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

Microbial Cell Factories

Research

Joeri Beauprez¹  Wim K. Soetaert¹  Sofie L. De Maeseneire¹  Marjan De Mey¹  Fatma G. Avci²  Veerle E. T. Maervoet³ 

[1] Department of Biochemical and Microbial Technology, Centre of Expertise-Industrial Biotechnology and Biocatalysis, Ghent University, Coupure links 653, 9000, Ghent, Belgium;Department of Biochemical and Microbial Technology, Centre of Expertise-Industrial Biotechnology and Biocatalysis, Ghent University, Coupure links 653, 9000, Ghent, Belgium;Bioengineering Department, Faculty of Engineering, Ege University, 35100, Bornova-Izmir, Turkey;Department of Biochemical and Microbial Technology, Centre of Expertise-Industrial Biotechnology and Biocatalysis, Ghent University, Coupure links 653, 9000, Ghent, Belgium;Department of Applied Bioscience Engineering, Laboratory of Applied Biotechnology, Ghent University, Valentin Vaerwyckweg 1, 9000, Ghent, Belgium;

关键词: 1,3-propanediol;    Glycerol;    Citrobacter werkmanii;    NADH;    Lactate dehydrogenase;    Ethanol dehydrogenase;    Multiple knock-out mutant;    Rational engineering;   

DOI  :  10.1186/s12934-016-0421-y

 received in 2015-07-01, accepted in 2016-01-13,  发布年份 2016

来源: Springer

【 摘 要 】

BackgroundImbalance 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 ∆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.ResultsBy 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 ∆dhaD resulted in the strain C. werkmanii ∆dhaD∆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.ConclusionsIn 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.

【 授权许可】

CC BY   
© Maervoet et al. 2016