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:Streptococcus gallolyticus subsp. gallolyticus is a commensal of the human gastrointestinal tract and a pathogen of infective endocarditis and other biofilm-associated infections with exposed collagen. Therefore, this study focuses on the characterization of the biofilm formation and collagen adhesion of S. gallolyticus subsp. gallolyticus under different conditions. It has been observed that lysozyme triggers biofilm formation divergently in the analyzed S. gallolyticus subsp. gallolyticus strains. The transcriptome analysis was performed for two strains which form more biofilm in the presence of lysozyme. Lysozyme leads to higher expression of genes of transcription and translation, of the dlt operon (cell wall modification), of hydrogen peroxide resistance proteins and of two immunity proteins which could be involved in biofilm formation. Furthermore, the adhesion ability of 73 different S. gallolyticus subsp. gallolyticus strains to collagen type I and IV was analyzed. High adhesion ability was observed for the strain UCN 34, whereas the strain DSM 16831 adhered only marginally to collagen. The full genome microarray analysis revealed strain-dependent gene expression due to adhesion. The expression of genes of a transposon and a phage region in strain DSM 16831 were increased, which corresponds to lateral gene transfer. Adherence to collagen leads to a change in the expression of genes of nutrients uptake in the strain UCN 34.
Project description:We report the 8.5-Mb genome sequence of Amycolatopsis decaplanina strain DSM 44594(T), isolated from a soil sample from India. The draft genome of strain DSM 44594(T) consists of 8,533,276 bp with a 68.6% G+C content, 7,899 protein-coding genes, and 57 RNAs.