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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

Plasmids construction via RADOM method.The red bars represent the homologous regions between different DNA fragments and vectors. The yellow and blue bars represent the promoter and terminator of each gene. The restriction sites are emphasized in red. (a) Plasmid pSW01 contains truncated HMG-CoA reductase (tHMG1) encoding gene and C-24 reductase (DHCR24) encoding gene. (b) Plasmid pSW02 contains alcohol dehydrogenase (ADH2) encoding gene, acetaldehyde dehydrogenase (ALD6) encoding gene, acetyl-CoA synthetase (ACS) variant encoding gene and ATP-dependent citrate lyase (ACL) encoding gene. (c) Plasmid pSW03 contains water-forming NADH oxidase (NOX) encoding gene and alternative oxidase (AOX1) encoding gene. (d) Plasmid pSW04 contains mannitol-1-phosphate 5-dehydrogenase encoding gene mtlD.
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pone.0130840.g001: Plasmids construction via RADOM method.The red bars represent the homologous regions between different DNA fragments and vectors. The yellow and blue bars represent the promoter and terminator of each gene. The restriction sites are emphasized in red. (a) Plasmid pSW01 contains truncated HMG-CoA reductase (tHMG1) encoding gene and C-24 reductase (DHCR24) encoding gene. (b) Plasmid pSW02 contains alcohol dehydrogenase (ADH2) encoding gene, acetaldehyde dehydrogenase (ALD6) encoding gene, acetyl-CoA synthetase (ACS) variant encoding gene and ATP-dependent citrate lyase (ACL) encoding gene. (c) Plasmid pSW03 contains water-forming NADH oxidase (NOX) encoding gene and alternative oxidase (AOX1) encoding gene. (d) Plasmid pSW04 contains mannitol-1-phosphate 5-dehydrogenase encoding gene mtlD.

Mentions: Each individual gene expression cassette was assembled by overlap extension PCR (OE-PCR) and gel purified by TIANgel Midi Purification Kit (TIANGEN Biotech, DP209). The resulting fragment TPI1p-mtlD-PGK1t was digested with NotI, and inserted into the corresponding site of pRS426. Other gene expression cassettes sharing 40bp homologous regions with the adjacent fragments or linearized vector were assembled based on RADOM method [20] (Fig 1). The correct clones were verified by PCR verification and restriction enzyme digestion. To integrate the mtlD into the genome of the strains, three fragments pAgTEF1-bleMX-tAgTEF1, TPI1p-mtlD-PGK1t and ERG5-down were obtained by OE-PCR. All plasmids and engineered yeast strains constructed were listed in Table 2 and Table 3.


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)

Plasmids construction via RADOM method.The red bars represent the homologous regions between different DNA fragments and vectors. The yellow and blue bars represent the promoter and terminator of each gene. The restriction sites are emphasized in red. (a) Plasmid pSW01 contains truncated HMG-CoA reductase (tHMG1) encoding gene and C-24 reductase (DHCR24) encoding gene. (b) Plasmid pSW02 contains alcohol dehydrogenase (ADH2) encoding gene, acetaldehyde dehydrogenase (ALD6) encoding gene, acetyl-CoA synthetase (ACS) variant encoding gene and ATP-dependent citrate lyase (ACL) encoding gene. (c) Plasmid pSW03 contains water-forming NADH oxidase (NOX) encoding gene and alternative oxidase (AOX1) encoding gene. (d) Plasmid pSW04 contains mannitol-1-phosphate 5-dehydrogenase encoding gene mtlD.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0130840.g001: Plasmids construction via RADOM method.The red bars represent the homologous regions between different DNA fragments and vectors. The yellow and blue bars represent the promoter and terminator of each gene. The restriction sites are emphasized in red. (a) Plasmid pSW01 contains truncated HMG-CoA reductase (tHMG1) encoding gene and C-24 reductase (DHCR24) encoding gene. (b) Plasmid pSW02 contains alcohol dehydrogenase (ADH2) encoding gene, acetaldehyde dehydrogenase (ALD6) encoding gene, acetyl-CoA synthetase (ACS) variant encoding gene and ATP-dependent citrate lyase (ACL) encoding gene. (c) Plasmid pSW03 contains water-forming NADH oxidase (NOX) encoding gene and alternative oxidase (AOX1) encoding gene. (d) Plasmid pSW04 contains mannitol-1-phosphate 5-dehydrogenase encoding gene mtlD.
Mentions: Each individual gene expression cassette was assembled by overlap extension PCR (OE-PCR) and gel purified by TIANgel Midi Purification Kit (TIANGEN Biotech, DP209). The resulting fragment TPI1p-mtlD-PGK1t was digested with NotI, and inserted into the corresponding site of pRS426. Other gene expression cassettes sharing 40bp homologous regions with the adjacent fragments or linearized vector were assembled based on RADOM method [20] (Fig 1). The correct clones were verified by PCR verification and restriction enzyme digestion. To integrate the mtlD into the genome of the strains, three fragments pAgTEF1-bleMX-tAgTEF1, TPI1p-mtlD-PGK1t and ERG5-down were obtained by OE-PCR. All plasmids and engineered yeast strains constructed were listed in Table 2 and Table 3.

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