β-nicotinamide mononucleotide (NMN) production in Escherichia coli

Abstract

Diabetes is a chronic and progressive disease with continuously increasing prevalence, rising financial pressure on the worldwide healthcare systems. Recently, the insulin resistance, hallmark of type 2 diabetes, was cured in mice treated with NAD+ precursor β-nicotinamide mononucleotide (NMN), no toxic effects being reported. However, NMN has a high price tag, more cost effective production methods are needed. This study proposes a biotechnological NMN production method in Escherichia coli. We show that bicistronic expression of recombinant nicotinamide phosphoribosyl transferase (Nampt) and phosphoribosyl pyrophosphate (PRPP) synthetase in the presence of nicotinamide (NAM) and lactose may be a successful strategy for cost effective NMN production. Protein expression vectors carrying NAMPT gene from Haemophilus ducreyi and PRPP synthetase from Bacillus amyloliquefaciens with L135I mutation were transformed in Escherichia coli BL21(DE3)pLysS. NMN production reached a maximum of 15.42 mg per L of bacterial culture (or 17.26 mg per gram of protein) in these cells grown in PYA8 medium supplemented with 0.1% NAM and 1% lactose.

Metabolic engineering ofEscherichia colifor optimized biosynthesis of nicotinamide mononucleotide, a noncanonical redox cofactor

“…NMN + production using S. cerevisiae Nrk1 or S. enterica NadR (Figure 2, Pathway 2, on the plasmids pWB304 and pWB305) with 200 μM NR supplementation was not efficient enough to restore growth ( Figure 2C, D). These results suggest that besides the well-established NadV route (Figure 1, Pathway 3) [19], the F. tularensis NadE*-dependent pathway is also effective in E. coli for NMN + biosynthesis.…”

“…This work represents the initial steps towards filling some of the fundamental knowledge gaps that remained open in previous work on NMN + biosynthesis in E. coli. The important work by Marinescu and coworkers [19] focused on the NadV pathway, but left many other naturally occurring NMN + biosynthetic routes unexplored. Our previous work [13] sought to simply recapitulate the NMN + metabolism of F. tularensis [23] by overexpressing both NadE* and NadV from this organism, without dissecting the role of each pathway.…”

“…solanacearum NadV strain performed significantly better than F. tularensis NadV, the NadV we used in our previous work [13], exhibiting a 2.8-fold increase in intracellular NMN + concentration. R. solanacearum NadV also performed better than H. ducreyi NadV [19].…”

“…Marinescu and coworkers demonstrated NMN + accumulation in E. coli by heterologously expressing three NadV homologs from Haemophilus ducreyi, Shewanella oneidensis, and Mus musculus while feeding NA [19], and they showed that H. ducreyi NadV performed the best [19]. We previously showed that NadV from F. tularensis, which belongs to a different clade in the NadV phylogenetic tree to H. ducreyi NadV [18], can also effectively produce NMN + in E. coli [13].…”

“…Compared to other noncanonical cofactors which are made through chemical synthesis [15][16][17], NMN + is particularly suited for fully renewable biomanufacturing processes because it is accessible through biosynthesis [18][19][20]. This feature is especially desirable in crude lysatebased cell-free biosynthesis and whole-cell biosynthesis, where NMN + produced in the cells does not need to be purified or exogenously supplied, and it can be directly used for downstream biocatalysis.…”