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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.

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Dual metabolic regulation in the cytoplasm and mitochondria for isoprene production.(a) Dual regulation strategy. (b) Isoprene production of recombinant strains YXM10 (ISPS-ISPS), BY4742-M-04 (MISPS-MISPS), BY4742-MC-01 (ISPS-MISPS), BY4742-C-05 (ISPS-ISPS) in aerobic batch fermentation. (c) Growth curves of the recombinant strains (in b) in aerobic batch fermentation. (d) Isoprene production of recombinant strains YXM10 (ISPS-ISPS), BY4742-M-04 (MISPS-MISPS), YXMH-01 (ISPS-ISPS), YXMH-02 (MISPS-MISPS), YXMH-03 (ISPS-MISPS), YXMH-04 (ISPS-MISPS) in aerobic batch fermentation. (e) Growth curves of the recombinant strains (in d) in aerobic batch fermentation. YXM10: strain with cytoplasm engineering; BY4742-M-04, strain with mitochondria engineering; BY4742-MC-01, haploid strain with a mixed cytosolic-mitochondrial strategy; BY4742-C-05, haploid strain with comprehensive regulation in the cytoplasm; YXMH-01, the control hybrid strain of YXM10 and BY4742-ΔGal80::HIS; YXMH-02, the control hybrid strain of BY4742-M-04-HIS and BY4741-ΔGal80::LEU; YXMH-03, the hybrid strain of YXM10 and BY4742-M-04-HIS; YXMH-04, the hybrid strain of BY4741-C-04-LEU and BY4742-M-04-HIS.
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f4: Dual metabolic regulation in the cytoplasm and mitochondria for isoprene production.(a) Dual regulation strategy. (b) Isoprene production of recombinant strains YXM10 (ISPS-ISPS), BY4742-M-04 (MISPS-MISPS), BY4742-MC-01 (ISPS-MISPS), BY4742-C-05 (ISPS-ISPS) in aerobic batch fermentation. (c) Growth curves of the recombinant strains (in b) in aerobic batch fermentation. (d) Isoprene production of recombinant strains YXM10 (ISPS-ISPS), BY4742-M-04 (MISPS-MISPS), YXMH-01 (ISPS-ISPS), YXMH-02 (MISPS-MISPS), YXMH-03 (ISPS-MISPS), YXMH-04 (ISPS-MISPS) in aerobic batch fermentation. (e) Growth curves of the recombinant strains (in d) in aerobic batch fermentation. YXM10: strain with cytoplasm engineering; BY4742-M-04, strain with mitochondria engineering; BY4742-MC-01, haploid strain with a mixed cytosolic-mitochondrial strategy; BY4742-C-05, haploid strain with comprehensive regulation in the cytoplasm; YXMH-01, the control hybrid strain of YXM10 and BY4742-ΔGal80::HIS; YXMH-02, the control hybrid strain of BY4742-M-04-HIS and BY4741-ΔGal80::LEU; YXMH-03, the hybrid strain of YXM10 and BY4742-M-04-HIS; YXMH-04, the hybrid strain of BY4741-C-04-LEU and BY4742-M-04-HIS.

Mentions: Since sufficient precursor supply is one of the most essential issues in bio-products accumulation, the complete utilization of acetyl-CoA within multiple compartments might benefit isoprene production in yeast. As previously reported for the cytoplasmic engineering of isoprene-producing S. cerevisiae27, enhancement of the precursor supply by overexpressing tHMG1 and downregulation of the competing pathway by weakening the farnesyl pyrophosphate synthetase gene (ERG20) led to significant enhancement in isoprene production (generating YXM10), which can be regarded as an effective strategy for cytoplasmic engineering. Therefore, to further improve production of isoprene, dual metabolic regulation of the isoprene synthetic pathway in both cytoplasm and mitochondria was explored (Fig. 4a). One method for dual regulation was to conduct cytoplasmic engineering in addition to mitochondrial engineering in the same strain by overexpressing tHMG1 and weakening ERG20 in BY4742-M-04, resulting in BY4742-MC-01. A modest increase in isoprene production (128 mg l−1) was obtained in BY4742-MC-01 (ISPS-MISPS), compared with strains BY4742-M-04 (MISPS-MISPS) (108 mg l−1) and YXM10 (ISPS-ISPS) (13 mg l−1) (Fig. 4b). The same strategy (overexpression of tHMG1 and downregulation of ERG20) was also performed in BY4742-C-04 to generate the cytoplasmic engineering-only control strain BY4742-C-05 (Supplementary Fig. 5D). In contrast to BY4742-MC-01 (ISPS-MISPS), BY4742-C-05 (ISPS-ISPS) produced as low as 25 mg l−1 of isoprene (Fig. 4b) and grew poorly (2.2 OD600) under aerobic fermentation condition (Fig. 4c), demonstrating the superiority of dual cytoplasmic-mitochondrial engineering over cytoplasmic engineering alone. However, the dual engineered strain BY4742-MC-01 (ISPS-MISPS) was unable to achieve high-density fermentation, especially in the fed phase (data not shown). Therefore, achieving comprehensive regulation of mitochondrial and cytoplasmic acetyl-CoA supply while still preserving high biomass warranted further investigation.


Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae
Dual metabolic regulation in the cytoplasm and mitochondria for isoprene production.(a) Dual regulation strategy. (b) Isoprene production of recombinant strains YXM10 (ISPS-ISPS), BY4742-M-04 (MISPS-MISPS), BY4742-MC-01 (ISPS-MISPS), BY4742-C-05 (ISPS-ISPS) in aerobic batch fermentation. (c) Growth curves of the recombinant strains (in b) in aerobic batch fermentation. (d) Isoprene production of recombinant strains YXM10 (ISPS-ISPS), BY4742-M-04 (MISPS-MISPS), YXMH-01 (ISPS-ISPS), YXMH-02 (MISPS-MISPS), YXMH-03 (ISPS-MISPS), YXMH-04 (ISPS-MISPS) in aerobic batch fermentation. (e) Growth curves of the recombinant strains (in d) in aerobic batch fermentation. YXM10: strain with cytoplasm engineering; BY4742-M-04, strain with mitochondria engineering; BY4742-MC-01, haploid strain with a mixed cytosolic-mitochondrial strategy; BY4742-C-05, haploid strain with comprehensive regulation in the cytoplasm; YXMH-01, the control hybrid strain of YXM10 and BY4742-ΔGal80::HIS; YXMH-02, the control hybrid strain of BY4742-M-04-HIS and BY4741-ΔGal80::LEU; YXMH-03, the hybrid strain of YXM10 and BY4742-M-04-HIS; YXMH-04, the hybrid strain of BY4741-C-04-LEU and BY4742-M-04-HIS.
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f4: Dual metabolic regulation in the cytoplasm and mitochondria for isoprene production.(a) Dual regulation strategy. (b) Isoprene production of recombinant strains YXM10 (ISPS-ISPS), BY4742-M-04 (MISPS-MISPS), BY4742-MC-01 (ISPS-MISPS), BY4742-C-05 (ISPS-ISPS) in aerobic batch fermentation. (c) Growth curves of the recombinant strains (in b) in aerobic batch fermentation. (d) Isoprene production of recombinant strains YXM10 (ISPS-ISPS), BY4742-M-04 (MISPS-MISPS), YXMH-01 (ISPS-ISPS), YXMH-02 (MISPS-MISPS), YXMH-03 (ISPS-MISPS), YXMH-04 (ISPS-MISPS) in aerobic batch fermentation. (e) Growth curves of the recombinant strains (in d) in aerobic batch fermentation. YXM10: strain with cytoplasm engineering; BY4742-M-04, strain with mitochondria engineering; BY4742-MC-01, haploid strain with a mixed cytosolic-mitochondrial strategy; BY4742-C-05, haploid strain with comprehensive regulation in the cytoplasm; YXMH-01, the control hybrid strain of YXM10 and BY4742-ΔGal80::HIS; YXMH-02, the control hybrid strain of BY4742-M-04-HIS and BY4741-ΔGal80::LEU; YXMH-03, the hybrid strain of YXM10 and BY4742-M-04-HIS; YXMH-04, the hybrid strain of BY4741-C-04-LEU and BY4742-M-04-HIS.
Mentions: Since sufficient precursor supply is one of the most essential issues in bio-products accumulation, the complete utilization of acetyl-CoA within multiple compartments might benefit isoprene production in yeast. As previously reported for the cytoplasmic engineering of isoprene-producing S. cerevisiae27, enhancement of the precursor supply by overexpressing tHMG1 and downregulation of the competing pathway by weakening the farnesyl pyrophosphate synthetase gene (ERG20) led to significant enhancement in isoprene production (generating YXM10), which can be regarded as an effective strategy for cytoplasmic engineering. Therefore, to further improve production of isoprene, dual metabolic regulation of the isoprene synthetic pathway in both cytoplasm and mitochondria was explored (Fig. 4a). One method for dual regulation was to conduct cytoplasmic engineering in addition to mitochondrial engineering in the same strain by overexpressing tHMG1 and weakening ERG20 in BY4742-M-04, resulting in BY4742-MC-01. A modest increase in isoprene production (128 mg l−1) was obtained in BY4742-MC-01 (ISPS-MISPS), compared with strains BY4742-M-04 (MISPS-MISPS) (108 mg l−1) and YXM10 (ISPS-ISPS) (13 mg l−1) (Fig. 4b). The same strategy (overexpression of tHMG1 and downregulation of ERG20) was also performed in BY4742-C-04 to generate the cytoplasmic engineering-only control strain BY4742-C-05 (Supplementary Fig. 5D). In contrast to BY4742-MC-01 (ISPS-MISPS), BY4742-C-05 (ISPS-ISPS) produced as low as 25 mg l−1 of isoprene (Fig. 4b) and grew poorly (2.2 OD600) under aerobic fermentation condition (Fig. 4c), demonstrating the superiority of dual cytoplasmic-mitochondrial engineering over cytoplasmic engineering alone. However, the dual engineered strain BY4742-MC-01 (ISPS-MISPS) was unable to achieve high-density fermentation, especially in the fed phase (data not shown). Therefore, achieving comprehensive regulation of mitochondrial and cytoplasmic acetyl-CoA supply while still preserving high biomass warranted further investigation.

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.


Related in: MedlinePlus