Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae

Abstract

Microbial production of isoprene from renewable feedstock is a promising alternative to traditional petroleum-based processes. Currently, efforts to improve isoprenoid production in Saccharomyces cerevisiae mainly focus on cytoplasmic engineering, whereas comprehensive engineering of multiple subcellular compartments is rarely reported. Here, we propose dual metabolic engineering of cytoplasmic and mitochondrial acetyl-CoA utilization to boost isoprene synthesis in S. cerevisiae. This strategy increases isoprene production by 2.1-fold and 1.6-fold relative to the recombinant strains with solely mitochondrial or cytoplasmic engineering, respectively. By combining a modified reiterative recombination system for rapid pathway assembly, a two-phase culture process for dynamic metabolic regulation, and aerobic fed-batch fermentation for sufficient supply of acetyl-coA and carbon, we achieve 2527, mg l−1 of isoprene, which is the highest ever reported in engineered eukaryotes. We propose this strategy as an efficient approach to enhancing isoprene production in yeast, which might open new possibilities for bioproduction of other value-added chemicals.

Development of a temperature‐responsive yeast cell factory using engineered Gal4 as a protein switch

“…YPD medium was used for routine cultivation of yeast cells, and YPD medium supplemented with 200 µg/ml geneticin (G418) was used for selection of the KanMX marker. SD were used for cultivation of the selected transformants carrying plasmids with the corresponding auxotroph marker, whereas SD‐FOA plate was used for counter‐selection of yeast strains with KanMX ‐ URA ‐ PRB322ori marker excision as previously described (Lv et al, ).…”

“…For directed evolution of Gal4, P416XWP‐P CYC1 /P GAL4 /P ERG9 /P ACT1 ‐ GAL4 plasmids containing promoters of different strength were constructed based on p416XWP‐P CYC1 ‐ crtE plasmid (Xie, Ye, et al, ). For lycopene pathway assembly, tHMG1 , crtE03M , crtI , and crtYB11M were sub‐cloned into pUMRI integration plasmids (Lv et al, ). All primers and recombinant plasmids are listed in Tables and S1, respectively.…”

Barriers and opportunities in bio-based production of hydrocarbons

“…Genencor has developed a gas-phase bioprocess for isoprene production of 60 g l -1 by engineering the endogenous DXP and heterologous MVA pathways in Escherichia coli (E. coli), resulting in a volumetric productivity of 2 g l -1 h -1 and a yield of 11% isoprene from glucose 14 . Other microbes, such as yeast and cyanobacteria, were also explored for isoprene production with much lower titre 15,16 . Cyanobacteria-based terpene production would provide a direct route for fixing of CO 2 and solar energy by photosynthesis 16,17 .…”

De NovoSynthesis of Plant Natural Products in Yeast

“…In addition to the cooperation of the MEP and the MVA pathways, taking full advantage of precursors synthesized in different organelles was helpful to boost the terpenoids producing. By improving the utilization of acetyl-CoA through the simultaneously introducing dual MVA pathways located in cytoplasmic and mitochondrial of yeast, 2.5 mg/L isoprene was obtained [37]. Moreover, HMG-CoA reductase catalyzing the conversion of HMG-CoA to mevalonate is a rate-limiting factor of MVA pathway.…”