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