POSTTRANSLATIONAL 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 expressi on 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 gro wth conditions led to an increase of production of active holo-protein for the actinorhodin (act) ACP, but this technique was ineffective fo r oxytetracycline (etc) and griseusin (gris) ACPs. Labelling experimen ts revealed that a low level of etc ACP expressed prior to induction w as produced mainly as active holo-protein, while post-induction N-15-l abelled protein was almost exclusively in the apo-ACP form. Limiting e ndogenous holo-acyl carrier protein synthase (ACPS) concentration was implicated as responsible for low apo-ACP to holo-ACP conversion, rath er than limiting substrate (coenzyme A) and cofactor (Mg2+) concentrat ions. Co-expression of act and gris ACPs with ACPS in E. cell led to h igh levels of production of active holo-ACPs and ACPS. We have also ma de the significant observation that ACPS is able to transfer acylated CoA moieties to act apo-ACP. (C) 1997 Federation of European Biochemic al Societies.