Ross Zirkle

The Role of Tandem Acyl Carrier Protein Domains in Polyunsaturated Fatty Acid Biosynthesis

Acyl carrier protein (ACP) plays an essential role in fatty acid and polyketide biosynthesis, and most of the fatty acid synthases (FASs) and polyketide synthases (PKSs) known to date are characterized with a single ACP for each cycle of chain elongation. Polyunsaturated fatty acid (PUFA) biosynthesis is catalyzed by the PUFA synthase, and all PUFA synthases known to date contain tandem ACPs (ranging from 5 to 9). Using the Pfa PUFA synthase from Shewanella japonica as a model system, we report here that these tandem ACPs are functionally equivalent regardless of their physical location within the PUFA synthase subunit, but the total number of ACPs controls the overall PUFA titer. These findings set the stage to interrogate other domains and subunits of PUFA synthase for their roles in controlling the final PUFA products and could potentially be exploited to improve PUFA production.

Biochemical characterization of polyunsaturated fatty acid synthesis in Schizochytrium: Release of the products as free fatty acids

In marine bacteria and some thraustochytrids (marine stramenopiles) long-chain polyunsaturated fatty acids (LC-PUFAs) such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are produced de novo by PUFA synthases. These large, multi-domain enzymes carry out the multitude of individual reactions required for conversion of malonyl-CoA to the final LC-PUFA products. Here we report on the release of fatty acids from the PUFA synthase found in Schizochytrium, a thraustochytrid that has been developed as a commercial source for DHA-enriched biomass and oil. Data from in vitro activity assays indicate that the PUFAs are released from the enzyme as free fatty acids (FFAs). Addition of ATP and Mg(2+) to in vitro assays facilitates appearance of radiolabel from (14)C-malonyl-CoA in a triacylglycerol fraction, suggesting the involvement of acyl-CoA synthetases (ACS). Furthermore, addition of triascin C, an inhibitor of ACSs, to the assays blocks this conversion. When the Schizochytrium PUFA synthase is expressed in Escherichia coli, the products of the enzyme accumulate as FFAs, suggesting that the thioesterase activity required for fatty acid release is an integral part of the PUFA synthase.

Canola engineered with a microalgal polyketide synthase-like system produces oil enriched in docosahexaenoic acid

Dietary omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), docosahexaenoic acid (DHA, C22:6) and eicosapentaenoic acid (EPA, C20:5) are usually derived from marine fish. Although production of both EPA and DHA has been engineered into land plants, including Arabidopsis, Camelina sativa and Brassica juncea, neither has been produced in commercially relevant amounts in a widely grown crop. We report expression of a microalgal polyketide synthase-like PUFA synthase system, comprising three multidomain polypeptides and an accessory enzyme, in canola (Brassica napus) seeds. This transgenic enzyme system is expressed in the cytoplasm, and synthesizes DHA and EPA de novo from malonyl-CoA without substantially altering plastidial fatty acid production. Furthermore, there is no significant impact of DHA and EPA production on seed yield in either the greenhouse or the field. Canola oil processed from field-grown grain contains 3.7% DHA and 0.7% EPA, and can provide more than 600 mg of omega-3 LC-PUFAs in a 14 g serving.

Vaccination against Influenza with Recombinant Hemagglutinin Expressed by Schizochytrium sp. Confers Protective Immunity

For the rapid production of influenza vaccine antigens in unlimited quantities, a transition from conventional egg-based production to cell-based and recombinant systems is required. The need for higher-yield, lower-cost, and faster production processes is critical to provide adequate supplies of influenza vaccine to counter global pandemic threats. In this study, recombinant hemagglutinin proteins of influenza virus were expressed in the microalga Schizochytrium sp., an established, fermentable organism grown in large scale for the manufacture of polyunsaturated fatty acids for animal and human health applications. Schizochytrium was capable of exporting the full-length membrane-bound proteins in a secreted form suitable for vaccine formulation. One recombinant hemagglutinin (rHA) protein derived from A/Puerto Rico/8/34 (H1N1) influenza virus was evaluated as a vaccine in a murine challenge model. Protective immunity from lethal challenge with homologous virus was elicited by a single dose of 1.7, 5 or 15 µg rHA with or without adjuvant at survival rates between 80–100%. Full protection (100%) was established at all dose levels with or without adjuvant when mice were given a second vaccination. These data demonstrate the potential of Schizochytrium sp. as a platform for the production of recombinant antigens useful for vaccination against influenza.