First Research Project
Polyketides are pharmacologically active, natural products biosynthesized by some soil bacteria using large, multI modular enzymes called polyketide synthase (PKS). A module of a PKS is made up of ketosynthase (KS), acyltransferase (AT), acyl carrier protein (ACP) - all of which are responsible for a single round of two carbon chain elongation- ketoreductase (KR), dehydratase (DH) and enoylreductase (ER). KR, ER and DH are responsible for functionalizing the growing polyketide chain. KR converts the β-carbonyl group on the polyketide chain to a hydroxy group, DH dehydrates the hydroxy product of KR activity into an olefin whiles ER reduces the olefinic DH product to a saturated group. By controlling the activity of these functionalization domains (i.e. KR, DH, ER), it is possible to produce a wide variety of polyketides distinct from those produced by the natural bacteria and hence increase the repertoire of compounds available for rational drug design.

As a proof of principle, a simple PKS module comprising all six domains is being used to biosynthesize four different δ-lactones. In one enzyme, all six domains are present and active. In the other three mutants, one of KR, DH or ER has been deactivated via active site mutagenesis. This simple PKS module utilizes 3-hydroxy-valeryl coenzyme-A as starter unit, malonyl coenzyme-A as extender unit and NADPH as cofactor for both KR and ER. The N-acetyl cysteamine thioester of 3-hydroxy valeric acid is used as an analog of 3-hydroxy valeryl coenzyme-A. This thioester was synthesized from methyl-3-hydroxy valerate by first hydrolyzing the ester to the acid followed by coupling to N-acetyl cysteamine. An assay has been developed for the wild type enzyme and 4-ethyl-δ-lactone was detected by GC/MS as the product. Currently, I am working on the biosynthesis of the other δ-lactones using the mutant enzymes.

Second Research Project
6-deoxyerythronolide B (6-dEB), the precursor of the antibiotic erythromycin, is biosynthesized by the enzyme 6-deoxyerythronolide B synthase (DEBS). DEBS requires a single starter unit (propionyl coenzyme A) and six extender units (methyl malonyl coenzyme) for the total biosynthesis of 6-dEB. The extender unit is thus amplified in the final 6-dEB product. Both the starter and extender units are also produced by the action of propionyl coenzyme A carboxylase (PCC) on propionic acid. It is therefore possible to place a tag on the propionic acid and this tag will be amplified in the final 6-dEB product.

To this end the propionic acid is tagged with fluorine. (The fluoropropionic acid was synthesized by the Jones oxidation of 3-flouropropanol) The 6-dEB product will thus be heavily fluorinated. The fluorine tag provides a
facile means for the isolation of the fluoro-6-dEB via fluorous extraction. Successful tagging of 6-dEB will be useful in the detection of methyl malonate derived metabolites in any bacteria. Fermentation of the bacteria in question with fluorinated propionic acid (in a bacteria which utilizes PCC) or fluoro methyl malonyl coenzyme-A (in a bacteria which lacks PCC) will produce fluorinated metabolites which can be isolated using the fluorous extraction technique.

I have been able to optimize the fermentation condition required for 6-dEB production by an engineered E. coli strain transformed with DEBS. These same conditions have also been used to produce the flouro-6-dEB. 6-dEB has been characterized by LC-MS. I am now working on developing a satisfactory method using the same technique to characterize my flouro-6-dEB.