November 2018, Volume 45, Issue 11, pp 939–950 | Cite as

Enhancement of NAD(H) pool for formation of oxidized biochemicals in Escherichia coli

Fermentation, Cell Culture and Bioengineering - Original Paper

The NAD+/NADH ratio and the total NAD(H) play important roles for whole-cell biochemical redox transformations. After the carbon source is exhausted, the degradation of NAD(H) could contribute to a decline in the rate of a desired conversion. In this study, methods to slow the native rate of NAD(H) degradation were examined using whole-cell Escherichia coli with two model oxidative NAD+-dependent biotransformations. A high phosphate concentration (50 mM) was observed to slow NAD(H) degradation. We also constructed E. coli strains with deletions in genes coding several enzymes involved in NAD+ degradation. In shake-flask experiments, the total NAD(H) concentration positively correlated with conversion of xylitol to l-xylulose by xylitol 4-dehydrogenase, and the greatest conversion (80%) was observed using MG1655 nadR nudC mazG/pZE12-xdh/pCS27-nox. Controlled 1-L batch processes comparing E. coli nadR nudC mazG with a wild-type background strain demonstrated a 30% increase in final l-xylulose concentration (5.6 vs. 7.9 g/L) and a 25% increase in conversion (0.53 vs. 0.66 g/g). MG1655 nadR nudC mazG was also examined for the conversion of galactitol to l-tagatose by galactitol 2-dehydrogenase. A batch process using 15 g/L glycerol and 10 g/L galactitol generated over 9.4 g/L l-tagatose, corresponding to 90% conversion and a yield of 0.95 g l-tagatose/g galactitol consumed. The results demonstrate the value of minimizing NAD(H) degradation as a means to improve NAD+-dependent biotransformations.

Biotransformation Cofactor engineering NAD+ degradation l-Tagatose l-Xylulose

The online version of this article ( https://doi.org/10.1007/s10295-018-2072-y) contains supplementary material, which is available to authorized users.

The authors thank Sarah Lee for the technical assistance.

10295_2018_2072_MOESM1_ESM.docx (19 kb)
Supplementary material 1 (DOCX 19 kb)
10295_2018_2072_MOESM2_ESM.docx (22 kb)
Supplementary material 2 (DOCX 22 kb)
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