Cloning and expression of key enzymes for NAD+ synthesis and optimization of fermentation in Escherichia coli

Abstract:

[Objective] Nicotinamide adenine dinucleotide (NAD⁺) plays a crucial role in controlling metabolism network. Improving its intracellular concentration or getting a high-NAD⁺-yield strain is of great significance for NAD⁺-dependent redox reaction rate.[Methods] First, we used endogenous regulation means to enhance the key genes of NAD⁺ synthesis pathway, such as over-expressing and co-expressing the key enzyme genes pncB, nadD and nadE. Second, we optimized NAD⁺ precursors supplement and fermentation conditions to increase NAD⁺ synthesis. Finally, we used Box-Bohnken method for optimal synthesis condition by 3 significant factors' interaction based on single-factor experiments and the response value of NAD⁺ content.[Results] According to different expression strategies, we constructed seven recombinant strains. Besides, the intracellular NAD⁺ content of the recombinant strain E. coli BL21/pET-21a-nadE-pncB was 405.2% higher than that of the original strain. Moreover, after optimization of induction conditions and NAD⁺ precursor concentration by Design Expert 8.0, NAD⁺ content reached 43.16 μmol/g DCW, 123.6% higher than that before optimization and 1029.8% higher than the original strain.[Conclusion] Co-expression the key enzyme genes pncB and nadE are essential to improve NAD⁺ synthesis. The recombinant strain with high NAD⁺ provides the feasibility to improve biocatalytic efficiency.

Key words:nicotinamide adenine dinucleotide;E.coli;pncB;nadD;nadE;NAD⁺ precursor;fermentation optimization