Journal
ANALYTICAL BIOCHEMISTRY
Volume 394, Issue 1, Pages 75-80Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2009.07.010
Keywords
Phosphopantetheinylation; Polyketide synthase; Heterologous expression; 6-Deoxyerythronolide B synthase; Lovastatin polyketide synthase
Funding
- National Science Foundation [BES-0432307]
- Kosan Biosciences
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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. (C) 2009 Elsevier Inc. All rights reserved.
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