Effects of transcription levels of key enzyme genes on NAD+ production in recombinant Escherichia coli

Abstract:

[Objective] Nicotinamide adenine dinucleotide (NAD⁺) is an important cofactor in organisms, and its intracellular concentration plays an important role in NAD⁺-dependent redox reaction and related biochemical synthesis. In order to strengthen the synthesis of cofactors, we optimized the induction conditions and increased the copies of key enzyme genes to elevate the intracellular NAD⁺ concentration.[Methods] First, the induction temperature, inducer concentration, and induction time were optimized for the starting strain NA016 of Escherichia coli. Meanwhile, the transcriptional levels of overexpressed genes in metabolic modification were determined by real-time fluorescence quantitative PCR. Subsequently, the correlations between NAD⁺ content and the transcription levels of these overexpressed genes were explored. Finally, the copies of the genes positively regulating NAD⁺ production were up-regulated to improve the intracellular NAD⁺ concentration.[Results] At the optimized induction conditions, the NAD⁺ concentration in NA016 increased by 35.37%. The genes nadE and pncB positively regulated the production of NAD⁺, and up-regulating the copies of these two genes in NA016 increased the NAD⁺ concentration by 22.46% to 41.66 μmol/g DCW.[Conclusion] Optimizing the induction conditions and up-regulating the copy number of key enzyme genes can increase the NAD⁺ concentration. These research findings provide reference for the study of NAD⁺synthesis.