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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.


Mitochondrial engineering for isoprene production.(a) Production of isoprene in recombinant strains. (b) Cell growth of recombinant strains. (c) Schematic diagram of a modified GAL regulation. (d) Two-stage process involved in the growth of GAL80-knockout strains. In the first stage, the genes under control of PGAL were expressed at a low level to sustain cell growth; while in the second stage, these genes were overexpressed at a high level. (e) Isoprene production and biomass of BY4742-M-04 MISPS-MISPS cultured in SG-URA (2% galactose), SD-URA (2% Dextrose) and SS-URA (2% Sucrose). The data in a,b,e are representative of three separate experiments. Bar represents mean±s.d.
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f2: Mitochondrial engineering for isoprene production.(a) Production of isoprene in recombinant strains. (b) Cell growth of recombinant strains. (c) Schematic diagram of a modified GAL regulation. (d) Two-stage process involved in the growth of GAL80-knockout strains. In the first stage, the genes under control of PGAL were expressed at a low level to sustain cell growth; while in the second stage, these genes were overexpressed at a high level. (e) Isoprene production and biomass of BY4742-M-04 MISPS-MISPS cultured in SG-URA (2% galactose), SD-URA (2% Dextrose) and SS-URA (2% Sucrose). The data in a,b,e are representative of three separate experiments. Bar represents mean±s.d.

Mentions: The effects of mitochondrial engineering were investigated. First, to examine the possible influence of gene disruption resulting from genomic integration at the corresponding sites, control experiments were conducted by knocking out LPP1, DPP1, HO and GAL80 genes, showing no visible effect on target product accumulation or cell growth. After localization of the isoprene synthetic pathway to the mitochondria, the isoprene production in recombinant strains was significantly improved, especially in BY4742-M-04 MISPS-MISPS (Fig. 2a), while the biomass of all BY4742-M-01/02/03/04 strains showed marked decrease (Fig. 2b and Supplementary Fig. 6A). In comparison, the strains overexpressing the corresponding genes in the cytoplasm (BY4742-C-01/02/03/04) mostly showed only slightly slower growth than the control except BY4742-C-04 (Supplementary Fig. 6B), indicating the growth decrease in mitochondria-engineered strains was not only caused by the metabolic burden resulting from multigene overexpression. We hypothesized that the accumulation of cytotoxic metabolic intermediates may have contributed to impaired cell growth. The intermediates of the MVA pathway in BY4742-M-01/02/03/04 could most likely not be transported out of the mitochondria or further converted due to the lack of downstream genes, leading to intermediate accumulation. To verify this hypothesis, pESC-URA-MISPS-MISPS was transformed into the mitochondria of BY4742-M-04 (generating BY4742-M-04 MISPS-MISPS), resulting in improved growth on day 4 and thereafter as compared with BY4742-M-04 MISPS-MISPS (N498D, inactive enzyme), although its cell growth was still not as good as the control strain (BY4742 ISPS-ISPS) (Supplementary Fig. 6C).


Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae
Mitochondrial engineering for isoprene production.(a) Production of isoprene in recombinant strains. (b) Cell growth of recombinant strains. (c) Schematic diagram of a modified GAL regulation. (d) Two-stage process involved in the growth of GAL80-knockout strains. In the first stage, the genes under control of PGAL were expressed at a low level to sustain cell growth; while in the second stage, these genes were overexpressed at a high level. (e) Isoprene production and biomass of BY4742-M-04 MISPS-MISPS cultured in SG-URA (2% galactose), SD-URA (2% Dextrose) and SS-URA (2% Sucrose). The data in a,b,e are representative of three separate experiments. Bar represents mean±s.d.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Mitochondrial engineering for isoprene production.(a) Production of isoprene in recombinant strains. (b) Cell growth of recombinant strains. (c) Schematic diagram of a modified GAL regulation. (d) Two-stage process involved in the growth of GAL80-knockout strains. In the first stage, the genes under control of PGAL were expressed at a low level to sustain cell growth; while in the second stage, these genes were overexpressed at a high level. (e) Isoprene production and biomass of BY4742-M-04 MISPS-MISPS cultured in SG-URA (2% galactose), SD-URA (2% Dextrose) and SS-URA (2% Sucrose). The data in a,b,e are representative of three separate experiments. Bar represents mean±s.d.
Mentions: The effects of mitochondrial engineering were investigated. First, to examine the possible influence of gene disruption resulting from genomic integration at the corresponding sites, control experiments were conducted by knocking out LPP1, DPP1, HO and GAL80 genes, showing no visible effect on target product accumulation or cell growth. After localization of the isoprene synthetic pathway to the mitochondria, the isoprene production in recombinant strains was significantly improved, especially in BY4742-M-04 MISPS-MISPS (Fig. 2a), while the biomass of all BY4742-M-01/02/03/04 strains showed marked decrease (Fig. 2b and Supplementary Fig. 6A). In comparison, the strains overexpressing the corresponding genes in the cytoplasm (BY4742-C-01/02/03/04) mostly showed only slightly slower growth than the control except BY4742-C-04 (Supplementary Fig. 6B), indicating the growth decrease in mitochondria-engineered strains was not only caused by the metabolic burden resulting from multigene overexpression. We hypothesized that the accumulation of cytotoxic metabolic intermediates may have contributed to impaired cell growth. The intermediates of the MVA pathway in BY4742-M-01/02/03/04 could most likely not be transported out of the mitochondria or further converted due to the lack of downstream genes, leading to intermediate accumulation. To verify this hypothesis, pESC-URA-MISPS-MISPS was transformed into the mitochondria of BY4742-M-04 (generating BY4742-M-04 MISPS-MISPS), resulting in improved growth on day 4 and thereafter as compared with BY4742-M-04 MISPS-MISPS (N498D, inactive enzyme), although its cell growth was still not as good as the control strain (BY4742 ISPS-ISPS) (Supplementary Fig. 6C).

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.