Unknown,Transcriptomics,Genomics,Proteomics

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Whole track RNAseq of the deletion mutant delta cgt in Actinoplanes sp. SE50/110 during growth phase and during transition into stationary phase

ABSTRACT: Actinoplanes sp. SE50/110 (ATCC 31044) is the wild type of industrial producer strains of acarbose. Acarbose is used since the early 1990s as an inhibitor of intestinal human alpha-glucosidases in the medical treatment of type II diabetes mellitus. The small secreted protein Cgt, which consists of a single carbohydrate-binding module (CBM) 20-domain, was found to be highly expressed in Actinoplanes sp. SE50/110 in previous studies, but neither its function nor a possible role in acarbose formation was explored, yet. Due to this and its high abundance in the extracellular proteome of Actinoplanes, a functional role within the sugar metabolism or in the environmental stress protection was assumed. However, the gene deletion mutant ∆cgt, constructed by CRISPR/Cas9 technology, displayed no apparent phenotype in screening experiments testing for pH and osmolarity stress, limited carbon source starch as well as excess of seven different sugars in liquid culture and further 97 carbon sources in the Omnilog Phenotypic Microarray system of Biolog. Therefore, a protective function as a surface protein or a function within the retainment and the utilization of carbon sources could not be experimentally validated. Remarkably, enhanced production of acarbose was determined yielding into 8-16 % higher product titers when grown in maltose-containing medium. Here the whole track RNAseq data of delta cgt and the wild type of Actinoplanes sp. SE50/110 during growth phase and during transition into stationary phase are provided, when grown in maltose minimal medium.

INSTRUMENT(S): Illumina MiSeq

ORGANISM(S): Actinoplanes sp. SE50/110

SUBMITTER: Lena Schaffert

PROVIDER: E-MTAB-8432 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Actinoplanes sp. SE50/110 is the wild type of industrial production strains of the fine-chemical acarbose (acarviosyl-maltose), which is used as α-glucosidase inhibitor in the treatment of type II diabetes. Although maltose is an important building block of acarbose, the maltose/maltodextrin metabolism has not been studied in Actinoplanes sp. SE50/110 yet. A PurR/LacI-type transcriptional regulator gene, named amlR (ACSP50_2475), is localized upstream of the maltase gene amlE (ACSP50_2474), which is organized in an operon with and a gene downstream (ACSP50_2473) encoding a GGDEF-EAL-domain-containing protein putatively involved in c-di-GMP signaling. A targeted gene deletion mutant of amlR was constructed by use of CRISPR/Cas9 technology. The transcription of the aml operon is significantly repressed in the wild type when growing on glucose and repression is absent in an ∆amlR deletion mutant. Although AmlR apparently is a local transcriptional regulator of the aml operon, the ∆amlR strain shows severe growth inhibitions on glucose and – concomitantly – differential transcription of several genes of various functional classes, which was shown by RNAseq. We used a pooled library for RNAseq for global pre-screening and validated these results for a total of 25 genes by RT-qPCR. RNA of triplicates of the wildtype and the deletion mutant was isolated from growing cultures and pooled equimolar separately for the wildtype and the deletion mutant (total amount of 2.5 µg RNA each in 26 µL RNase-free water). rRNA was depleted using the Ribo-Zero rRNA Removal Kit for bacteria (Illumina, San Diego, USA). Successful depletion of rRNA was verified by an Agilent RNA 6000 Pico chip in the Bioanalyzer (Agilent, Böblingen, Germany). cDNA libraries were prepared following protocols from Pfeifer-Sancar et al. (2013) and Irla et al. (2015) by use of the TruSeq stranded mRNA kit (Illumina, San Diego, CA, USA). The libraries were quantified by a DNA High Sensitivity Assay chip in the Bioanalyzer (Agilent, Böblingen, Germany) and sequenced on a 2 x 75 nt HiSeq 1500 run (Illumina, San Diego, CA, USA). Sequencing yielded about 10.7 million read pairs for the wildtype library and 10.2 million read pairs for the deletion mutant library, respectively. Raw reads were quality-trimmed using Trimmomatic v0.3.5 (Bolger et al., 2014). Trimmed reads were mapped to the respective reference sequence (GenBank: LT827010.1) using bowtie2 in the paired-end mode (Langmead and Salzberg, 2012), resulting in 20.7 and 19.5 million mappings. ReadXplorer was used for visualization and differential gene expression analysis (Hilker et al., 2016).

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Whole track RNAseq of the deletion mutant delta cgt in Actinoplanes sp. SE50/110 during growth phase and during transition into stationary phase

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