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

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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.


Fed-batch fermentation of YXMH-03 (ISPS-MISPS).Fermentation was performed in a 5-l fermentor containing 2.5-l fermentation medium at 30 °C with an airflow rate of 1–3 v.v.m. The pH was controlled automatically at 5.0 with the addition of 5 M NH4OH. The isoprene concentration in the off-gas was determined by GC every 3 hours. Error bars represent s.d. from three independent experiments.
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f5: Fed-batch fermentation of YXMH-03 (ISPS-MISPS).Fermentation was performed in a 5-l fermentor containing 2.5-l fermentation medium at 30 °C with an airflow rate of 1–3 v.v.m. The pH was controlled automatically at 5.0 with the addition of 5 M NH4OH. The isoprene concentration in the off-gas was determined by GC every 3 hours. Error bars represent s.d. from three independent experiments.

Mentions: Comparison of the two cytoplasmic engineering strains revealed higher isoprene production from BY4742-C-04 than YXM10, suggesting that overexpression of the entire MVA pathway in the cytoplasm could be an alternative strategy for improving isoprene overproduction. To check whether mating of strains respectively overexpressing the entire MVA pathway in the cytoplasm and the mitochondria led to even higher isoprene yield compared with YXMH-03, the entire MVA pathway was reconstructed in BY4741, resulting in BY4741-C-04, complemented with LEU marker, and then mated with BY4742-M-04-HIS, generating YXMH-04. Genotype analysis of BY4741-C-04 (Supplementary Fig. 5D) confirmed the successful reconstruction of the MVA pathway in BY4741. YXMH-04 (ISPS-MISPS) produced 195 mg l−1 of isoprene, which was 80% higher than BY4742-M-04 (MISPS-MISPS) but 20% lower than YXMH-03 (ISPS-MISPS) (Fig. 4d). In addition, the cell growth of YXMH-04 (ISPS-MISPS) (OD600=11) was improved by 140% compared with BY4742-M-04 (MISPS-MISPS) (OD600=7.8), but was still lower than YXMH-03 (ISPS-MISPS) (OD600=14.3) (Fig. 4e). The lower isoprene production and cell growth of YXMH-04 compared with YXMH-03 might be ascribed to metabolic burden from overexpression of sixteen genes, including four copies of tHMG1 and two copies of the other six genes involved in the MVA pathway. Finally, fed-batch fermentation was performed with the dual engineered strain YXMH-03 (ISPS-MISPS) under aerobic conditions, producing 2527, mg l−1 of isoprene within 120 h (Fig. 5).


Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae
Fed-batch fermentation of YXMH-03 (ISPS-MISPS).Fermentation was performed in a 5-l fermentor containing 2.5-l fermentation medium at 30 °C with an airflow rate of 1–3 v.v.m. The pH was controlled automatically at 5.0 with the addition of 5 M NH4OH. The isoprene concentration in the off-gas was determined by GC every 3 hours. 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

f5: Fed-batch fermentation of YXMH-03 (ISPS-MISPS).Fermentation was performed in a 5-l fermentor containing 2.5-l fermentation medium at 30 °C with an airflow rate of 1–3 v.v.m. The pH was controlled automatically at 5.0 with the addition of 5 M NH4OH. The isoprene concentration in the off-gas was determined by GC every 3 hours. Error bars represent s.d. from three independent experiments.
Mentions: Comparison of the two cytoplasmic engineering strains revealed higher isoprene production from BY4742-C-04 than YXM10, suggesting that overexpression of the entire MVA pathway in the cytoplasm could be an alternative strategy for improving isoprene overproduction. To check whether mating of strains respectively overexpressing the entire MVA pathway in the cytoplasm and the mitochondria led to even higher isoprene yield compared with YXMH-03, the entire MVA pathway was reconstructed in BY4741, resulting in BY4741-C-04, complemented with LEU marker, and then mated with BY4742-M-04-HIS, generating YXMH-04. Genotype analysis of BY4741-C-04 (Supplementary Fig. 5D) confirmed the successful reconstruction of the MVA pathway in BY4741. YXMH-04 (ISPS-MISPS) produced 195 mg l−1 of isoprene, which was 80% higher than BY4742-M-04 (MISPS-MISPS) but 20% lower than YXMH-03 (ISPS-MISPS) (Fig. 4d). In addition, the cell growth of YXMH-04 (ISPS-MISPS) (OD600=11) was improved by 140% compared with BY4742-M-04 (MISPS-MISPS) (OD600=7.8), but was still lower than YXMH-03 (ISPS-MISPS) (OD600=14.3) (Fig. 4e). The lower isoprene production and cell growth of YXMH-04 compared with YXMH-03 might be ascribed to metabolic burden from overexpression of sixteen genes, including four copies of tHMG1 and two copies of the other six genes involved in the MVA pathway. Finally, fed-batch fermentation was performed with the dual engineered strain YXMH-03 (ISPS-MISPS) under aerobic conditions, producing 2527, mg l−1 of isoprene within 120 h (Fig. 5).

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.