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High yield 1,3-propanediol production by rational engineering of the 3-hydroxypropionaldehyde bottleneck in Citrobacter werkmanii

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Abstract
Background: Imbalance in cofactors causing the accumulation of intermediates in biosynthesis pathways is a frequently occurring problem in metabolic engineering when optimizing a production pathway in a microorganism. In our previous study, a single knock-out Citrobacter werkmanii Delta dhaD was constructed for improved 1,3-propanediol (PDO) production. Instead of an enhanced PDO concentration on this strain, the gene knock-out led to the accumulation of the toxic intermediate 3-hydroxypropionaldehyde (3-HPA). The hypothesis was emerged that the accumulation of this toxic intermediate, 3-HPA, is due to a cofactor imbalance, i.e. to the limited supply of reducing equivalents (NADH). Here, this bottleneck is alleviated by rationally engineering cell metabolism to balance the cofactor supply. Results: By eliminating non-essential NADH consuming enzymes (such as lactate dehydrogenase coded by ldhA, and ethanol dehydrogenase coded by adhE) or by increasing NADH producing enzymes, the accumulation of 3-HPA is minimized. Combining the above modifications in C. werkmanii Delta dhaD resulted in the strain C. werkmanii Delta dhaD Delta ldhA.adhE::ChlFRT which provided the maximum theoretical yield of 1.00 +/- 0.03 mol PDO/mol glycerol when grown on glucose/glycerol (0.33 molar ratio) on flask scale under anaerobic conditions. On bioreactor scale, the yield decreased to 0.73 +/- 0.01 mol PDO/mol glycerol although no 3-HPA could be measured, which indicates the existence of a sink of glycerol by a putative glycerol dehydrogenase, channeling glycerol to the central metabolism. Conclusions: In this study, a multiple knock-out was created in Citrobacter species for the first time. As a result, the concentration of the toxic intermediate 3-HPA was reduced to below the detection limit and the maximal theoretical PDO yield on glycerol was reached.
Keywords
1_3-propanediol, Glycerol, NADH, ARCA, GENES, CONVERSION, STRAIN, METABOLISM, GLYCEROL, ALDEHYDE DEHYDROGENASE, MICROAEROBIC CONDITIONS, KLEBSIELLA-PNEUMONIAE, ESCHERICHIA-COLI, Rational engineering, Multiple knock-out mutant, Lactate dehydrogenase, Ethanol dehydrogenase, Citrobacter werkmanii DSM17579

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MLA
Maervoet, Veerle, et al. “High Yield 1,3-Propanediol Production by Rational Engineering of the 3-Hydroxypropionaldehyde Bottleneck in Citrobacter Werkmanii.” MICROBIAL CELL FACTORIES, vol. 15, 2016, doi:10.1186/s12934-016-0421-y.
APA
Maervoet, V., De Maeseneire, S., Avcı, F. G., Beauprez, J., Soetaert, W., & De Mey, M. (2016). High yield 1,3-propanediol production by rational engineering of the 3-hydroxypropionaldehyde bottleneck in Citrobacter werkmanii. MICROBIAL CELL FACTORIES, 15. https://doi.org/10.1186/s12934-016-0421-y
Chicago author-date
Maervoet, Veerle, Sofie De Maeseneire, Fatma Gizem Avcı, Joeri Beauprez, Wim Soetaert, and Marjan De Mey. 2016. “High Yield 1,3-Propanediol Production by Rational Engineering of the 3-Hydroxypropionaldehyde Bottleneck in Citrobacter Werkmanii.” MICROBIAL CELL FACTORIES 15. https://doi.org/10.1186/s12934-016-0421-y.
Chicago author-date (all authors)
Maervoet, Veerle, Sofie De Maeseneire, Fatma Gizem Avcı, Joeri Beauprez, Wim Soetaert, and Marjan De Mey. 2016. “High Yield 1,3-Propanediol Production by Rational Engineering of the 3-Hydroxypropionaldehyde Bottleneck in Citrobacter Werkmanii.” MICROBIAL CELL FACTORIES 15. doi:10.1186/s12934-016-0421-y.
Vancouver
1.
Maervoet V, De Maeseneire S, Avcı FG, Beauprez J, Soetaert W, De Mey M. High yield 1,3-propanediol production by rational engineering of the 3-hydroxypropionaldehyde bottleneck in Citrobacter werkmanii. MICROBIAL CELL FACTORIES. 2016;15.
IEEE
[1]
V. Maervoet, S. De Maeseneire, F. G. Avcı, J. Beauprez, W. Soetaert, and M. De Mey, “High yield 1,3-propanediol production by rational engineering of the 3-hydroxypropionaldehyde bottleneck in Citrobacter werkmanii,” MICROBIAL CELL FACTORIES, vol. 15, 2016.
@article{7064021,
  abstract     = {{Background: Imbalance in cofactors causing the accumulation of intermediates in biosynthesis pathways is a frequently occurring problem in metabolic engineering when optimizing a production pathway in a microorganism. In our previous study, a single knock-out Citrobacter werkmanii Delta dhaD was constructed for improved 1,3-propanediol (PDO) production. Instead of an enhanced PDO concentration on this strain, the gene knock-out led to the accumulation of the toxic intermediate 3-hydroxypropionaldehyde (3-HPA). The hypothesis was emerged that the accumulation of this toxic intermediate, 3-HPA, is due to a cofactor imbalance, i.e. to the limited supply of reducing equivalents (NADH). Here, this bottleneck is alleviated by rationally engineering cell metabolism to balance the cofactor supply. 
Results: By eliminating non-essential NADH consuming enzymes (such as lactate dehydrogenase coded by ldhA, and ethanol dehydrogenase coded by adhE) or by increasing NADH producing enzymes, the accumulation of 3-HPA is minimized. Combining the above modifications in C. werkmanii Delta dhaD resulted in the strain C. werkmanii Delta dhaD Delta ldhA.adhE::ChlFRT which provided the maximum theoretical yield of 1.00 +/- 0.03 mol PDO/mol glycerol when grown on glucose/glycerol (0.33 molar ratio) on flask scale under anaerobic conditions. On bioreactor scale, the yield decreased to 0.73 +/- 0.01 mol PDO/mol glycerol although no 3-HPA could be measured, which indicates the existence of a sink of glycerol by a putative glycerol dehydrogenase, channeling glycerol to the central metabolism. 
Conclusions: In this study, a multiple knock-out was created in Citrobacter species for the first time. As a result, the concentration of the toxic intermediate 3-HPA was reduced to below the detection limit and the maximal theoretical PDO yield on glycerol was reached.}},
  articleno    = {{23}},
  author       = {{Maervoet, Veerle and De Maeseneire, Sofie and Avcı, Fatma Gizem and Beauprez, Joeri and Soetaert, Wim and De Mey, Marjan}},
  issn         = {{1475-2859}},
  journal      = {{MICROBIAL CELL FACTORIES}},
  keywords     = {{1_3-propanediol,Glycerol,NADH,ARCA,GENES,CONVERSION,STRAIN,METABOLISM,GLYCEROL,ALDEHYDE DEHYDROGENASE,MICROAEROBIC CONDITIONS,KLEBSIELLA-PNEUMONIAE,ESCHERICHIA-COLI,Rational engineering,Multiple knock-out mutant,Lactate dehydrogenase,Ethanol dehydrogenase,Citrobacter werkmanii DSM17579}},
  language     = {{eng}},
  pages        = {{11}},
  title        = {{High yield 1,3-propanediol production by rational engineering of the 3-hydroxypropionaldehyde bottleneck in Citrobacter werkmanii}},
  url          = {{http://dx.doi.org/10.1186/s12934-016-0421-y}},
  volume       = {{15}},
  year         = {{2016}},
}

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