Post-translational modification of heterologously expressed Streptomyces type II polyketide synthase acyl carrier proteins

Expression in Escherichia cell of Streptomyces acyl carrier proteins (ACPs) associated with polyketide biosynthesis using the pT7-7 expression system of Tabor and Richardson led to the production predominantly of inactive apo-proteins lacking the 4'-phosphopantetheinyl prosthetic group essential for polyketide synthase activity. Modification of growth conditions led to an increase of production of active holo-protein for the actinorhodin (act) ACP, but this technique was ineffective for oxytetracycline (etc) and griseusin (gris) ACPs. Labelling experiments revealed that a low level of etc ACP expressed prior to induction was produced mainly as active holo-protein, while post-induction N-15-labelled protein was almost exclusively in the apo-ACP form. Limiting endogenous holo-acyl carrier protein synthase (ACPS) concentration was implicated as responsible for low apo-ACP to holo-ACP conversion, rather than limiting substrate (coenzyme A) and cofactor (Mg2+) concentrations. Co-expression of act and gris ACPs with ACPS in E. cell led to high levels of production of active holo-ACPs and ACPS. We have also made the significant observation that ACPS is able to transfer acylated CoA moieties to act apo-ACP. (C) 1997 Federation of European Biochemical Societies.