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Polyunsaturated fatty acids (PUFAs) are essential membrane components in higher eukaryotes and are important components of human health and nutrition.1,2,3 In addition, PUFAs are important ingredients in biodiesel preparations and other industrial applications.4,5 PUFAs from deep-sea bacteria are synthesized by a modular polyketide synthase, which contains several domains including two distinct dehydratase (DH) domains, DH1 and DH2, which are responsible for the introduction of cis or trans double bonds into the final product.6,7 In an effort to understand how double bonds are introduced into PUFAs we will focus our research on the activity and specificity of the DH domains. Individual proteins containing the dehydratase domains of Photobacterium Profundum will be designed in order to ensure the expression of soluble and enzymatically active DH constructs. Bioinformatic tools will be employed for the prediction of protein linkers and protein domains.8 The resulting designs comprising the putative DH domains will be cloned and expressed in Escherichia coli containing one or both DH domains. Expressed proteins will be purified by affinity and anion exchange chromatography. In order to elucidate substrate specificity, purified protein will be assayed against a variety of substrates by monitoring the consumption of substrates by UV spectroscopy and corroborated by mass spectrometry. The enzymatic mechanism of dehydration will be studied by site-directed mutagenesis and enzyme assays using a variety of surrogate substrates. Finally we will assess the effect of overexpression of DH domains on the fatty acid profile of E coli with the view of exploring the potential of these enzymes in the production of fatty acids in bacterial fermentation.
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