Physiologic and metabolic characterization of Saccharomyces cerevisiae reveals limitations in the synthesis of the triterpene squalene

Abstract

Heterologous synthesis of triterpenoids in Saccharomyces cerevisiae from its native metabolite squalene has been reported to offer an alternative to chemical synthesis and extraction from plant material if productivities can be increased.Here, we physiologically characterized a squalene overproducing S. cerevisiae CEN.PK strain to elucidate the effect of cultivation conditions on the production of this central triterpenoid precursor. The maximum achievable squalene concentration was substantially influenced by nutritional conditions, medium composition and cultivation mode. Batch growth on glucose resulted in minimal squalene accumulation, while squalene only significantly accumulated during ethanol consumption (up to 59 mg/gCDW), probably due to increased acetyl-CoA supply on this carbon source. Likewise, low squalene concentrations were observed in glucose-limited chemostat cultivations and improved up to 8-fold upon increasing the ethanol fraction in the feed. In those experiments, a constant, growth-rate-independent specific squalene accumulation rate (2.2 mg/gCDW/h) was recorded resulting in a maximal squalene loading of 30 mg/gCDW at low dilution rates with longer residence times. Coenzyme A availability was identified as possible bottleneck as increased vitamin concentrations, including the Coenzyme A precursor pantothenate, improved squalene titers in batch and chemostat cultivations. This analysis demonstrates that thorough physiologic characterization of production strains is valuable for the identification of bottlenecks already in early stages of strain development and for guiding further optimization efforts.

Combined Biosynthetic Pathway Engineering and Storage Pool Expansion for High-Level Production of Ergosterol in Industrial Saccharomyces cerevisiae

“…Glucose and ethanol are commonly used carbon sources for production of terpenoids in yeast ( Zhang et al, 2015 ; Qiao et al, 2019 ). Compared with glucose, ethanol can be utilized to directly supply cytosolic acetyl-CoA, resulting in significantly increased metabolic flux to the MVA pathway and accordingly terpenoid biosynthesis ( De Jong-Gubbels et al, 1995 ; Ebert et al, 2018 ). In addition, NADPH generated from ethanol catabolism can provide sufficient reducing power for ergosterol biosynthesis in S. cerevisiae .…”

Fermentation Strategies for Production of Pharmaceutical Terpenoids in Engineered Yeast

“…[61] employed continuous cultivation and achieved α-santalene productivity of 0.036 Cmmol/(g biomass)/h at the dilution rate of 0.05 h −1 . In another study, squalene accumulation of 30 mg/g (product/dry cell weight) was obtained at low dilution rates between 0.05 h −1 and 0.20 h −1 in glucose limited chemostat cultivation [120]. Despite demonstrated applicability in terpenoids production, continuous fermentation has been poorly explored, mainly because fed-batch fermentation has been shown to be the most effective way for obtaining meaningful terpene titers for industry, combining the advantages of batch and continuous mode at once.…”

Heterologous Production of Squalene from Glucose in Engineered Corynebacterium glutamicum Using Multiplex CRISPR Interference and High-Throughput Fermentation

“…9 Recently, Coenzyme A availability has been identified as a potential bottleneck for improving squalene production. 10 In contrast, wild-type E. coli does not produce squalene. However, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which can be synthesized via the methylerythritol 4-phosphate (MEP) pathway in E. coli, are further converted to FPP by the native FPP synthase (the ispA gene).…”

“…In addition, xylose, as alternative carbon source, has been demonstrated to accumulate cytosolic acetyl-coA-derived squalene in engineered S. cerevisiae . Recently, Coenzyme A availability has been identified as a potential bottleneck for improving squalene production …”