Sterane molecular fossils are broadly interpreted as eukaryotic biomarkers, although diverse bacteria also produce sterols. Steranes with side-chain methylations can act as more specific biomarkers if their sterol precursors are limited to particular eukaryotes and are absent in bacteria. One such sterane, 24-isopropylcholestane, has been attributed to demosponges and potentially represents the earliest evidence for animals on Earth, but enzymes that methylate sterols to give the 24-isopropyl side-chain remain undiscovered. Here, we show that sterol methyltransferases from both sponges and yet-uncultured bacteria function in vitro and identify three methyltransferases from symbiotic bacteria each capable of sequential methylations resulting in the 24-isopropyl sterol side-chain. We demonstrate that bacteria have the genomic capacity to synthesize side-chain alkylated sterols, and that bacterial symbionts may contribute to 24-isopropyl sterol biosynthesis in demosponges. Together, our results suggest bacteria should not be dismissed as potential contributing sources of side-chain alkylated sterane biomarkers in the rock record.

Fossilized sterols, or steranes, are broadly interpreted as biomarkers for past eukaryotic life. Steranes with side chain methylations can act as more specific eukaryotic biomarkers given the limited taxonomic distribution of their sterol precursors in extant eukaryotes. One side chain methylated sterane, 24-isopropylcholestane, has been interpreted as a biomarker for marine sponges of the Demospongiae class as they are the only organisms known to produce 24-isopropyl sterols as their major sterols. The presence of 24-isopropylcholestane in late Neoproterozoic rocks may therefore represent some of the earliest evidence for animals on Earth. However, the use of 24-isopropylcholestane as a sponge biomarker has been questioned for a variety of reasons including the potential for alternative sources and a lack of information on the biochemical mechanism behind the formation of its sterol precursors. Specifically, enzymes capable of methylating sterols to give the 24-isopropyl side chain have not been identified, and the function of putative sponge sterol 24-C-methyltransferases has not been demonstrated experimentally. In this study, we verified the functionality of sponge sterol methyltransferases and explored the potential for bacterial sterol side chain methylation through in vitro protein expression and lipid analyses. We identified three bacterial sterol methyltransferases each capable of sequential methylations resulting in the 24-isopropyl side chain structure of the 24-isopropylcholestane biomarker. Two of these side chain propylating enzymes were identified in a metagenome from a demosponge microbiome, suggesting bacterial symbionts may play a role in 24-isopropyl sterol biosynthesis in the demosponge holobiont.Significance StatementAccording to the sponge biomarker hypothesis, geologic 24-isopropylcholestane can act as an indicator for ancient demosponges and may represent some of the first evidence for animal life on Earth. However, enzymes capable of methylating sterols to give the 24-isopropyl sterol side chain have not been identified. We therefore investigated the functionality of sponge sterol methyltransferases, which alkylate the sterol side chain, through in vitro protein expression. We also analyzed the function of several bacterial sterol methyltransferases, three of which produced 24-isopropyl sterols. Our results indicate yet-uncultured bacteria, including demosponge symbionts, have the genomic capacity to synthesize side chain alkylated sterols and should therefore be considered when interpreting ergostane, stigmastane, and 24-isopropylcholestane biomarkers in the geologic record.