Engineering NAD+ availability for Escherichia coli whole-cell biocatalysis: a case study for dihydroxyacetone production

Microbial Cell Factories

Research

Wei Yang¹  Zhiwei Zhu¹  Lei Wang¹  Sufang Zhang²  Zongbao K Zhao³  Yongjin J Zhou⁴ 

[1] Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, 116023, Dalian, China;Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, 116023, Dalian, China;Dalian National Laboratory for Clean Energy, Dalian Institute of Chemical Physics, CAS, 116023, Dalian, China;Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, 116023, Dalian, China;Dalian National Laboratory for Clean Energy, Dalian Institute of Chemical Physics, CAS, 116023, Dalian, China;State Key Laboratory of Catalysis, Dalian Institute of Chemical Physics, CAS, 116023, Dalian, China;Division of Biotechnology, Dalian Institute of Chemical Physics, CAS, 116023, Dalian, China;Department of Chemical and Biological Engineering, Chalmers University of Technology, Kemivägen 10, SE-412 96, Gothenburg, Sweden;

关键词: Cofactor engineering;    NAD(H) level;    NAD transporter;    Escherichia coli;    Dihydroxyacetone;    Whole-cell biocatalysis;   

DOI  :  10.1186/1475-2859-12-103

 received in 2013-07-10, accepted in 2013-11-05,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundWhole-cell redox biocatalysis has been intensively explored for the production of valuable compounds because excellent selectivity is routinely achieved. Although the cellular cofactor level, redox state and the corresponding enzymatic activity are expected to have major effects on the performance of the biocatalysts, our ability remains limited to predict the outcome upon variation of those factors as well as the relationship among them.ResultsIn order to investigate the effects of cofactor availability on whole-cell redox biocatalysis, we devised recombinant Escherichia coli strains for the production of dihydroxyacetone (DHA) catalyzed by the NAD+-dependent glycerol dehydrogenase (GldA). In this model system, a water-forming NAD+ oxidase (NOX) and a NAD+ transporter (NTT4) were also co-expressed for cofactor regeneration and extracellular NAD+ uptake, respectively. We found that cellular cofactor level, NAD+/NADH ratio and NOX activity were not only strain-dependent, but also growth condition-dependent, leading to significant differences in specific DHA titer among different whole-cell biocatalysts. The host E. coli DH5α had the highest DHA specific titer of 0.81 g/gDCW with the highest NAD+/NADH ratio of 6.7 and NOX activity of 3900 U. The biocatalyst had a higher activity when induced with IPTG at 37°C for 8 h compared with those at 30°C for 8 h and 18 h. When cells were transformed with the ntt4 gene, feeding NAD+ during the cell culture stage increased cellular NAD(H) level by 1.44 fold and DHA specific titer by 1.58 fold to 2.13 g/gDCW. Supplementing NAD+ during the biotransformation stage was also beneficial to cellular NAD(H) level and DHA production, and the highest DHA productivity reached 0.76 g/gDCW/h. Cellular NAD(H) level, NAD+/NADH ratio, and NOX and GldA activity dropped over time during the biotransformation process.ConclusionsHigh NAD+/NADH ratio driving by NOX was very important for DHA production. Once cofactor was efficiently cycled, high cellular NAD(H) level was also beneficial for whole-cell redox biocatalysis. Our results indicated that NAD+ transporter could be applied to manipulate redox cofactor level for biocatalysis. Moreover, we suggested that genetically designed redox transformation should be carefully profiled for further optimizing whole-cell biocatalysis.

【 授权许可】

Unknown   
© Zhou et al.; licensee BioMed Central Ltd. 2013. This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.