Advanced search
1 file | 1.75 MB

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

Author
Organization
Project
IWT-Vlaanderen: B/14045/07
Project
Biotechnology for a sustainable economy (Bio-Economy)
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

Downloads

  • final.pdf
    • full text
    • |
    • open access
    • |
    • PDF
    • |
    • 1.75 MB

Citation

Please use this url to cite or link to this publication:

Chicago
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.
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.
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.
MLA
Maervoet, Veerle, Sofie De Maeseneire, Fatma Gizem Avcı, et al. “High Yield 1,3-propanediol Production by Rational Engineering of the 3-hydroxypropionaldehyde Bottleneck in Citrobacter Werkmanii.” MICROBIAL CELL FACTORIES 15 (2016): n. pag. Print.
@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 Avc{\i}, Fatma Gizem and Beauprez, Joeri and Soetaert, Wim and De Mey, Marjan},
  issn         = {1475-2859},
  journal      = {MICROBIAL CELL FACTORIES},
  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},
}

Altmetric
View in Altmetric
Web of Science
Times cited: