Introduction
Yarrowia lipolytica is an industrial oleaginous yeast that has been extensively engineered to synthesize lipophilic compounds, including lipids (Qiao, Wasylenko, Zhou, Xu, & Stephanopoulos, 2017), oleochemicals (P. Xu, Qiao, Ahn, & Stephanopoulos, 2016), carotenoids (Gao et al., 2017; Macarena Larroude et al., 2018), terpenoids (Jin, Zhang, Song, & Cao, 2019) and aromatic polyketides (Lv, Marsafari, Koffas, Zhou, & Xu, 2019) et al. The lipogeneity of this yeast makes it a superior host to produce chemicals that are derived from acetyl-CoA, malonyl-CoA, HMG-CoA and NADPHs. The compartmentalization of oil droplets into lipid bodies provides a hydrophobic environment to sequestrate many lipid-related compounds and mitigate the toxicity issues associated with lipophilic membrane damages. In addition, the ease of genetic manipulation, substrate flexibility and robust growth present us tremendous opportunity to upgrade low-value renewable feedstocks to high-value compounds. It has also been recognized as a ‘generally regarded as safe’ (GRAS) organism (Groenewald et al., 2014) in the food and nutraceutical industry. A large collection of customized genetic toolboxes, including YaliBricks gene assembly (Wong, Engel, Jin, Holdridge, & Xu, 2017), CRISPR-Cas9 (Bae, Park, Kim, & Hahn, 2020; Macarena Larroude, Trabelsi, Nicaud, & Rossignol, 2020) or CRISPR-Cpf1 (Yang, Edwards, & Xu, 2020) genome editing, Cre-LoxP-based iterative chromosomal integrations (Lv, Edwards, Zhou, & Xu, 2019), transposon-based mutagenesis (Wagner, Williams, & Alper, 2018) and Golden-gate cloning (Celińska et al., 2017; Egermeier, Sauer, & Marx, 2019; M. Larroude et al., 2019), enabled us to rapidly modify its genome and evaluate many metabolic events to explore the catalytic diversity of this yeast beyond its regular portfolio of fatty acids, fatty alcohols, biofuels et al . Recent metabolic engineering effort in this yeast has allowed us to access more specialized secondary metabolites with pharmaceutical values, including sesquiterpenes (Marsafari & Xu, 2020), triterpenoids (Jin et al., 2019) and flavonoids (Lv, Marsafari, et al., 2019; Palmer, Miller, Nguyen, & Alper, 2020) et al .
Isoprenoids are a large group of natural products with diverse biological functions. An estimation of more than 70,000 isoprenoids, ranging from monoterpenes, sesquiterpenes, diterpenes and triterpenes have been discovered from nature (Moser & Pichler, 2019). Isoprenoids play a major role in maintaining membrane homeostasis, protein prenylation for subcellular targeting (Palsuledesai & Distefano, 2015), signal transduction, the deployment of plant defense pathways, and controlling the transcriptional activity of sterol-responsive-element-binding-proteins (SREBPs) (Shimano, 2001). The yeast-based mevalonate (MVA) pathway starts with acetyl-CoA condensation reactions, proceeds through the reduction of intermediate HMG-CoAvia HMG-CoA reductase, which is the rate-limiting step and the molecular target to design many statins-related anti-cholesterol drugs (Xie & Tang, 2007). The universal five-carbon precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), derived from mevalonate, are condensed to make the farnesyl pyrophosphate (FPP), which later can be diversified to many sesquiterpenes or triterpenes. Squalene is a 30-carbon triterpene hydrocarbon synthesized from the condensation of two FPPs, which serve as the gateway molecule for all triterpenoids with tens of thousands of structural variations. Squalene possess strong antioxidant and anti-inflammatory activity and is widely used in the cosmetic industry as skin-compatible super-lubricant and hydration protectors (Spanova & Daum, 2011). Squalene emulsions were used as efficient adjuvants to enhance the immune response of certain vaccines (Spanova & Daum, 2011). Squalene is primarily sourced from shark liver, which poses significant ecological or ethical concerns related with shark-hunting. Reconstitution of squalene pathway in microbes may provide an alternative route to sustainably produce squalene from renewable feedstocks. A number of metabolic engineering studies have set the effort to engineer bacteria or bakers’ yeast to produce squalene, with improved yield and process efficiency. For example, a recent work identified that yeast peroxisome may serves as a dynamic depot to store squalene up to 350 mg/g dry cell weight (G.-S. Liu et al., 2020), despite the highly oxidative nature of peroxisome. In this work, we report the systematic optimization and engineering of the endogenous mevalonate pathway in Yarrowia lipolytica for efficient synthesis of squalene from simple synthetic media. We identified the bottlenecks of the mevalonate pathway and discovered alternative reducing equivalents (NADPH) pathways to improve squalene production. Our engineered strain produced up to 502.7 mg/L of squalene in shake flask. This work may set a foundation for us to explore oleaginous yeast as a chassis for cost-efficient production of squalene and triterpenoids in a long-term run.
Methods and Materials