Enhancing production of nicotinamide mononucleotide by metabolic engineering and flow cytometry screening of Escherichia coli
摘要
β-Nicotinamide mononucleotide (NMN) is a nucleotide that serves as a vital source of cellular energy and a direct precursor of nicotinamide adenine dinucleotide (NAD+), a crucial cofactor in human metabolism. Given its significant value in the fields of nutrition and healthcare, NMN biosynthesis has recently become a prominent area of research globally. In this study, we constructed the recombinant plasmid pETDuet-NPk encoding three intracellular proteins (Prs, Rk, and Nampt) and the recombinant plasmid pCDFDuet-NPC, containing the ribose ABC transporter system, the nicotinamide nucleotide transporter protein (PnuC), and nicotinic acid (NA) transporter protein (NiaP). The double plasmids were used to develop the engineered strain E. coli 2d, which facilitated a whole-cell catalytic reaction pathway for NMN production using D-ribose and nicotinamide as substrates. This approach yielded 0.139 g/L, which is 3.6 times higher than that obtained using glucose as a substrate. Additionally, the mutant strain E. coli 2d-C1, identified through UV mutagenesis and flow cytometry screening, yielded 0.91 g/L of NMN, representing a 6.5-fold increase compared to the unmutated strain E. coli 2d. This study introduces a novel approach for high-throughput NMN screening and highlights the use of cost-effective synthetic substrates to enhance NMN production.