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.
Highlights
According to International Diabetes Federation, the number of adults with type 2 diabetes was 425 million in 2017, and it is expected to hit 649 million by 2045
Transformed E. coli cells with nadV (NAMPT) genes carried by pET28a(+) plasmids formed colonies on the LB Agar plates supplemented with kanamycin
The DNA bands matched the bands from E. coli BL21(DE3)pLysS cells (Supplementary Fig. S2A, lane 6, 7, 8), which were transformed with corresponding plasmids extracted directly from E. coli DH5α
Summary
According to International Diabetes Federation, the number of adults with type 2 diabetes was 425 million in 2017, and it is expected to hit 649 million by 2045. NAD+-dependent protein deacetylases such as SIRT1 and SIRT6 serve as metabolic sensors, and regulate downstream pathways, which eventually restore mitochondrial function and insulin sensitivity[6,7,8,9,10,11,12,13]. This finding extends the research domain of NAD+ effects to other degenerative diseases associated with aging, such as: cardiovascular, cancer, arthritis, osteoporosis or Alzheimer’s diseases[14,15,16]. As bacterial metabolism is versatile, the variation of carbon source[39] and concentration of medium supplemented enzyme substrates are key factors to take into consideration in a biotechnological process
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