Biosynthesis Of Nicotinamide Mononucleotide And Preliminary Analysis Of The Crystal Structure Of Phosphoribosyl Pyrophosphate Synthetase From Bacillus Amyloliquefaciens

Level of nicotinamide adenine dinucleotide(NAD~+)in the human body declines with age increasing,leading to a variety of age-related symptoms.Exogenous NAD~+supplementation is not directly absorbed and utilized by human cells due to its large molecular weight.However,it has been found that the main precursor of NAD~+,β-nicotinamide mononucleotide(NMN),can be absorbed by human cells and then converted to NAD~+through the Salvage pathway in vivo,restoring the physiological activities of the body to some extent.The intake of NMN has shown a good safety profile in both animal and human studies up to now.The existing methods of NMN synthesis include chemical synthesis,whole cell biotransformation and enzymatic catalysis,among which chemical synthesis requires multi-step reactions and generates by-products,while synthesis of NMN by enzymatic catalysis or using microbial cell culture is relatively simple,safe and environmentally friendly,and has obvious advantages.In order to simulate the pathway of NMN synthesis in living organisms,the biosynthesis of NMN was carried out using adenosine triphosphate,ribose 5-phosphate and nicotinamide as raw materials by using phosphate pyrophosphate synthase from Bacillus amyloliquefaciens(ba PRS)and nicotinamide phosphoribosyltransferase from Haemophilus ducreyi(hd NAMPT).In order to obtain the maximum yield,a combination of genetic engineering,molecular biology and structural biology were carried out to obtain the maximum yield as follows.(1)Both of two single-promoter recombinant plasmids,pET28a-ba PRS and p ET28a-hd NAMPT,were successfully constructed using homologous recombination technology,with 8×His tags and protease cleavage sites at the N-terminal of the target proteins for purification conveniently.Then,conditions for protein expression were optimized,in which the best expression conditions for ba PRS were:E.coli BL21(DE3),0.5 m M IPTG,and induction for 4 hours at 37°C,and the best expression conditions for hd NAMPT were:E.coli BL21(DE3),0.5 m M IPTG,and induction for 24 hours at 16°C.In addition,the p ETDuet-1 plasmid was used as a vector for dual-promoter expression vector p ETDuet-hdNAMPT-baPRS was constructed by inserting the hd NAMPT and ba PRS gene sequences after the T7 promoter at two multiple cloning sites,respectively.hd NAMPT was highly expressed and much higher than ba PRS when induced by IPTG of0.5 m M at 28°C and for 20 hours.After affinity chromatography and gel filtration chromatography in Ni-NTA column.After purification,high purity ba PRS and hd NAMPT were obtained,and single bands were identified by SDS-PAGE.(2)NMN was synthesized by both whole cell biotransformation and extracellular enzymatic methods in E.coli and the yields were compared.In the whole-cell biotransformation method,the optimal concentration of nicotinamide added to the medium was 0.5%,at which time there was an intracellular accumulation of NMN in E.coli,and more NMN was accumulated in E.coli transfected with p ET28a-hd NAMPT and p ETDuet-hd NAMPT-ba PRS plasmids relative to the p ET28a-ba PRS plasmid,with a maximum of 39.96μM(13.36 mg/L).In the enzymatic method,the activities of the purified two proteins were measured and the reaction conditions were optimized.Under the optimal reaction system,NMN concentration could reach 86μM(28.7 mg/L)after 30min of the dual enzyme-catalyzed reaction,and the conversion rate of both ribose5-phosphate and NAM reached 86%,which exceeded the conversion rate of the chemically synthesized substrate and the yield of the whole-cell biotransformation method.(3)After the initial screening of the kit and optimization of the conditions,ba PRS protein crystals of suitable shape and size were successfully obtained.X-ray diffraction data were collected before and after immersion of the substrate ribose 5-phosphate,and the three-dimensional crystal structures with resolutions of 2.37(?)and 3.51(?),respectively,were obtained after software processing.Preliminary comparison and analysis revealed that the flexible loop formed from Arg102 to Ile113 was in two different states before and after soaking ribose 5-phosphate.In addition,the binding sites of both phosphate and ribose 5-phosphate were near the loop formed from Ile222 to Thr231,and it could be inferred that high concentration of phosphate could compete with ribose5-phosphate for the binding site and inhibit the catalytic activity of ba PRS.Finally,in the study,three recombinant expression vectors,including p ET28a-ba PRS,were successfully constructed based on the E.coli prokaryotic expression system,and highly active and pure ba PRS and hd NAMPT were obtained after purification in various ways.compared with the E.coli whole-cell biotransformation method,the enzymatic reaction synthesis produced a higher content of NMN than the E.coli whole-cell biotransformation method.For ba PRS,its three-dimensional structure was successfully obtained by X-ray diffraction and preliminary analysis was performed,which laid the theoretical foundation for subsequent modification at the molecular level,increasing the enzyme activity and stability,and thus improving the NMN yield.