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

Veerle E T Maervoet,Marjan De Mey,Joeri Beauprez,Wim K Soetaert,Sofie L De Maeseneire,Fatma G Avci

doi:10.1186/s12934-016-0421-y

Veerle E T Maervoet, Marjan De Mey + Show 4 more

Open Access

https://doi.org/10.1186/s12934-016-0421-y

Copy DOI

Journal: Microbial Cell Factories	Publication Date: Jan 28, 2016
Citations: 30	License type: cc-by

Affiliation: Ghent University, Ege University

Abstract

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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0421-y) contains supplementary material, which is available to authorized users.

Highlights

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
Zhang et al [6] and Luo et al [7] have increased the yield of PDO on glycerol using an in vivo cofactor regeneration system which converts NAD+ into NADH, while, respectively, CO2 is produced from formate by formate dehydrogenase, and 3-HPA is changed into 3-hydroxypropionic acid by an aldehyde dehydrogenase AldH
When the triple mutant was grown under anaerobic conditions in medium with glycerol as sole carbon source, the specific GDH activity is three times higher than when the strain is grown under the same conditions in medium with glycerol and glucose. These results indicate that a promiscuous dehydrogenase is responsible for the utilization of glycerol for cell growth and maintenance, or that a second glycerol dehydrogenase coding gene is present in the genome of C. werkmanii DSM17579

Summary

Introduction

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. 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) This bottleneck is alleviated by rationally engineering cell metabolism to balance the cofactor supply. For the deletion of the genes which products consume NADH, the focus was laid on the lactate dehydrogenase gene ldhA in Klebsiella oxytoca M5a1 [8] and K. pneumoniae HR526 [9] and on an aldehyde dehydrogenase gene aldA in K. pneumoniae YMU2 [10]. In both cases one NADH is consumed per reaction. Deletion of the genes resulted in a significantly enhanced NADH/NAD+ ratio, and an increased yield on glycerol of PDO and improved final PDO titer in Klebsiella sp. [8,9,10,11]

Methods

Results

Conclusion