Tandem acyl carrier protein domains in polyunsaturated fatty acid synthases

Polyunsaturated fatty acids (PUFAs) can be biosynthesized via aerobic pathways that rely on combinations of desaturases and elongases to convert saturated fatty acids to PUFAs or anaerobic pathways that exploit polyketide synthase (PKS)-like enzymes known as PUFA synthases for de novo synthesis from acyl CoA precursors. In contrast to most fatty acid synthases (FASs) and PKSs that contain a single acyl carrier protein (ACP) domain for each cycle of fatty acid or polyketide chain elongation, all PUFA synthases known to date contain tandem ACPs (ranging from five to nine). The roles and engineering potential of such tandem ACPs in PUFA synthases remain largely unknown, although the growing demand for PUFAs and decline of current sources dictate that a greater understanding of these PUFA synthases is not only warranted, but urgently needed. This chapter describes methods and protocols developed to dissect the role and underlying biochemistry of each of the PfaA-ACPs in the Shewanella japonica PUFA synthase for eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA) biosynthesis. These studies have set the stage to interrogate the roles of the other domains and subunits of the Pfa PUFA synthase in EPA and DPA biosynthesis. Applications of the methods and protocols described here to other PUFA synthases are therefore envisioned to help close the knowledge gap currently limiting microbial production of PUFAs via PUFA synthase engineering and heterologous expression.