A geranylgeranyl reductase homolog required for cholesterol production in Myxococcota

Myxococcota is a phylum of sterol producing bacteria. They exhibit a clade depth for sterol biosynthesis unparalleled in the bacterial domain and produce sterols of a biosynthetic complexity that rivals eukaryotes. Additionally, the sterol biosynthesis pathways found in this phylum have been proposed as a potential source for sterol biosynthesis in the last eukaryotic common ancestor, lending an evolutionary importance to our understanding of this pathway in Myxococcota. However, sterol production has only been characterized in a few species and outstanding questions about the evolutionary history of this pathway remain. Here, we identify two myxobacteria, Minicystis rosea and Sandaracinus amylolyticus , capable of cholesterol biosynthesis. These two myxobacteria possess a cholesterol biosynthesis pathway that differs in both the ordering and enzymes involved in biosynthesis compared to Enhygromyxa salina , a myxobacterium previously demonstrated to produce cholesterol, as well as the canonical pathways found in eukaryotes. We characterize an alternative bacterial reductase responsible for performing C-24 reduction, further delineating bacterial cholesterol production from eukaryotes. Finally, we examine the distribution and phylogenetic relationships of sterol biosynthesis proteins across both cultured and uncultured Myxococcota species, providing evidence for multiple acquisition events and instances of both horizontal and vertical transfer at the family level. Altogether, this work further demonstrates the capacity of myxobacteria to synthesize eukaryotic sterols but with an underlying diversity in the biochemical reactions which govern sterol synthesis, suggesting a complex evolutionary history and refining our understanding of how myxobacterial cholesterol production relates to their eukaryotic counterparts.