WILLIAMS, JAYME, M.S. Overexpression, Purification, and Characterization of AcpK, PksG, and MmgA from Bacillus subtilis Strain 168. (2007)

Polyketides are a diverse class of natural products that have varying therapeutic values. Polyketide synthases (PKSs) use different domains within the larger enzyme to vary the amount of reduction seen at each ketide position. The ability to manipulate PKSs would make the synthesis of unnatural polyketides possible, potentially creating novel biologically active molecules. Bacillus subtilis strains 39320 and 39374 produce the polyketide antibiotic difficidin, which has a subunit that can not be explained by well-understood polyketide synthesis. Studies of the synthesis of difficidin may be useful for diversifying the synthesis of new unnatural polyketides. Sfp, a phosphopantetheinyl transferase (PPTase) activates ACPs. AcpK carries an acyl unit to react with PksG, an enzyme that synthesizes an intermediate that may lead to the unusual subunit. An enzyme expressed during sporulation, MmgA, is homologous to acetyl coenzyme A acetyltransferases. AcpK, PksG, and MmgA were successfully cloned, overexpressed, and purified from B. subtilis strain 168. Sfp was successfully cloned, overexpressed, and purified from a donated overexpression vector. PPT reactions with Sfp and AcpK were successfully performed. Biochemical characterization of PksG was carried out and defined by other unrelated research. MmgA was biochemically determined to be a β-keto acyl thiolase. OVEREXPRESSION, PURIFICATION, AND CHARACTERIZATIO