Engineering NAD availability for Escherichia coli whole-cell biocatalysis: a case study for dihydroxyacetone productionReportar como inadecuado

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Microbial Cell Factories

, 12:103

First Online: 09 November 2013Received: 10 July 2013Accepted: 05 November 2013


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 NADH 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 NADH level and DHA production, and the highest DHA productivity reached 0.76 g-gDCW-h. Cellular NADH 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 NADH 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.

KeywordsCofactor engineering NADH level NAD transporter Escherichia coli Dihydroxyacetone Whole-cell biocatalysis Electronic supplementary materialThe online version of this article doi:10.1186-1475-2859-12-103 contains supplementary material, which is available to authorized users.

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Autor: Yongjin J Zhou - Wei Yang - Lei Wang - Zhiwei Zhu - Sufang Zhang - Zongbao K Zhao


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