Modification of isoprene synthesis to enable production of curcurbitadienol synthesis inSaccharomyces cerevisiae

Abstract

Cucurbitane-type triterpenoids such as mogrosides and cucurbitacins that are present in the plants of Cucurbitaceae are widely used in Asian traditional medicine. Cucurbitadienol is the skeleton of cucurbitane-type triterpenoids. As an alternative production strategy, we developed baker’s yeast Saccharomyces cerevisiae as a microbial host for the eventual transformation of cucurbitadienol. The synthetic pathway of cucurbitadienol was constructed in Saccharomyces cerevisiae by introducing the cucurbitadienol synthase gene from different plants, resulting in 7.80 mg cucurbitadienol from 1 L of fermentation broth. Improving supplies of isoprenoid precursors was then investigated for increasing cucurbitadienol production. Cucurbitadienol production increased to 21.47 mg/L through the overexpression of a global regulatory factor (UPC2) gene of triterpenoid synthase. In addition, knockout of the ERG7 gene increased cucurbitadienol production from 21.47 to 61.80 mg/L. Finally, fed-batch fermentation was performed, and 63.00 mg/L cucurbitadienol was produced. This work is an important step towards the total biosynthesis of valuable cucurbitane-type triterpenoids and demonstrates the potential for developing a sustainable and secure yeast biomanufacturing platform for triterpenoids.

Efficient biosynthesis of antitumor ganoderic acid HLDOA using a dual tunable system for optimizing the expression of CYP5150L8 and a Ganoderma P450 reductase

“…Impaired cell growth was observed particularly when the dual tunable system was adopted (Figure ). As for production of other triterpenoids via metabolic engineering strategies, cell growth retardation was also observed in many engineered S. cerevisiae strains (J. Li & Zhang, ; Madsen et al, ; Qiao et al, ; Zhang et al, ; Zhu et al, ). Possible explanations are (a) metabolic burden arising from expression of multiple copies of CYP5150L8 and iGLCPR, (b) GA‐HLDOA toxicity, (c) the uncoupling events, or (d) the supply of the inadequate equivalent as mentioned above, which may need comprehensive host engineering.…”

Improved Production and In Situ Recovery of Sesquiterpene (+)-Zizaene from Metabolically-Engineered E. coli

“…As usually, the scale-up of the fermentation is performed by optimizing the bioreactor variables such as pH, oxygen supply, and stirring [11]. Additionally, scale-up studies can be performed using fed-batch fermentation by feeding of a carbon source, such as glucose, to reach high cell density cultures (HCDC), as is the case with the production of farnesene, santalene, cucurbitadienol, and (-)-α-bisabolol [12,13,14].…”

Identification of a Novel Specific Cucurbitadienol Synthase Allele in Siraitia grosvenorii Correlates with High Catalytic Efficiency

“…Residues 50R and 573L in SgCS correspond to 52R and 578L, respectively, in CcCS and CpCS and 70R and 599L, respectively, in CsCS. Previous study has shown that SgCS, which had the highest cucurbitadienol yield, is the wild-type CS enzyme with highest catalytic efficiency [36]. Therefore, SgCS can be used as a guideline for other CS enzyme modification.…”

“…Four different copies of SgCS genes were codon-optimized for synthesis according to the codon bias of yeast and cloned into the Bam HI/ Eco RI sites of the pCEV-G4-Km yeast expression vector under the control of the TEF1 promoter to construct pCEV-50R573L, pCEV-50C573L, pCEV-50R573Q and pCEV-50C573Q, respectively. The sequences that showed in National Center for Biotechnology Information (NCBI) database was obtained and codon-optimized as described by Qiao previously [36].…”