Determination of the extent of phosphopantetheinylation of polyketide synthases expressed in Escherichia coli and Saccharomyces cerevisiae

• Published in: Analytical biochemistry Vol. 394; no. 1; pp. 75 - 80
• Main Authors: Lee, Ka Kit Michael, Silva, Nancy A. Da, Kealey, James T.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.2009
• Subjects: 6-Deoxyerythronolide B synthase; Acyl Carrier Protein - metabolism; Acyltransferases - metabolism; Bacterial Proteins - metabolism; Biocatalysis; Escherichia coli - genetics; Escherichia coli - metabolism; Fluorescent Dyes - metabolism; Gene Expression; Heterologous expression; Ligases - metabolism; Lovastatin - metabolism; Lovastatin polyketide synthase; Macrolides - metabolism; Multienzyme Complexes - metabolism; Oxidoreductases - metabolism; Pantetheine - analogs & derivatives; Pantetheine - metabolism; Phosphopantetheinylation; Polyketide synthase; Polyketide Synthases - biosynthesis; Polyketide Synthases - chemistry; Polyketide Synthases - genetics; Polyketide Synthases - metabolism; Protein Structure, Tertiary; Saccharomyces cerevisiae - genetics; Transferases (Other Substituted Phosphate Groups) - metabolism; Polyketide synthase; Lovastatin polyketide synthase; 6-Deoxyerythronolide B synthase; Phosphopantetheinylation; Heterologous expression
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2009.07.010

A sensitive fluorescent assay was developed to measure the extent of phosphopantetheinylation of polyketide synthase (PKS) acyl carrier protein (ACP) domains in polyketide production strains. The in vitro assay measures PKS fluorescence after transfer of fluorescently labeled phosphopantetheine from coenzyme A to PKS ACP domains in crude protein extracts. The assay was used to determine the extent of phosphopantetheinylation of ACP domains of the erythromycin precursor polyketide synthase, 6-deoxyerythronolide B synthase (DEBS), expressed in a heterologous Escherichia coli polyketide production strain. The data showed that greater than 99.9% of DEBS is phosphopantetheinylated. The assay was also used to interrogate the extent of phosphopantetheinylation of the lovastatin nonaketide synthase (LNKS) heterologously expressed in Saccharomyces cerevisiae. The data showed that LNKS was efficiently phosphopantetheinylated in S. cerevisiae and that lack of production of the lovastatin precursor polyketide was not due to insufficient phosphopantetheinylation of the expressed synthase.