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Expression of a Tetradomain Fragment from a Polyunsaturated Fatty Acid Synthase with Dehydratase Activity
Author(s) -
Oyola-Robles Delise J,
Rodriguez-Guilbe Maria M,
Bermudez Mei-Ling,
Rivera-Diaz Monica,
Carballeira Nestor M,
Baerga-Ortiz Abel
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.756.19
Subject(s) - biochemistry , polyunsaturated fatty acid , fatty acid synthase , dehydratase , chemistry , atp synthase , enzyme , protein engineering , polyketide synthase , biosynthesis , escherichia coli , biology , fatty acid , polyketide , gene
Polyunsaturated fatty acids (PUFAs) are important components of human health and 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. In order to study double bond formation in PUFAs, we have expressed and purified the individual protein fragments from a deep‐sea PUFA synthase. 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 two putative DH domains in tandem was expressed in E coli , purified by chromatography and assayed against surrogate substrates by UV spectroscopy. Results showed that the designed construct is more active against CoA‐linked substrates than for N‐acetylcysteamine thioester, as revealed by reaction kinetics. Additionally, E coli strains engineered to over‐express DH1‐DH2 can produce 3–5 times more FAs than the wild‐type strain, suggesting that dehydration may be rate‐limiting. We anticipate this result can be implemented to drive the production of FAs in bacteria and be a viable option for the production of biodiesel precursors. This work was funded by Grant CHE0953254 from the NSF and MBRS‐RISE Program (R25GM061838) of the UPR‐MSC.