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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

Effects of NADH regeneration system on 7-DHC production, by-product accumulation and cytosolic free NADH/NAD+ ratio.(a) Cofactor regeneration system consisted of the H2O-forming NADH oxidase (NOX) and the alternative oxidase (AOX1). The NOX localizes in the cytosol, whereas AOX1 is directed to the mitochondria. NOX primarily impacts glycerol production, while AOX1 influences ethanol formation. SyBE_Sc01100011 was ΔERG5 with the 7-DHC biosynthesis pathway (the introduction of dhcr24 and the over-expression of thmg1) and the acetyl-CoA provision enhancement module (the over-expression of adh2 and ald6 together with the introduction of acs and acl). SyBE_Sc01100020 was SyBE_Sc01100011 with the cofactor regeneration system (the introduction of nox and aox1). All strains were cultivated in shake flasks and sampled after 24h after they were inoculated into the fresh YPD medium. (b) 7-DHC production was measured after 36 h shake flask culture.
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pone.0130840.g004: Effects of NADH regeneration system on 7-DHC production, by-product accumulation and cytosolic free NADH/NAD+ ratio.(a) Cofactor regeneration system consisted of the H2O-forming NADH oxidase (NOX) and the alternative oxidase (AOX1). The NOX localizes in the cytosol, whereas AOX1 is directed to the mitochondria. NOX primarily impacts glycerol production, while AOX1 influences ethanol formation. SyBE_Sc01100011 was ΔERG5 with the 7-DHC biosynthesis pathway (the introduction of dhcr24 and the over-expression of thmg1) and the acetyl-CoA provision enhancement module (the over-expression of adh2 and ald6 together with the introduction of acs and acl). SyBE_Sc01100020 was SyBE_Sc01100011 with the cofactor regeneration system (the introduction of nox and aox1). All strains were cultivated in shake flasks and sampled after 24h after they were inoculated into the fresh YPD medium. (b) 7-DHC production was measured after 36 h shake flask culture.

Mentions: In this study, the NADH regeneration system consisted of two NADH oxidases: NOX and AOX1 (Fig 4A). H2O-forming NADH oxidase (NOX) purified from Streptococcus pneumoniae is a soluble flavoprotein that reoxidizes NADH and reduces molecular O2 to water [17]. Histoplasma capsulatum alternative oxidase (AOX1) possesses cyanide-insensitive oxygen-consuming activities [18]. When the heterologous NADH oxidases were expressed in S. cerevisiae, the NOX localized in the cytosol, whereas AOX1 was directed to the mitochondria [4]. NOX primarily impacts glycerol production by mediating the non-respiratory dissipation of NADH [4]. The glycerol synthesis pathway is activated in S. cerevisiae as the outlet for NADH consumption [35] and NOX relieves the need to generating glycerol [4]. On the other hand, aerobic ethanol formation in S. cerevisiae is the result of a limitation in electron transport from NADH to oxygen [36]. AOX1 up-regulates almost every step of TCA cycle and recruits parts of the respiratory system in the transfer of electrons from NADH to oxygen [4]. Both of these two NADH oxidases were incorporated into SyBE_Sc01100011 to obtain SyBE_Sc01100020 in our study. Consequently, the cytosolic free NADH/NAD+ ratio and the concentration of glycerol and ethanol were reduced by 78.0% (from 0.010±0.001 to 0.002±0.001), 50.7% (from 1.006±0.003 g/L to 0.496±0.003 g/L) and 7.9% (from 8.463±0.077 g/L to 7.788±0.035 g/L) respectively (Fig 4A). The production of 7-DHC in SyBE_Sc01100020 was increased by 74.4% (from 4.84±0.75 mg/L to 8.44±0.42 mg/L) (Fig 4B). And when the SyBE_Sc01100020 strain was cultivated in a 5-L bioreactor under glucose restriction strategy (<2 g/L), the production of 7-DHC reached 44.49±9.63 mg/L, which was the highest reported titer as known so far (Fig 5).


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)

Effects of NADH regeneration system on 7-DHC production, by-product accumulation and cytosolic free NADH/NAD+ ratio.(a) Cofactor regeneration system consisted of the H2O-forming NADH oxidase (NOX) and the alternative oxidase (AOX1). The NOX localizes in the cytosol, whereas AOX1 is directed to the mitochondria. NOX primarily impacts glycerol production, while AOX1 influences ethanol formation. SyBE_Sc01100011 was ΔERG5 with the 7-DHC biosynthesis pathway (the introduction of dhcr24 and the over-expression of thmg1) and the acetyl-CoA provision enhancement module (the over-expression of adh2 and ald6 together with the introduction of acs and acl). SyBE_Sc01100020 was SyBE_Sc01100011 with the cofactor regeneration system (the introduction of nox and aox1). All strains were cultivated in shake flasks and sampled after 24h after they were inoculated into the fresh YPD medium. (b) 7-DHC production was measured after 36 h shake flask culture.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130840.g004: Effects of NADH regeneration system on 7-DHC production, by-product accumulation and cytosolic free NADH/NAD+ ratio.(a) Cofactor regeneration system consisted of the H2O-forming NADH oxidase (NOX) and the alternative oxidase (AOX1). The NOX localizes in the cytosol, whereas AOX1 is directed to the mitochondria. NOX primarily impacts glycerol production, while AOX1 influences ethanol formation. SyBE_Sc01100011 was ΔERG5 with the 7-DHC biosynthesis pathway (the introduction of dhcr24 and the over-expression of thmg1) and the acetyl-CoA provision enhancement module (the over-expression of adh2 and ald6 together with the introduction of acs and acl). SyBE_Sc01100020 was SyBE_Sc01100011 with the cofactor regeneration system (the introduction of nox and aox1). All strains were cultivated in shake flasks and sampled after 24h after they were inoculated into the fresh YPD medium. (b) 7-DHC production was measured after 36 h shake flask culture.
Mentions: In this study, the NADH regeneration system consisted of two NADH oxidases: NOX and AOX1 (Fig 4A). H2O-forming NADH oxidase (NOX) purified from Streptococcus pneumoniae is a soluble flavoprotein that reoxidizes NADH and reduces molecular O2 to water [17]. Histoplasma capsulatum alternative oxidase (AOX1) possesses cyanide-insensitive oxygen-consuming activities [18]. When the heterologous NADH oxidases were expressed in S. cerevisiae, the NOX localized in the cytosol, whereas AOX1 was directed to the mitochondria [4]. NOX primarily impacts glycerol production by mediating the non-respiratory dissipation of NADH [4]. The glycerol synthesis pathway is activated in S. cerevisiae as the outlet for NADH consumption [35] and NOX relieves the need to generating glycerol [4]. On the other hand, aerobic ethanol formation in S. cerevisiae is the result of a limitation in electron transport from NADH to oxygen [36]. AOX1 up-regulates almost every step of TCA cycle and recruits parts of the respiratory system in the transfer of electrons from NADH to oxygen [4]. Both of these two NADH oxidases were incorporated into SyBE_Sc01100011 to obtain SyBE_Sc01100020 in our study. Consequently, the cytosolic free NADH/NAD+ ratio and the concentration of glycerol and ethanol were reduced by 78.0% (from 0.010±0.001 to 0.002±0.001), 50.7% (from 1.006±0.003 g/L to 0.496±0.003 g/L) and 7.9% (from 8.463±0.077 g/L to 7.788±0.035 g/L) respectively (Fig 4A). The production of 7-DHC in SyBE_Sc01100020 was increased by 74.4% (from 4.84±0.75 mg/L to 8.44±0.42 mg/L) (Fig 4B). And when the SyBE_Sc01100020 strain was cultivated in a 5-L bioreactor under glucose restriction strategy (<2 g/L), the production of 7-DHC reached 44.49±9.63 mg/L, which was the highest reported titer as known so far (Fig 5).

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