Illuminating Novel Terpenoid Biosynthesis Pathways in Yarrowia lipolytica by Metabolomics
Authors:
Glenn Nurwono* ([email protected]), Zuodong Sun, Edward Hu, Yi Tang, Junyoung O. Park (PI)
Institutions:
University of California–Los Angeles
Abstract
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, this research group aimed to illuminate novel terpenoid biosynthetic pathways and quantify metabolic flux and free energy therein by employing metabolomics, isotope tracing, and genetic engineering. The group 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, the group observed a lower amount of IPP during the second and third days, suggesting the activation of secondary metabolism and prompting an investigation how the isoprenoid backbone was being utilized.
Upon untargeted metabolomic analysis using liquid chromatography-mass spectrometry (LC-MS), researchers 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, the group hypothesized that these molecules were oxygenated terpenoids. After compound purification and nuclear magnetic resonance (NMR) spectroscopy, the group confirmed that these compounds were terpenoids, with a farnesyl scaffold and bifunctionalized with carboxylic acids. To the group’s knowledge, this is the first observed biosynthesis of such diacid compounds. To map the novel terpenoid biosynthetic pathway, the group reconstituted the putative enzymatic steps in Saccharomyces cerevisiae and successfully conferred full biosynthetic capabilities.
Furthermore, isotope tracing and direct farnesol feeding were utilized to elucidate biosynthetic intermediates. Notably, a P450 enzyme previously shown to be involved in alkane assimilation was responsible for the hydroxylation of the allylic carbon-hydrogen bond, demonstrating the substrate promiscuity and multifunctionality of involved enzymes. This work demonstrates the utility of increasing precursor availability to activate untapped metabolic pathways for the discovery of new natural products. Furthermore, the new compounds and their biosynthetic intermediates represent an exciting pool of organic building blocks that can be accessed for renewable fuel, polymer, and natural product synthesis.