Biosynthesis Of Nicotinamide Mononucleotide In Saccharomyces Cerevisiae

As a precursor molecule of nicotinamide adenine dinucleotide(NAD~+),β-nicotinamide mononucleotides(NMN)have received considerable attention in recent years due to their anti aging activity.NMN is a molecule that naturally exists in biological cells,but its content is very small and purification is difficult.Currently,there are two methods for synthesizing NMN:chemical synthesis and biosynthesis.The cost of chemical synthesis is high,while there are many by-products resulting in low safety.In contrast,biosynthesis is more green and safe,mainly including enzymatic and fermentation methods.The method of enzymatic synthesis of NMN has been relatively mature and has a high yield,but its required substrate costs such as ribose and ATP are relatively high.In contrast,fermentation using low-cost substrates such as nicotinamide and glucose to synthesize NMN has the potential for large-scale industrial synthesis.Fermentation methods usually use Escherichia coli as a production strain,but this method has certain safety risks.Using Saccharomyces cerevisiae as a production strain is a safer and environmentally friendly production method.Therefore,this study uses Saccharomyces cerevisiae as a production strain and adopts genetic engineering strategies to transform Saccharomyces cerevisiae,enabling the modified recombinant yeast to have a certain NMN production capacity,providing strong support for the production of higher quality NMN products.(1)First,single gene expression strains of yeast were constructed.The codon-optimized nicotinamide phosphoribosyltransferase gene(Nampt)a nd nicotinamide transporter protein gene(Nia P)and NMN transporter protein gene(Pnu C)were introduced into Saccharomyces cerevisiae by genetic engineering methods to obtain yeast single gene expres sion strains of the three genes,the shake flask fermentation results showed that all three genes significantly increased the yield of NMN in yeast under the action of inducible promoter(GAL1)an d constitutive promoter(PGK1),respectively.And the highest yield of NMN is 49.13mg/L through shake flask fermentation for 24 hours,produced by the Nampt single gene expression strain(p ESC-Nampt-TRP)under the action of inducible promoter.(2)Then,polygenes expression strains of yeast were constructed.Firstly,three genes were combined in pairs,and a vector with two genes was introduced into Saccharomyces cerevisiae using genetic engineering methods to obtain a two-genes expression strains.The shake flask fermentation results showed that the combined expression of Nampt and Pnu C with inducible promoters in yeast(p ESC-Nampt-Pnu C-TRP)had the highest yield,reaching 50.43 mg/L through shake flask fermentation for 21 hours;After that,three genes were connected to a vector,and then introduced into yeast to obtain a three-genes expression strains(p ESC-Nampt-Pnu C-Nia P-TRP).The shake flask fermentation results showed that the NMN production of this strain was 50.07 mg/L through shake flask fermentation for 24 hours.(3)Optimization of shake flask fermentation conditions for modified yeast.The modified yeast with the highest NMN production efficiency obtained in the above experiments is p ESC-Nampt-Pnu C-TRP expression strain,so this strain was selected for shake flask fermentation in this experiment.In shake flask fermentation experiments,the ability of yeast to produce NMN was fur ther improved by optimizing fermentation time,substrate concentration,inducer concentration,and adding sodium citrate in order to increasing the flux of the pentose phosphate pathway in yeast.Finally,under the action of a nicotinamide substrate concentration of 3 g/L,sodium citrate of 0.5 g/L,and a galactose inducer of 2%,p ESC-Nampt-Pnu C-TRP expression strain obtained a NMN yield of 52.26mg/L through shake flask fermentation for 20 hours.