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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


Comparison of cytoplasm-engineered and mitochondria-engineered strains.(a) Schematic representation for cytoplasmic engineering and mitochondrial engineering of the complete isoprene synthetic pathway. Gene and pathway reconstruction is marked in magenta. Blue box represents mitochondria. (b) Isoprene production of strains constructed by cytoplasmic engineering and mitochondrial engineering of the complete isoprene synthetic pathway in aerobic batch fermentation. (c) Squalene production of strains constructed by cytoplasmic engineering and mitochondrial engineering of the MVA pathway. In b,c, error bars represent s.d. from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5036000&req=5

f3: Comparison of cytoplasm-engineered and mitochondria-engineered strains.(a) Schematic representation for cytoplasmic engineering and mitochondrial engineering of the complete isoprene synthetic pathway. Gene and pathway reconstruction is marked in magenta. Blue box represents mitochondria. (b) Isoprene production of strains constructed by cytoplasmic engineering and mitochondrial engineering of the complete isoprene synthetic pathway in aerobic batch fermentation. (c) Squalene production of strains constructed by cytoplasmic engineering and mitochondrial engineering of the MVA pathway. In b,c, error bars represent s.d. from three independent experiments.

Mentions: To compare the effect of cytoplasmic engineering and mitochondrial engineering of the MVA pathway (Fig. 3a) on isoprene production, the endogenous MVA pathway was also overexpressed in cytoplasm using pUMRI plasmids, constructing the BY4742-C-01/02/03/04 strains in which all the genes were under control of the same promoters and terminators as BY4742-M-01/02/03/04, with the mitochondrial target sequence as the only difference. The fermentation data demonstrated that the strain overexpressing mitochondria targeted enzymes of the complete MVA pathway together with ISPS, BY4742-M-04 MISPS-MISPS, outperformed the corresponding cytoplasm-engineered strain BY4742-C-04 ISPS-ISPS in isoprene production (108 mg l−1 versus 64 mg l−1) (Fig. 3b). To check whether the increased isoprene production in BY4742-M-04 was resulted from the lack of competing downstream pathways in mitochondria, the production of squalene in both strains was analysed. As shown in Fig. 3c, the production of squalene in BY4742-M-04 was lower than that of BY4742-C-04, although the squalene production in both BY4742-M-04 (0.03 mg ml−1 and 9.0 mg g−1 DCW, Dry Cell Weight) and BY4742-C-04 (0.25 mg ml−1 and 37.5 mg g−1 DCW) was improved compared with that of the wild-type strain BY4742 (0.001 and 0.12 mg g−1 DCW). In addition, expression levels of the pathway enzymes in BY4742-M-04 and BY4742-C-04 were estimated by western blot, with no obvious distinction found in most of the proteins (Supplementary Fig. 7). The above results indicate that the higher isoprene production in mitochondria-engineered strain might be ascribed to the reduction of flux loss into competing pathways by physical separation via the mitochondrial membrane, thus maximizing the flux into the target pathway rather than increasing local enzyme concentrations in the mitochondria.


Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae
Comparison of cytoplasm-engineered and mitochondria-engineered strains.(a) Schematic representation for cytoplasmic engineering and mitochondrial engineering of the complete isoprene synthetic pathway. Gene and pathway reconstruction is marked in magenta. Blue box represents mitochondria. (b) Isoprene production of strains constructed by cytoplasmic engineering and mitochondrial engineering of the complete isoprene synthetic pathway in aerobic batch fermentation. (c) Squalene production of strains constructed by cytoplasmic engineering and mitochondrial engineering of the MVA pathway. In b,c, error bars represent s.d. from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036000&req=5

f3: Comparison of cytoplasm-engineered and mitochondria-engineered strains.(a) Schematic representation for cytoplasmic engineering and mitochondrial engineering of the complete isoprene synthetic pathway. Gene and pathway reconstruction is marked in magenta. Blue box represents mitochondria. (b) Isoprene production of strains constructed by cytoplasmic engineering and mitochondrial engineering of the complete isoprene synthetic pathway in aerobic batch fermentation. (c) Squalene production of strains constructed by cytoplasmic engineering and mitochondrial engineering of the MVA pathway. In b,c, error bars represent s.d. from three independent experiments.
Mentions: To compare the effect of cytoplasmic engineering and mitochondrial engineering of the MVA pathway (Fig. 3a) on isoprene production, the endogenous MVA pathway was also overexpressed in cytoplasm using pUMRI plasmids, constructing the BY4742-C-01/02/03/04 strains in which all the genes were under control of the same promoters and terminators as BY4742-M-01/02/03/04, with the mitochondrial target sequence as the only difference. The fermentation data demonstrated that the strain overexpressing mitochondria targeted enzymes of the complete MVA pathway together with ISPS, BY4742-M-04 MISPS-MISPS, outperformed the corresponding cytoplasm-engineered strain BY4742-C-04 ISPS-ISPS in isoprene production (108 mg l−1 versus 64 mg l−1) (Fig. 3b). To check whether the increased isoprene production in BY4742-M-04 was resulted from the lack of competing downstream pathways in mitochondria, the production of squalene in both strains was analysed. As shown in Fig. 3c, the production of squalene in BY4742-M-04 was lower than that of BY4742-C-04, although the squalene production in both BY4742-M-04 (0.03 mg ml−1 and 9.0 mg g−1 DCW, Dry Cell Weight) and BY4742-C-04 (0.25 mg ml−1 and 37.5 mg g−1 DCW) was improved compared with that of the wild-type strain BY4742 (0.001 and 0.12 mg g−1 DCW). In addition, expression levels of the pathway enzymes in BY4742-M-04 and BY4742-C-04 were estimated by western blot, with no obvious distinction found in most of the proteins (Supplementary Fig. 7). The above results indicate that the higher isoprene production in mitochondria-engineered strain might be ascribed to the reduction of flux loss into competing pathways by physical separation via the mitochondrial membrane, thus maximizing the flux into the target pathway rather than increasing local enzyme concentrations in the mitochondria.

View Article: PubMed Central - PubMed

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.

No MeSH data available.