Sunday, July 24, 2011
Grand Ballroom, 5th fl (Sheraton New Orleans)
Polyunsaturated fatty acids (PUFAs) are important components of human health and nutrition as well as important ingredients in biodiesel preparations. PUFAs from deep-sea bacteria are synthesized by a modular polyketide synthase, which contains several domains including two dehydratase (DH) domains responsible for the introduction of double bonds into the final product. In order to study the mechanism and substrate specificity of DH domains, we have sought to express and purify individual protein fragments with DH activity from a deep-sea PUFA synthase enzyme complex. Protein constructs were designed using a bioinformatic tool for the prediction of protein linkers which revealed the presence of two previously uncharacterized pseudo-domains. The resulting design comprising the two putative DH domains in tandem (DH1-DH2-UMA) was expressed in E coli, purified by chromatography and assayed against surrogate substrates by UV spectroscopy. Results show that the DH1-DH2-UMA construct is more active against CoA-linked substrates than for the N-acetylcysteamine (NAC) thioester as revealed by kinetic analysis of the hydration reaction. Additionally, we have shown that E coli strains engineered to over-express DH1-DH2 can produce 3-5 times more FAs than the wild-type strain, suggesting that dehydration is rate-limiting in E coli. We anticipate this result can be implemented to drive the production of FAs in E coli and be a viable option for the production of biodiesel precursors. This work was funded by seeds funds from the Grant CHE0953254 from the National Science Foundation and MBRS-RISE Program (R25GM061838) of the UPR-MSC.