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Abstract
Squalene is the precursor for triterpene-based natural products and steroids-based drugs. It has been widely used as pharmaceutical intermediates and personal care products. The aim of this work is to test the feasibility of engineering Yarrowia lipolytica as a potential host for squalene production. The bottleneck of the pathway was removed by overexpressing native HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase. With the recycling of NADPH from the mannitol cycle, the engineered strain produced about 180.3 mg/L and 188.2 mg/L squalene from glucose or acetate minimal media. By optimizing the C/N ratio, controlling the media pH and mitigating acetyl-CoA flux competition from lipogenesis, the engineered strain produced 502.7 mg/L squalene, a 28-fold increase over the parental strain (17.2 mg/L). This work may serve as a baseline to harness Y. lipolytica as an oleaginous cell factory for sustainable production of squalene or terpenoids-based chemicals and natural products.
Highlights
Staring with acetyl-CoA condensation, yeast uses a number of critical enzymes to synthesize squalene, including acetoacetyl-CoA thiolase (Erg10, YALI0B08536g), hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (YALI0E04807g), mevalonate kinase (Erg12, YALI0B16038g), phosphomevalonate kinase (Erg8, YALI0E06193g), mevalonate pyrophosphate decarboxylase (MVD1, YALI0F05632g), farnesyl pyrophosphate synthase (Erg20, YALI0E05753g), geranyl pyrophosphate synthase (YALI0D17050g) and squalene synthase (SQS1, YALI0A10076g)
When co-expressed with endogenous squalene synthase gene (SQS), the strain with the truncated HMG1 (SctHMG1) led to squalene production at 83.76 mg/L (Fig. 2A), indicating that overexpression of HMG-CoA reductase was beneficial for squalene production
Yarrowia lipolytica was used as the microbial host for squalene production
Summary
Yarrowia lipolytica is an industrial oleaginous yeast that has been extensively engineered to synthesize lipophilic compounds, including lipids (Qiao et al, 2017), oleochemicals (Xu et al, 2016), carotenoids (Larroude et al, 2018), terpenoids (Jin et al, 2019) and aromatic polyketides (Czajka et al, 2018; Lv et al, 2019b) et al The lipogeneity of this yeast makes it a superior host to produce chemicals that are derived from acetyl-CoA, malonyl-CoA, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) and NADPHs (Gu et al, 2020; Ma et al, 2020). 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 been recognized as a ‘generally regarded as safe’ (GRAS) organism (Groenewald et al, 2014) in the food and nutraceutical industry. The yeast-based mevalonate (MVA) pathway starts with acetyl-CoA condensation reactions, proceeds through the reduction of intermediate HMG-CoA via HMG-CoA reductase, which is the rate-limiting step and the molecular target to design many statins-related anti-cholesterol drugs (Xie & Tang, 2007).
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