Efficient Synthesis Of β-Nicotinamide Mononucleotide In Escherichia Coli By Metabolic Engineering

β-nicotinamide mononucleotide（NMN）is a biologically active nucleotide.NMN plays an active role in resisting individual aging,preventing and treating neurodegenerative diseases and diabetes.Whole cell catalysis is economical,environmental friendly and has a high specificity in the reaction process,so it has a good application prospect in the production of NMN.In this study,Escherichia coli BL21（DE3）was used as the starting strain,and the gene engineering strategy was used to properly modify it,and the substrate glucose and niacinamide were used to efficiently synthesize NMN,so as to explore the biosynthesis of NMN.The main results are as follows:（1）To screen highly active nicotinamide phosphoribosyltransferase（NAMPT/NadV）.Nampt/NadV from Haemophilus ducreyi,Shewanella oneidensis,Vibrio bacteriophage KVP40 and Chitinophaga pinensis were used as enzyme sources to express in Escherichia coli BL21（DE3）respectively,and the recombinant bacteria were studied.The experimental results showed that the activity of nicotinamide phosphoribosyltransferase（VpNadV）from Vibrio bacteriophage KVP40 was the highest,and the yield of NMN of the recombinant strain was 26.2±0.75 mg·L^-1.Therefore,VpNadV is selected for the follow-up study.（2）To construct the co-expression plasmid pETDuet-VpnadV-Ecprs of VpNadV and ribose phosphate phosphokinase of E.coli（EcPRS）,and to improve EcPRS activity and optimize its expression level.The results showed that the recombinant strain E.coli/pETDuet-VpnadV-Ecprs had better NMN synthesis ability than the strain expressed by VpnadV alone.Among all the mutants of EcPRS,the mutant EcPRS^D115 with single amino acid residue mutation showed the best enzyme activity.In the optimization of EcPRS expression level,the combination of promoter P_J23119 and RBS_Ⅲperformed best in increasing the yield of NMN.（3）Enhance the supply of precursor 5-phosphoribosyl diphosphate（PRPP）and weaken the degradation of product NMN.The synthesis pathway of PRPP was reformed,and the gene zwf（encoding glucose-6-phosphate dehydrogenase）and gene gnd A（encoding 6-phosphogluconate dehydrogenase）were replaced by zwf^A243T and gnd^S361F from Corynebacterium glutamicum,and the genes rpiA and rpiB（encoding ribose-5-phosphate isomerase）were overexpressed,which enhanced the synthesis of PRPP.Knock out the gene pfk A（encoding fructose-6-phosphate kinase）in the branch metabolic pathway,and obtain the recombinant strain ZY14,and the synthesis amount of NMN is increased again.Knocking out the gene pnc C（encoding NMN aminohydrolase）and gene ush A（encoding 5’-nucleotidase）in the degradation pathway of NMN,the degradation of NMN was weakened,and the NMN yield of the obtained strain ZY16 reached 925.3±12.5 mg·L^-1,which was 1.74 times higher than that of the control strain ZY14.（4）For the recombinant strain ZY17 with the best performance,the whole cell catalytic conditions were optimized at shake flask level.The optimum catalytic conditions are:p H 8.0,temperature 35℃,shaking speed 200 r·min^-1,Mg²⁺concentration 6 mmol·L^-1.The recombinant strain ZY17 was biotransformed for 12 h under the optimal conditions,and a total of NMN2.37±0.03 g·L^-1 was produced,which was 1.47 times higher than that before optimization.The scale-up test of the reaction system showed that the yield of NMN reached 13.3±0.35 g·L^-1 and the molar conversion rate of nicotinamide was 85.3%in a 2 L bioreactor.