(129b) Illuminating Novel Terpenoid Biosynthetic Pathways in Yarrowia Lipolytica through Metabolomics

Yarrowia lipolytica is an emerging microbial host for the bioconversion of low-value carbon into natural products, but its endogenous terpenoid metabolism has yet to be fully mapped. Here we aimed to illuminate novel terpenoid biosynthetic pathways by employing metabolomics, isotope tracing, and genetic engineering. We engineered a strain to push increased carbon flux through the mevalonate pathway and to farnesyl pyrophosphate (FPP) by overexpression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and FPP synthase (FPPS). Overexpression of HMGR and FPPS led to a 150-fold increase in mevalonate production and a 1.5-fold increase in isopentenyl diphosphate (IPP) production after one day of growth, indicative of increased metabolic activity through the mevalonate pathway and terpenoid metabolism. However, we observed a lower amount of IPP during the second and third days, suggesting the activation of secondary metabolism and prompting us to investigate how the isoprenoid backbone was being utilized. Upon untargeted metabolomic analysis using liquid chromatography-mass spectrometry (LC-MS), we discovered several new metabolites being produced in the engineered strain but absent in the wild-type strain. Based on measured monoisotopic mass-to-charge ratios and proposed molecular formulas, we hypothesized that these molecules were oxygenated terpenoids. After compound purification and nuclear magnetic resonance (NMR) spectroscopy, we confirmed that these compounds were terpenoids, with a linear isoprenoid backbone of various lengths and bifunctionalized with carboxylic acids. To our knowledge, this is the first observed biosynthesis of such diacid compounds. To map the novel terpenoid biosynthetic pathway, we reconstituted the putative enzymatic steps in Saccharomyces cerevisiae and successfully conferred full biosynthetic capabilities. Heterologous stepwise expression of these putative enzymes further allowed for identification of biosynthetic intermediates. Notably, a P450 enzyme previously shown to be involved in alkane assimilation was responsible for the hydroxylation of the allylic C-H bond, demonstrating the multifunctionality of involved enzymes for size, desaturation, and branching of permissible substrates. Lastly, isotope tracing and direct diacid feeding were utilized to elucidate potential degradation products to investigate the biological utility of these terpenoid diacids. Our work thus demonstrates the utility of increasing precursor availability to activate untapped metabolic pathways for the discovery of new natural products and novel enzyme promiscuity. Furthermore, the new compounds and their biosynthetic intermediates represent an exciting pool of organic building blocks that can be accessed for renewable fuels, bio-derived polymers, and natural product synthesis.