Simultaneous Analysis of Spatio-Temporal Gene Expression for Cephamycin Biosynthesis in Streptomyces clavuligerus

Abstract

Linkage between structural and regulatory genes implies that a direct correlation should exist between the spatio-temporal distribution of their expression. Green fluorescent protein (GFP) and cyan fluorescent protein (CFP) were used as reporters to analyze simultaneously expression of lysine-epsilon-aminotransferase (LAT) and its corresponding genetic regulator, CcaR. The isogenic strain containing lat::gfp and ccaR::cfp in the chromosome produced cephamycin C at levels similar to wild type Streptomyces clavuligerus. Confocal laser scanning microscopy revealed that expression of both LAT and CcaR in liquid culture was temporally dynamic and spatially heterogeneous in S. clavuligerus mycelia. During the early culture stage only a part of the mycelia began to express LAT and CcaR at low levels. As the culture aged, expression levels and the population of mycelia expressing LAT and CcaR increased and were followed late in the growth cycle by a reduction of the mycelia population expressing LAT and CcaR. The approach provides a precise simultaneous temporal-spatial expression profile and corroborates the regulatory linkage between ccaR and lat in S. clavuligerus.

Heterologous production of the antifungal polyketide antibiotic soraphen A of Sorangium cellulosum So ce26 in Streptomyces lividans

“…lividans chromosome for added stability, and supplying the sorR oxidase and the methoxymalonate biosynthetic operon sorCDFE on an autonomously replicating plasmid for added convenience. All these genes were placed under the control of the thiostrepton-inducible promoter PtipA (Murakami et al, 1989) to provide for regulated expression of the cluster during fermentation (Ali et al, 2002; aspects under investigation in our laboratory) that is also decoupled from the developmental regulation of secondary metabolic promoters (Kyung et al, 2001).…”