Abstract
During industrial fermentation, Streptomyces clavuligerus F613-1 simultaneously produces primary product clavulanic acid (CA) and cephamycin C. The cephamycin C biosynthetic gene cluster and pathway have been basically elucidated and the CcaR positive regulator was found to control the cephamycin genes expression. However, additional mechanisms of regulation cannot be excluded. The BB341_RS13780/13785 gene pair in S. clavuligerus F613-1 (annotated as SCLAV_2960/2959 in S. clavuligerus ATCC27064) encodes a bacterial two-component system (TCS) and were designated as CepRS (for cephamycin regulator/sensor). CepRS significantly affects cephamycin C production but only slightly affects CA production. To further understand the regulation of cephamycin C biosynthesis, the cepRS genes were deleted from S. clavuligerus F613-1. The deletion mutant resulted in decreased cephamycin C production but had no phenotypic effects. Real-time quantitative polymerase chain reaction analysis revealed that CepRS regulates the expression of most genes involved in cephamycin C biosynthesis, with electrophoretic mobility shift assays showing that CepR interacts with the cefD-cmcI intergenic region. These results demonstrate that the CepR response regulator serves as a transcriptional activator of cephamycin C biosynthesis, which may provide an approach for metabolic engineering methods for CA production by S. clavuligerus F613-1 in future.
Highlights
The actinomycete Streptomyces clavuligerus produces a variety of secondary metabolites, including cephamycin C (7-methoxy-3ʹ-carbamoyl-deacetyl-cephalosporin C) and clavulanic acid (CA)
Disruption of cepRS leads to a significant reduction in cephamycin C production by S. clavuligerus F613‐1 cephamycin regulator/sensor (CepRS) is one of the uncharacterized two-component systems in S. clavuligerus F613-1
While cephamycin C production was reduced for both ΔcepR and ΔcepS strains (Additional file 1: Figure S1C), suggesting that CepR and CepS are critical for cephamycin C production
Summary
The actinomycete Streptomyces clavuligerus produces a variety of secondary metabolites, including cephamycin C (7-methoxy-3ʹ-carbamoyl-deacetyl-cephalosporin C) and clavulanic acid (CA). Genes responsible for cephamycin C and CA biosynthesis are clustered in the genome, forming a β-lactam supercluster (Ward and Hodgson 1993). The cephamycin C biosynthetic gene cluster mainly includes ten genes: pcd, lat, pcbAB, pcbC, cefD, cefE, cmcI, cmcJ, cefF, and cmcH. The four genes pcd, lat, pcbAB, pcbC are involved in the early stages of cephamycin C biosynthesis. CefD and cefE are involved in the intermediate stages of cephamycin C biosynthesis, and are responsible for forming deacetylcephalosporin C. The four genes cmcI, cmcJ, cefF, and cmcH are involved in the late stages of cephamycin C biosynthesis and are responsible for the specific C-7 methoxylation and carbamoylation steps (Coque et al 1995a, b; Enguita et al 1996)
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