Intracellular redox state of electricity active cells (EAC) is one of the most important physiological traits of extracellular electron transfer efficiency. In particular, the NAD+(H) pool size plays a central role of most metabolic pathways. By overexpressing the NAD synthetase, encoded be gene nadE and catalyzes the final step in de novo synthesis and salvage pathway of NAD biosynthesis (Fig. 1), the NAD+ level is increased thereby up-regulating genes whose products catalyze NADH synthesis. Therefore the augmented pool size of NAD+(H) result in promotion of NADH(the carrier of electrons)level, leading to high generation of intracellular releasable electrons and better electricity performance of EAC. [1]
Fig. 1 De novo synthesis and salvage pathway of NAD biosynthesis.
HUST-China 2023
We codon optimized the sequence of nadE for S. cerevisiae and determined its effect on intracellular NAD(H/+) concentration when co-expressed with nadD[BBa_K4595014], nadM[BBa_K4595015], and the experimental results proved that it could effectively increase the intracellular NAD(H/+) concentration.
Fig.2 The NAD(H/+) concentration of S. oneidensis MR-1, S. oneidensis MR-1(ycel-pncB), S. oneidensis MR-1(nadE-nadD-nadM).
We Compared to the wild type, the total amount of NAD(H/+) in S. oneidensis MR-1(nadD-nadE-nadM) increased by 27.34%. This indicates that, S. oneidensis MR-1(nadD-nadE-nadM) facilitates more efficient electron transfer.
In addition, we also determined the effect of nadE on the electroproduction capacity of S. cerevisiae when co-expressed with nadD and nadM, and the results showed that the three co-expression could effectively increase the electroproduction efficiency of S. cerevisiae.
Fig.3 The out put voltage of S.oneidensis MR-1, S.oneidensis MR-1(ycel-pncB), S.oneidensis MR-1(nadD-nadE-nadM) when the anoditic solution is M9 buffer and 18mM lactate.
The results showed that S. oneidensis MR-1(nadD-nadE-nadM) significantly higher discharge peak and prolonged high-efficiency discharge duration compared to the wild type. The highest out put voltage was up to 150.7 mV, with a 42.32% increase in the hightest power output .. It is speculated that this could be attributed to the ability ofS. oneidensis MR-1(nadD-nadE-nadM) to accelerate intracellular NADH synthesis, resulting in a higher power output.
Fig. 1 De novo synthesis and salvage pathway of NAD biosynthesis.
