Identification of a novel Streptomyces chattanoogensis L10 and enhancing its natamycin production by overexpressing positive regulator ScnRII

Abstract

A novel Streptomyces strain, L10, which is capable of producing natamycin, was isolated from a soil sample collected from Zhejiang province, China. On the basis of phylogenetic analysis of rpoB gene and 16S rDNA sequences, as well as phenotypic comparison, strain L10 (CGMCC 2644) is proposed to be a previously uncharacterized strain of S. chattanoogensis. By screening a cosmid library of strain L10 and primer walking, a partial sequence of scnRI and the entire sequence of scnRII were obtained, which are orthologues to the pathway-specific positive regulator genes of natamycin biosynthesis in S. natalensis. The engineered S. chattanoogensis Dl, generated by inserting an additional copy of scnRII into the chromosome of strain L10, increased its natamycin production by 3.3 fold in YSG medium and 4.6 fold in YEME medium without sucrose.

The pleitropic regulator AdpAch is required for natamycin biosynthesis and morphological differentiation in Streptomyces chattanoogensis

“…The NTM biosynthetic gene cluster of S. chattanoogensis was identified by genomic walking using a cosmid library and DNA segments from scnRI, a pimR homologue in S. chattanoogensis L10 (Du et al, 2009). Sequencing of the inserts in the five cosmids obtained from the genomic library revealed 18 ORFs putatively responsible for NTM biosynthesis, spanning 86 kb of DNA sequence, as shown in Fig.…”

“…Primers (adpA-COF and adpA-COR) were selected using protein alignments of the previously characterized adpA genes of S. coelicolor and S. griseus. These primers were used to amplify part of the adpA ch gene and screen a cosmid library of S. chattanoogensis L10 (Du et al, 2009) by PCR. The PCR product used as a probe for Southern hybridization with the BglII-digested cosmids was prepared by PCR in the presence of biotin-11-dUTP (Fermentas).…”

“…The main cultivation was carried out for 5 days for NTM production under the same conditions as for seed culture preparation. Quantification of NTM production was performed as described previously (Du et al, 2009).…”

“…Using these primers, a single 0.4 kb DNA fragment was obtained by PCR from the genomic DNA of S. chattanoogensis. After confirmation by DNA sequencing, these primers were used to screen the cosmid library of S. chattanoogensis L10 (Du et al, 2009). Three recombinant cosmids giving the expected product were selected and analysed by Southern blotting using the 0.4 kb DNA fragment as probe.…”

“…In a previous study (Du et al, 2009), we reported a new strain of S. chattanoogensis. After optimization of media components and culture conditions, NTM production reaches a maximum of 4.5 g l 21 in shake-flask experiments (our unpublished data).…”