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Alleviating Redox Imbalance Enhances 7-Dehydrocholesterol Production in Engineered Saccharomyces cerevisiae.

Su W, Xiao WH, Wang Y, Liu D, Zhou X, Yuan YJ - PLoS ONE (2015)

Bottom Line: Maintaining redox balance is critical for the production of heterologous secondary metabolites, whereas on various occasions the native cofactor balance does not match the needs in engineered microorganisms.In the meanwhile, the ratio of free NADH/NAD+ and the concentration of glycerol and ethanol were reduced by 78.0%, 50.7% and 7.9% respectively.In a 5-L bioreactor, the optimal production of 7-DHC reached 44.49(±9.63) mg/L.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, China.

ABSTRACT
Maintaining redox balance is critical for the production of heterologous secondary metabolites, whereas on various occasions the native cofactor balance does not match the needs in engineered microorganisms. In this study, 7-dehydrocholesterol (7-DHC, a crucial precursor of vitamin D3) biosynthesis pathway was constructed in Saccharomyces cerevisiae BY4742 with endogenous ergosterol synthesis pathway blocked by knocking out the erg5 gene (encoding C-22 desaturase). The deletion of erg5 led to redox imbalance with higher ratio of cytosolic free NADH/NAD+ and more glycerol and ethanol accumulation. To alleviate the redox imbalance, a water-forming NADH oxidase (NOX) and an alternative oxidase (AOX1) were employed in our system based on cofactor regeneration strategy. Consequently, the production of 7-dehydrocholesterol was increased by 74.4% in shake flask culture. In the meanwhile, the ratio of free NADH/NAD+ and the concentration of glycerol and ethanol were reduced by 78.0%, 50.7% and 7.9% respectively. In a 5-L bioreactor, the optimal production of 7-DHC reached 44.49(±9.63) mg/L. This study provides a reference to increase the production of some desired compounds that are restricted by redox imbalance.

No MeSH data available.


Related in: MedlinePlus

The cytosolic free NADH/NAD+ ratio and metabolites accumulation in strains from BY4742 and the ergosterol defected strains (ΔERG5) strains.All strains were cultivated in shake flasks and sampled after 24 h after they were inoculated into the fresh YPD medium. Columns with different shades of red color represent the strains from the wild type BY4742 and those with different shades of gray color represent the strains from ΔERG5. Significance levels of Students t-test: *P< 0.05, **P< 0.01, ***P< 0.001.
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pone.0130840.g003: The cytosolic free NADH/NAD+ ratio and metabolites accumulation in strains from BY4742 and the ergosterol defected strains (ΔERG5) strains.All strains were cultivated in shake flasks and sampled after 24 h after they were inoculated into the fresh YPD medium. Columns with different shades of red color represent the strains from the wild type BY4742 and those with different shades of gray color represent the strains from ΔERG5. Significance levels of Students t-test: *P< 0.05, **P< 0.01, ***P< 0.001.

Mentions: During the fermentation in shake flasks, the cytosolic free NADH/NAD+ ratio and the amounts of glycerol and ethanol produced by strains derived from BY4742 and ΔERG5 were measured at 24 h in the stationary phase. The free NADH/NAD+ ratios and glycerol and ethanol accumulation in the ergosterol defected strains were higher than those of the strains from BY4742 with the same alterations. Especially, the free NADH/NAD+ ratio of SyBE_Sc01100002 was 0.011(±0.003), while the ratio of strains engineered from BY4742 were less than half of that value. At the same time, the glycerol produced by SyBE_Sc01100002 reached 1.402 g/L, which was the highest among all yeast strains in this study (Fig 3). Because excessive NADH in cytosol gave rise to the redox imbalance, decreasing the ratio of NADH/NAD+ by cofactor engineering became a rational approach to alleviate redox imbalance in the ergosterol defected strains.


Alleviating Redox Imbalance Enhances 7-Dehydrocholesterol Production in Engineered Saccharomyces cerevisiae.

Su W, Xiao WH, Wang Y, Liu D, Zhou X, Yuan YJ - PLoS ONE (2015)

The cytosolic free NADH/NAD+ ratio and metabolites accumulation in strains from BY4742 and the ergosterol defected strains (ΔERG5) strains.All strains were cultivated in shake flasks and sampled after 24 h after they were inoculated into the fresh YPD medium. Columns with different shades of red color represent the strains from the wild type BY4742 and those with different shades of gray color represent the strains from ΔERG5. Significance levels of Students t-test: *P< 0.05, **P< 0.01, ***P< 0.001.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4476719&req=5

pone.0130840.g003: The cytosolic free NADH/NAD+ ratio and metabolites accumulation in strains from BY4742 and the ergosterol defected strains (ΔERG5) strains.All strains were cultivated in shake flasks and sampled after 24 h after they were inoculated into the fresh YPD medium. Columns with different shades of red color represent the strains from the wild type BY4742 and those with different shades of gray color represent the strains from ΔERG5. Significance levels of Students t-test: *P< 0.05, **P< 0.01, ***P< 0.001.
Mentions: During the fermentation in shake flasks, the cytosolic free NADH/NAD+ ratio and the amounts of glycerol and ethanol produced by strains derived from BY4742 and ΔERG5 were measured at 24 h in the stationary phase. The free NADH/NAD+ ratios and glycerol and ethanol accumulation in the ergosterol defected strains were higher than those of the strains from BY4742 with the same alterations. Especially, the free NADH/NAD+ ratio of SyBE_Sc01100002 was 0.011(±0.003), while the ratio of strains engineered from BY4742 were less than half of that value. At the same time, the glycerol produced by SyBE_Sc01100002 reached 1.402 g/L, which was the highest among all yeast strains in this study (Fig 3). Because excessive NADH in cytosol gave rise to the redox imbalance, decreasing the ratio of NADH/NAD+ by cofactor engineering became a rational approach to alleviate redox imbalance in the ergosterol defected strains.

Bottom Line: Maintaining redox balance is critical for the production of heterologous secondary metabolites, whereas on various occasions the native cofactor balance does not match the needs in engineered microorganisms.In the meanwhile, the ratio of free NADH/NAD+ and the concentration of glycerol and ethanol were reduced by 78.0%, 50.7% and 7.9% respectively.In a 5-L bioreactor, the optimal production of 7-DHC reached 44.49(±9.63) mg/L.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin, China; SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and Technology, Tianjin University, Tianjin, China.

ABSTRACT
Maintaining redox balance is critical for the production of heterologous secondary metabolites, whereas on various occasions the native cofactor balance does not match the needs in engineered microorganisms. In this study, 7-dehydrocholesterol (7-DHC, a crucial precursor of vitamin D3) biosynthesis pathway was constructed in Saccharomyces cerevisiae BY4742 with endogenous ergosterol synthesis pathway blocked by knocking out the erg5 gene (encoding C-22 desaturase). The deletion of erg5 led to redox imbalance with higher ratio of cytosolic free NADH/NAD+ and more glycerol and ethanol accumulation. To alleviate the redox imbalance, a water-forming NADH oxidase (NOX) and an alternative oxidase (AOX1) were employed in our system based on cofactor regeneration strategy. Consequently, the production of 7-dehydrocholesterol was increased by 74.4% in shake flask culture. In the meanwhile, the ratio of free NADH/NAD+ and the concentration of glycerol and ethanol were reduced by 78.0%, 50.7% and 7.9% respectively. In a 5-L bioreactor, the optimal production of 7-DHC reached 44.49(±9.63) mg/L. This study provides a reference to increase the production of some desired compounds that are restricted by redox imbalance.

No MeSH data available.


Related in: MedlinePlus