Glycerol improves heterologous biosynthesis of betulinic acid in engineered Yarrowia lipolytica

Boosting the biosynthesis of betulinic acid and related triterpenoids in Yarrowia lipolytica via multimodular metabolic engineering

“…Previous studies have shown that in S. cerevisiae , recombinant plant CYPs work well with endogenous CPRs [21, 23]. However, our results and those of several other studies [20, 46, 47] indicated that in Y. lipolytica , plant CYPs might require the coexpression of heterogenous CPRs for exogenous terpenoid production. Accordingly, five CPR genes from different sources, namely, ATR2 from Arabidopsis thaliana , CrCPR from C .…”

“…Appropriate concentrations of lupeol, betulin (Aladdin Industrial Corporation, US) and betulinic acid (MedChemExpress, US) were dissolved in IPM to prepare standard curves via GC–MS analysis. The methods used for betulinic acid detection were based on previous studies with some modification [15–17, 20]. A Rts-5 MS (30 m × 0.25 mm × 0.5 μm) GC column was used on a SHIMADZU GCMS-QP2020 system.…”

“…The first step in the exogenous biosynthesis of betulinic acid from endogenous 2,3-oxidosqualene is catalyzed by lupeol synthase (LUS), which is followed by three sequential oxidations catalyzed by lupeol C-28 oxidases (cytochrome P450 monooxygenases, CYPs), leading to the generation of betulin, betulinic aldehyde, and betulinic acid. As increasing CYPs have been mined by bioinformatics analysis [14, 15], amino acid sequence alignment [16–18], and the combination approach of activation tagging and reverse genetic TILLING [19], heterologous betulinic acid production has been established in microbes [16, 20–23]. Li et al demonstrated that attenuation of the competitive fatty acid biosynthesis pathway [23] and increasing the NADPH and oxygen supply [21] were beneficial for improvement of the production capability of betulinic acid in Saccharomyces cerevisiae .…”

“…Moreover, multicopy integration of AtLUP1 and CYP716Al1 with single-copy integration of ATR2 and overexpression of endogenous ERG1 and tHMG1 could improve betulinic acid production to ~ 28 mg/L in shake flask cultures in S. cerevisiae [22]. Furthermore, by coexpression of LUS and the fusion protein of CYP with CPR and multicopy integration of tHMG1 and ERG9 , betulinic acid biosynthesis was achieved in Y. lipolytica and the yield was improved to 26.53 mg/L by substitution of glycerol for glucose as a carbon source [20]. Although the exogenous production pathway of betulinic acid has been introduced in yeast, no considerable yield was observed, and the engineering of heterologous production of betulinic acid was not performed as systematically as that of the production of other terpenoids.

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A roadmap to engineering antiviral natural products synthesis in microbes

“…The engineered S. cerevisiae with two copies of CYP716A155 improved the titer of betulinic acid by ninefold from 21.7 mg/L to 193.5 mg/L, yielding a final titer of 1.5 g/L in a 5-L fermenter [ 48 ]. The titer of betulinic acid in Y. lipolytica was improved by twofold using glycerol as carbon source [ 49 ]. Glycyrrhetinic acid, a pentacyclic triterpenoid, is synthesized from β -amyrin by the sequential actions of two CYPs, CYP88D6 and CYP72A154.…”