3.3 Glucose and acetate as media for squalene production
Y. lipolytica can grow on a broad range of substrates and convert various organic waste to high-value chemicals (Dobrowolski, Mituła, Rymowicz, & Mirończuk, 2016; Rakicka, Biegalska, Rymowicz, Dobrowolski, & Mirończuk, 2017). For example, it has been reported that Y. lipolytica possessed strong acetate utilization pathway that is equivalent or even superior to the hexose utilization pathway, which led to an improvement of triacylglyceride (TAG) production from 100 g/L to 115 g/L in bench-top bioreactors, when the cultivation was switched from glucose media to acetate media (Qiao et al., 2017; J. Xu, Liu, Qiao, Vogg, & Stephanopoulos, 2017) . In another work, Y. lipolyticawas reported to efficiently uptake acetic acid as sole carbon source to produce polyketides up to 4.76 g/L, indicating that acetate may serve as a metabolic “shortcut” to acetyl-CoA with improved carbon conversion efficiency and pathway yield (Huan Liu, Monireh Marsafari, Fang Wang, et al., 2019). In this study, a similar strategy was explored to investigate the conversion process of acetate to squalene by the engineered strain HLYaliS01 , HLYaliS02 andHLYaliS03 (Supplementary Fig. S1). 41 g/L sodium acetate (NaAc), equivalently to 29.5 g/L acetic acid (HAc, 0.5 M) was used to cultivate the engineered strains. In situ pH indicator (bromocresol purple) was used to track the pH change and 6 M HCl was used to adjust the pH in the shake flask. Among the engineered strains, the highest squalene titer reached 191.68 mg/L at 168 h in acetate-YNB medium by strainHLYaliS01 , with 99% of acetic acid depleted and 6.6 g/L biomass produced, yielding of squalene at 29.04 mg/g DCW (Supplementary Fig. S1 B and Table S3 ). Strain HLYaliS02 produced 180.28 mg/L squalene at 140 h in acetate-YNB medium with the highest productivity (Supplementary Fig. S1 D). When both engineered strains (HLYaliS01 and HLYaliS02 ) were cultivated in glucose-YNB medium, 157.81 mg/L and 188.18 mg/L squalene was achieved by strainHLYaliS01 and HLYaliS02 , with a yield of 16.53 mg/g DCW and 15.91 mg/g DCW, respectively (Supplementary Fig. S1 A and C, Table S3). This indicates that the mannitol cycle (which is engineered in strain HLYaliS02 with SQS-ylHMG-ylMnDH2 ) may function well when glucose is used as carbon source. Compared with HLYaliS01and HLYaliS02 , HLYaliS03 (the strain withSQS-ylHMG-ylACL2 ) produced less squalene in glucose (138.33 mg/L), but similar amount of squalene in acetate (176.8 mg/L) (Supplementary Fig. S1 E and F). The data demonstrated that both glucose and acetate could be utilized as carbon sources to produced squalene byY. lipolytica and acetate as a potential and cheap industrial chemicals had a promising application and commercial value for squalene and terpene production.
To further improve squalene synthesis, we assembled ylACL2 to the plasmid harboring SQS, ylHMG and ylMnDH2. But the engineered strain (HLYaliS04 ) didn’t result in an improved squalene production from neither glucose nor acetate as substrate (Supplementary Fig. S2), possibly due to the metabolic imbalance or gene expression overloading causing burdensome effects to the cell factory.