High yield 1, 3-propanediol production by rational engineering of the 3-hydroxypropionaldehyde bottleneck in Citrobacter werkmanii. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Maervoet, Veerle; De Maeseneire, Sofie; Avci, Fatma; Beauprez, Joeri; Soetaert, Wim; De Mey, Marjan
• Is Part Of: Microbial cell factories. Volume 15:Issue 1(2016)
• Page Start: 1
• Page End: 11
• Publication Date: 2016-12
• Subjects: 1, 3-propanediol -- Glycerol -- Citrobacter werkmanii DSM17579 -- NADH -- Lactate dehydrogenase -- Ethanol dehydrogenase -- Multiple knock-out mutant -- Rational engineering; Microbial biotechnology -- Periodicals; Recombinant proteins -- Synthesis -- Periodicals; 660.62
• DOI: 10.1186/s12934-016-0421-y
• Languages: English
• ISSNs: 1475-2859

Abstract

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-outCitrobacter 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. Results By eliminating non-essential NADH consuming enzymes (such as lactate dehydrogenase coded byldhA, and ethanol dehydrogenase coded byadhE ) or by increasing NADH producing enzymes, the accumulation of 3-HPA is minimized. Combining the above modifications inC. werkmanii ∆dhaD resulted in the strainC. 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. Conclusions In this study, a multiple knock-out was created inCitrobacter 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.