk, which encodes a polyphosphate kinase and was previously shown to play a negative role in the control of antibiotic biosynthesis in Streptomyces lividans; ii.) a missense mutation in argS2 affecting one of the two arginyl tRNA synthetases of A. mediterranei; iii.) a missense mutation in rifN coding for a protein belonging to the ROK family of transcriptional regulators with kanosamine kinase activity. Microarray analysis of the transcriptome of HP-130 as compared to that of S699 was useful to understand the effects of several mutations on global gene expression profile. Genomic and transcriptomic data were used to improve rifamycin B production in S699 by genetic engineering thus proving the causative effect of the above-mentioned mutations on the overproducing phenotype. In this study by using comparative genomic analysis we have identified all genetic changes that have occurred during development of a rifamycin B overproducer (HP-130), which was obtained by the traditional mutate-and-screen method. To gain insight about the mechanisms underlying improved rifamycin B production in HP-130, DNA microarray of A. mediterranei were manufactured and used for comparative analysis of the gene expression profile in HP-130 and S699. In DNA microarray experiments the two strains were cultivated under standard batch-culture conditions in modified RFB 2244 fermentation medium, and samples were collected at different time points (24-72 h) for RNA extraction. Gene expression data were analyzed to identify transcripts modulated during the growth curve. Amycolatopsis mediterranei S699 (WT) and the improved rifamycin B producer (HP-130) were profiled at 5 time points. For each time point two biological replicates are provided.

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Transcriptome analysis using RNA-Seq reveals complex acid responsive mechanisms in 3-hydroxypropionic aicd producing strains of E. coli

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