Project description:We reported transcriptome profiles of rifamycin SV-produced strain Amycolatopsis mediterranei U32 in the Bennet medium with or without 80 mM nitrate in different cultivation stage. By comparative analysis of genome-wide gene expression in these conditions, we found that the mechanism of high production of rifamycin SV induced by nitrate could be elucidated.
Project description:Amycolatopsis is an important source of diverse valuable bioactive natural products. The CRISPR/Cas-mediated gene editing tool has been established in some Amycolatopsis species and has accomplished the deletion of single gene or two genes. The goal of this study was to develop a high-efficient CRISPR/Cas9-mediated genome editing system in vancomycin-producing strain A. keratiniphila HCCB10007 and enhance the production of vancomycin by deleting the large fragments of ECO-0501 BGC. By adopting the promoters of gapdhp and ermE*p which drove the expressions of scocas9 and sgRNA, respectively, the all-in-one editing plasmid by homology-directed repair (HDR) precisely deleted the single gene gtfD and inserted the gene eGFP with the efficiency of 100%. Furthermore, The CRISPR/Cas9-mediated editing system successfully deleted the large fragments of cds13-17 (7.7 kb), cds23 (12.7 kb) and cds22-23 (21.2 kb) in ECO-0501 biosynthetic gene cluster (BGC) with high efficiencies of 81%-97% by selecting the sgRNAs with a suitable PAM sequence. Finally, a larger fragment of cds4-27 (87.5 kb) in ECO-0501 BGC was deleted by a dual-sgRNA strategy. The deletion of the ECO-0501 BGCs revealed a noticeable improvement of vancomycin production, and the mutants, which were deleted the ECO-0501 BGCs of cds13-17, cds22-23 and cds4-27, all achieved a 30%-40% increase in vancomycin yield. Therefore, the successful construction of the CRISPR/Cas9-mediated genome editing system and its application in large fragment deletion in A. keratiniphila HCCB10007 might provide a powerful tool for other Amycolatopsis species.
Project description:We reported transcriptome profiles of rifamycin SV-produced strain Amycolatopsis mediterranei U32 in the Bennet medium with or without 80 mM nitrate in different cultivation stage. By comparative analysis of genome-wide gene expression in these conditions, we found that the mechanism of high production of rifamycin SV induced by nitrate could be elucidated. Examination of 2 time-course transcription in 2 different nitrogen source conditions
Project description:Amycolatopsis sp. BX17 is an actinobacterium isolated from milpa soils that antagonizes the phytopathogenic fungus Fusarium graminearum. Metabolites secreted by the actinobacterium cultured in medium without glucose inhibited 100% the mycelial growth of F. graminearum RH1, while in medium supplemented with 20 g/L of glucose inhibition was 65%. With the aim of studying how the metabolism of strain BX17 is modulated by glucose, as the main carbon source, media with 0 and 20 g/L glucose were selected to analyze the intracellular proteins by quantitative label-free proteomic analysis.