Research studies on NAD + have proven its crucial role in aging and disease. Nicotinamide mononucleotide (NMN), as the key intermediate of NAD + , plays a significant role in supplying and maintaining NAD + levels. In the present study, a biocatalytic method for the efficient synthesis of NMN was established. First, Escherichia coli was systematically modified to make it more conducive to the biosynthesis and accumulation of NMN. Next, the performance of nicotinamide phosphoribosyltransferase from Vibrio bacteriophage KVP40 (VpNadV) was determined, which has the best catalytic activity to produce NMN from nicotinamide. The accumulation of extracellular NMN was further increased after the introduction of an NMN transporter. Fine-tuning of gene expression and copy number led to the synthesis of NMN at the yield of 2.6 g/L at the shake flask level. The introduction of a nicotinamide transporter, BcniaP, could not obviously increase the production of NMN at the shake flask level, but it decreased the production of NMN at the bioreactor level. Finally, the titer of NMN reached 16.2 g/L with a conversion ratio of 97.0% from nicotinamide, both of which are highest according to currently available reports. The fed-batch fermentation with direct supplementation of nicotinamide could facilitate the industrialscale production of NMN compared to that achieved by the whole-cell catalysis process. These results also represent the highest reported yield of NMN synthesized from nicotinamide in E. coli.

(S)-Equol is the terminal metabolite of daidzein and plays important roles in human health. However, due to anaerobic inefficiency, limited productivity in (S)-equol-producing strains often hinders (S)-equol mass production. Here, a multi-enzyme cascade system was designed to generate a higher (S)-equol titer. First, full reversibility of the (S)-equol synthesis pathway was found and a blocking reverse conversion strategy was established. As biosynthetic genes are present in the microbial genome, an effective daidzein reductase was chosen using evolutionary principles. And our analyses showed that NADPH was crucial for the pathway. In response to this, a novel NADPH pool was redesigned after analyzing a cofactor metabolism model. By adjusting synthesis pathway genes at the right expression level, the entire synthesis pathway can take place smoothly. Thus, the cascade system was optimized by regulating the gene expression intensity. Finally, after optimizing fermentation conditions, a 5 L bioreactor was used to generate a high (S)-equol production titer (3418.5 mg/L), with a conversion rate of approximately 85.9%. This study shows a feasible green process route for the production of (S)-equol.