Hereby we investigated the role of sgcR3 in C-1027 biosynthesis, and provided an initial understanding of pathway-specific regulatory network of sgcR1, sgcR2 and sgcR3 CHIR-99021 manufacturer in S. globisporus C-1027. OSI-027 in vivo Results Overexpression of sgcR3 increased the production of C-1027 Computer-assisted analysis
of the sgcR3 gene product (395 aa) showed a high sequence similarity (33% identities and 47% positives) within the whole length of protein TylR of S. fradiae (Fig. 2B), a pathway-specific global activator of tyl cluster [20, 23]. To investigate the function of sgcR3, the expression plasmid of sgcR3 associated with its native promoter, named pKCR3 (see Methods), was constructed based on the multi-copy pKC1139  and then introduced into S. globisporus C-1027 by conjugation. Thereafter, the resultant sgcR3 overexpression strains were fermented by incubation in liquid medium FMC-1027-1 (see Methods). The antibacterial bioassay against Bacillus subtilis CMCC(B) 63501 (data not shown) and the HPLC analysis indicated that the pKCR3 led to a 30–40% increase in C-1027 production (Fig. 3c)
in comparison to that in wild type strain (Fig. 3b), whereas C-1027 production level detected in the wild type strain with the parental vector pKC1139 had no difference. Therefore, the result suggested that the function of sgcR3 could be positive for C-1027 biosynthesis in selleck inhibitor S. globisporus C-1027. Figure 3 Determination of C-1027 production in sgcR3 overexpression strain and disruption strain R3KO. HPLC analysis of C-1027 chromophore standard (a), C-1027 produced by wild type strain (b), one of sgcR3 overexpression strains (c) and R3KO mutant (d) are shown. Inactivation and complementation of sgcR3 In order to ascertain the contribution of sgcR3 to
the regulation of C-1027 biosynthesis, a part of coding region of sgcR3 (507 bp) was replaced Digestive enzyme with a thiostrepton resistant gene (tsr) to create the sgcR3 disrupted strain S. globisporus R3KO (Fig. 4A). Successful disruption of the intended target was confirmed by PCR using primers complementary to one end of tsr and to untouched DNA outside the disruption constructs (data not shown). Southern blot analyses authenticated the site-specific disruptions of sgcR3 using left arm for crossover and deleted part of sgcR3 gene as probes respectively (Fig. 4B, 4C). The antibacterial bioassay against B. subtilis (Fig. 4D, b) and HPLC analysis (Fig. 3d) showed that disruption of sgcR3 completely abolished C-1027 production. Figure 4 Inactivation and complementation of sgcR3. A, The plasmid pOJR3KO, constructed for sgcR3 inactivation as described in Methods, was used for gene disruption. Predicted restriction enzyme polymorphism caused by gene replacement is shown. B, BamHI; Bc, BclI; E, EcoRV. B, Southern blot hybridization of BamHI-digested chromosomal DNA of wild type strain (lane 1) and R3KO mutant (lane 2). Left arm for crossover is used as hybridization probe.