Enhancing Geranylgeraniol Production by Metabolic Engineering and Utilization of Isoprenol as a Substrate in Saccharomyces cerevisiae

Abstract

The amount of geranylgeranyl diphosphate (GGPP) is vital for microbial production of geranylgeraniol (GGOH) in Saccharomyces cerevisiae. In this study, a GGPP synthase with stronger catalytic ability was used to increase the supply of GGPP, and an engineered strain producing 374.02 mg/L GGOH at the shake flask level was constructed. Then, by increasing the metabolic flux of the mevalonate (MVA) pathway and the supply of isopentenyl pyrophosphate (IPP), the titer was further increased to 772.98 mg/L at the shake flask level, and we achieved the highest GGOH titer to date of 5.07 g/L in a 5 L bioreactor. This is the first report on the utilization of isoprenol for increasing the amount of IPP and enhancing GGOH production in S. cerevisiae. In the future, these strategies and engineered strains can be used to enhance the production of other terpenoids in S. cerevisiae.

Improving isoprenol productionviasystematic CRISPRi screening in engineeredEscherichia coli

“…To further alleviate isoprenol toxicity while minimizing isoprenol loss from evaporation, the combination of host strain optimization and in situ isoprenol recovery would boost isoprenol yields and simplify the purification process in the future. 15,56,57 In conclusion, this work exemplified the feasibility of harnessing CRISPRi screening for the optimization of synthetic pathway performance using the isoprenol pathway as a demonstration. Without genetic intervention, CRISPRi screening allows rapid identification of endogenous gene targets involved in the isoprenol biosynthesis pathway in the microbial host E. coli and optimization of isoprenol pro-duction performance.…”

“…To further alleviate isoprenol toxicity while minimizing isoprenol loss from evaporation, the combination of host strain optimization and in situ isoprenol recovery would boost isoprenol yields and simplify the purification process in the future. 15,56,57…”

The Terpene Mini-Path, a New Promising Alternative for Terpenoids Bio-Production

“…Using Saccharomyces cerevisiae as a chassis, the production of geranylgeraniol was studied by Wang et al [ 34 ]. The optimization of an initial strain by overexpression of a truncated version of the 3-hydroxy-3-methylglutaryl-coenzyme A reductase gene ( tHMG1 ), the use of a GGPPS-FPPS fusion protein, the use of a dodecane layer, and the deletion of the gene ROX1 (transcriptional repressor of the sterol pathway) allowed, after 7 days, a final GGOH titer of 3.66 g/L in a fed-batch cultivation.…”

Recent Advances in the Biosynthesis of Farnesene Using Metabolic Engineering

“…48 In addition, the reaction rate from IPP to FPP can be "pulled" through the screening and fusion expression of the enzyme involved in the subsequent reaction to reduce the toxic effect of IPP/DMAPP on cells. 98 Eukaryotes, such as yeast, contain peroxisomes in their cells. The peroxisome membrane could prevent the diffusion of IPP in cells.…”

“…Thus, the expression of high levels of IDI and farnesyl diphosphate synthase (IspA) is an effective method for managing IPP/DMAPP toxic on cells . In addition, the reaction rate from IPP to FPP can be “pulled” through the screening and fusion expression of the enzyme involved in the subsequent reaction to reduce the toxic effect of IPP/DMAPP on cells . Eukaryotes, such as yeast, contain peroxisomes in their cells.…”