Functional Analysis of Three Dehydratase Domains, DH _PKS , DH1 _FabA , and DH2 _FabA , in Microalgal Docosahexaenoic Acid Synthase

Schizochytrium sp., a eukaryotic microalga, biosynthesizes docosahexaenoic acid (DHA; C22:6 ω 3) by DHA synthase, which is composed of three subunits (OrfA to C) possessing multiple catalytic domains. The enzyme has three dehydratase (DH) domains, DH PKS in OrfA and tandem DH FabA (DH1 FabA and DH2 FabA ) domains in OrfC. In this study, the function of each of the DH domains was investigated by in vivo heterologous expression experiments in Escherichia coli and in vitro studies with recombinant truncated OrfA containing DH PKS and full‐length OrfC, in which one of the tandem DH FabA domains was inactivated by site‐directed mutagenesis, and acyl–acyl carrier protein (ACP) intermediates. Following heterologous expression, the DH1 FabA ‐ or DH2 FabA ‐inactivated enzyme produced no DHA, revealing that both domains are essential, while the DH PKS ‐inactivated enzyme showed significantly reduced DHA productivity, confirming that DH PKS is required for full production of DHA. Moreover, in vitro experiments showed that the C4‐ACP substrate was accepted by all of the DHs, but DH PKS and DH2 FabA showed higher activities than DH1 FabA . DH1 FabA catalyzed two types of reactions: (de)hydrations of C10‐, C16‐, and C22‐ACP substrates and (de)hydrations/2‐ trans to 2‐ cis isomerization of C8‐, C14‐, and C20‐ACP substrates. By contrast, DH2 FabA catalyzed (de)hydrations/2‐ trans to 3‐ cis isomerization of C6‐, C12‐, and C18‐ACP substrates.