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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

Genetic modification in 7-DHC producing engineered yeast strains.(a) Genetic modification of the wild type BY4742 strain including the disruption of ergosterol synthesis (erg5 deletion), the construction of the 7-DHC biosynthesis (the introduction of dhcr24 and the over-expression of thmg1) and the enhancement of acetyl-CoA provision (the over-expression of adh2 and ald6 together with the introduction of acs and acl). Genes in red represent endogenous genes and genes in blue represent heterologous genes. (b) Intracellular acetyl-CoA in SyBE_Sc01100002 (before the enhancement in acetyl-CoA provision) and SyBE_Sc01100011 (after the enhancement in acetyl-CoA provision) was quantified in the mid-logarithmic phase (8 h) and stationary phase (16 h). (c) The amount of 7-dehydrocholesterol produced by engineered yeast was measured after 36 h shake flask culture.
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pone.0130840.g002: Genetic modification in 7-DHC producing engineered yeast strains.(a) Genetic modification of the wild type BY4742 strain including the disruption of ergosterol synthesis (erg5 deletion), the construction of the 7-DHC biosynthesis (the introduction of dhcr24 and the over-expression of thmg1) and the enhancement of acetyl-CoA provision (the over-expression of adh2 and ald6 together with the introduction of acs and acl). Genes in red represent endogenous genes and genes in blue represent heterologous genes. (b) Intracellular acetyl-CoA in SyBE_Sc01100002 (before the enhancement in acetyl-CoA provision) and SyBE_Sc01100011 (after the enhancement in acetyl-CoA provision) was quantified in the mid-logarithmic phase (8 h) and stationary phase (16 h). (c) The amount of 7-dehydrocholesterol produced by engineered yeast was measured after 36 h shake flask culture.

Mentions: The dhcr24 gene encodes a FAD-dependent oxidoreductase, which catalyzes the reduction of the delta-24 double bond of sterol intermediates [15]. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) reductase is known as the rate- limiting enzyme in early sterol biosynthesis of eukaryotic cells. A truncated form of the hmg1 gene, which encodes a protein that contains the catalytically active domain in C-terminal but lacks the N-terminal membrane-spanning region is proved to be effective to overcome the regulatory effects and to avoid karmella formation [25]. To construct a functional 7-DHC biosynthesis pathway, a Homo sapiens C-24 reductase (DHCR24) was introduced and the endogenous HMG-CoA reductase without membrane-binding region (tHMG1) was over-expressed in S.cerevisiae BY4742, obtaining SyBE_Sc01100001 (Fig 2A). However, ergosterol rather than 7-DHC was observed as the main product, indicating the natural ergosterol biosynthesis pathway competed much with 7-DHC formation pathway [26]. Thus the S.cerevisiae BY4742 erg5 mutant strain ΔERG5 was then chosen as chassis in this study to eliminate the competition of ergosterol biosynthesis pathway, and the result of GC-TOF-MS showed no detection of ergosterol (S2 Fig) accordingly. When the dhcr24 gene and thmg1 gene were introduced into ΔERG5 strain, obtaining SyBE_Sc01100002, a titer of 2.61±0.19 mg/L 7-DHC was obtained in this strain (Fig 2A and 2C).


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)

Genetic modification in 7-DHC producing engineered yeast strains.(a) Genetic modification of the wild type BY4742 strain including the disruption of ergosterol synthesis (erg5 deletion), the construction of the 7-DHC biosynthesis (the introduction of dhcr24 and the over-expression of thmg1) and the enhancement of acetyl-CoA provision (the over-expression of adh2 and ald6 together with the introduction of acs and acl). Genes in red represent endogenous genes and genes in blue represent heterologous genes. (b) Intracellular acetyl-CoA in SyBE_Sc01100002 (before the enhancement in acetyl-CoA provision) and SyBE_Sc01100011 (after the enhancement in acetyl-CoA provision) was quantified in the mid-logarithmic phase (8 h) and stationary phase (16 h). (c) The amount of 7-dehydrocholesterol produced by engineered yeast was measured after 36 h shake flask culture.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130840.g002: Genetic modification in 7-DHC producing engineered yeast strains.(a) Genetic modification of the wild type BY4742 strain including the disruption of ergosterol synthesis (erg5 deletion), the construction of the 7-DHC biosynthesis (the introduction of dhcr24 and the over-expression of thmg1) and the enhancement of acetyl-CoA provision (the over-expression of adh2 and ald6 together with the introduction of acs and acl). Genes in red represent endogenous genes and genes in blue represent heterologous genes. (b) Intracellular acetyl-CoA in SyBE_Sc01100002 (before the enhancement in acetyl-CoA provision) and SyBE_Sc01100011 (after the enhancement in acetyl-CoA provision) was quantified in the mid-logarithmic phase (8 h) and stationary phase (16 h). (c) The amount of 7-dehydrocholesterol produced by engineered yeast was measured after 36 h shake flask culture.
Mentions: The dhcr24 gene encodes a FAD-dependent oxidoreductase, which catalyzes the reduction of the delta-24 double bond of sterol intermediates [15]. The enzyme 3-hydroxy-3-methylglutaryl-coenzyme-A (HMG-CoA) reductase is known as the rate- limiting enzyme in early sterol biosynthesis of eukaryotic cells. A truncated form of the hmg1 gene, which encodes a protein that contains the catalytically active domain in C-terminal but lacks the N-terminal membrane-spanning region is proved to be effective to overcome the regulatory effects and to avoid karmella formation [25]. To construct a functional 7-DHC biosynthesis pathway, a Homo sapiens C-24 reductase (DHCR24) was introduced and the endogenous HMG-CoA reductase without membrane-binding region (tHMG1) was over-expressed in S.cerevisiae BY4742, obtaining SyBE_Sc01100001 (Fig 2A). However, ergosterol rather than 7-DHC was observed as the main product, indicating the natural ergosterol biosynthesis pathway competed much with 7-DHC formation pathway [26]. Thus the S.cerevisiae BY4742 erg5 mutant strain ΔERG5 was then chosen as chassis in this study to eliminate the competition of ergosterol biosynthesis pathway, and the result of GC-TOF-MS showed no detection of ergosterol (S2 Fig) accordingly. When the dhcr24 gene and thmg1 gene were introduced into ΔERG5 strain, obtaining SyBE_Sc01100002, a titer of 2.61±0.19 mg/L 7-DHC was obtained in this strain (Fig 2A and 2C).

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