ALGAL HOMOLOGS OF THE PLANT CER1 AND CER3 PROTEINS ARE FUNCTIONAL HYDROCARBON–FORMING ENZYMES

In plants, very-long-chain (VLC) alkanes (C25-C35) are secreted onto the epidermal surface of aerial organs, often forming the primary component of the waterproof cuticular wax layer. This secretion plays a vital role in preventing desiccation in terrestrial environments. The key to VLC alkane biosynthesis in plants lies in the complex formed by two homologous membrane-bound proteins, ECERIFERUM 3 (CER3) and ECERIFERUM 1 (CER1). This complex transforms an acyl-CoA substrate into an aldehyde intermediate, which is then converted into an alkane. The ability to synthesize and secrete alkanes is believed to have been a pivotal event in the evolution of land plants from their green algal ancestors. Interestingly, a single homolog of CER1 and CER3, known as CER1/3, has been identified in certain algae. However, the functionality of this protein remains to be investigated. In this study, we present the functional characterization in yeast of CER1/3 proteins from various algal species belonging to the green lineage. We demonstrate that CER1/3 proteins alone can efficiently mediate hydrocarbon biosynthesis. Furthermore, we demonstrate that point mutations in conserved motifs in the N- or C-terminal domains of CER1/3 lead to impaired hydrocarbon biosynthesis. Additionally, we show that coexpressing plant CER3 with algal CER1/3 results in longer alkanes being formed in yeast. Together, these findings support the hypothesis that the alkane-forming CER1/CER3 complex found in land plants evolved from a green algal CER1/3 bifunctional enzyme through a process of gene duplication followed by protein specialization.