The algal homolog of the plant <scp>CER1</scp> and <scp>CER3</scp> proteins is a bifunctional hydrocarbon‐forming enzyme

The ability to synthesize and secrete hydrophobic compounds is believed to have been a pivotal event in the evolution of land plants from their aquatic green algal ancestors. The key to biosynthesis of plant surface alkanes is a heterodimeric complex consisting of two homologous membrane-bound proteins, ECERIFERUM 3 (CER3) and ECERIFERUM 1 (CER1), which bear distinct enzyme activities. A single homolog of CER1 and CER3, referred to as CER1/3, has long been identified in some algae. However, it has remained unknown whether CER1/3 exhibits CER1 and/or CER3 activity or another ancestral activity. Here we investigate the function of CER1/3 by using CRISPR-Cas9-mediated knockout mutants in the early-branching chlorophyte Ostreococcus tauri and by yeast heterologous expression. Genome mining shows that in green algal genomes the presence of CER1/3 is correlated with the absence of fatty acid photodecarboxylase. Knockouts provide evidence that CER1/3 is necessary for synthesizing a C21:6 alkene in Ostreococcus. Yeast expression experiments demonstrate that algal CER1/3 are bifunctional enzymes with aldehyde- and hydrocarbon-forming domains, corresponding to CER3 and CER1 activities, respectively. These findings support the idea that the land plant alkane-forming CER1/CER3 complex evolved from a bifunctional hydrocarbon-forming CER1/3 enzyme found in some of the earliest-diverging green algal lineages.