Synthesis of Nicotinamide Mononucleotide from Xylose via Coupling Engineered Escherichia coli and a Biocatalytic Cascade

Abstract

β‐Nicotinamide mononucleotide (NMN) has recently gained attention for a nutritional supplement because it is an intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD+). In this study, we developed NMN synthesis by coupling two modules. The first module is to culture E. coli MG1655 ▵tktA ▵tktB ▵ptsG to metabolize xylose to generate D‐ribose in the medium. The supernatant containing D‐ribose was applied in the second module which is composed of EcRbsK‐EcPRPS‐CpNAMPT reaction to synthesize NMN, that requires additional enzymes of CHU0107 and EcPPase to remove feedback inhibitors ADP and pyrophosphate. The second module can be rapidly optimized by comparing NMN production determined by the cyanide assay. Finally, 10 mL optimal biocascade reaction generated NMN with a good yield of 84 % from 1 mM D‐ribose supplied from the supernatant of E. coli MG1655 ▵tktA ▵tktB ▵ptsG. Our results can further guide researchers to metabolically engineer E. coli for NMN synthesis.

Rapid prototyping enzyme homologs to improve titer of nicotinamide mononucleotide using a strategy combining cell-free protein synthesis with split GFP

“…As expected, a further decrease in the NMN yield was observed (Figure S5b), indicating that PPi indeed has the inhibitory effects on NMN production. This result was well consistent with a recent study that suggested that PPi has feedback inhibition on Nampt (Ngivprom et al, 2022). Next, we tested whether the NMN production could be improved by removing the PPi inhibition.…”

Make or break: the thermodynamic equilibrium of polyphosphate kinase-catalysed reactions

“…Consequently, we decided to use the enzymes with the tags, as this likely is the most pragmatic and straightforward preparation of the enzymes for their use in chemical synthesis. The assay setup used for all PPKs is similar to established ATP regeneration systems with PPK2 enzymes, with a 10:1 excess of polyP (calculated as single phosphates [49]) over the nucleotide (default concentration in this work 2 mM) [39,[50][51][52]. This is also a realistic scenario for biosynthetic reactions using PPKs for the production of NTPs [22,52].…”

“…The assay setup used for all PPKs is similar to established ATP regeneration systems with PPK2 enzymes, with a 10:1 excess of polyP (calculated as single phosphates [49]) over the nucleotide (default concentration in this work 2 mM) [39,[50][51][52]. This is also a realistic scenario for biosynthetic reactions using PPKs for the production of NTPs [22,52]. The excess of polyP should prevent depletion of sufficiently long polyP chains since acceptance of very short chains (n < 10) might differ between different PPKs [10,15].…”

“…Therefore, the results presented here can serve to further tune and improve these multi-enzyme reactions and the yield of biomimetic NTP synthesis systems. Usually, reactions for polyP-dependent ATP regeneration as well as NTP biosynthesis are carried out under conditions comparable to the ones used for this study [ 22 , 39 , 50 , 52 ]. Overall, we can clearly see a preference of the thermodynamic system towards ATP.…”

Biomimetic S-Adenosylmethionine Regeneration Starting from Different Byproducts Enables Biocatalytic Alkylation with Radical SAM Enzymes

“…This approach has been successfully used before as one-pot reactions to produce different types of nucleotides from their corresponding nucleobase and sugars. [69][70][71][72][73] Both, RK and RPPK, use ATP for the phosphorylation reactions. Different regeneration systems have been used for the rephosphorylation of ADP (RK reaction) or AMP (RPPK reaction).…”