| Streptomyces is a kind of prokaryotic microorganism between bacteria and fungi.It can produce many biologically active secondary metabolites,such as anti-tumor,anti-viral,anti-diabetes,etc.,which is receiving increasing attention from researchers.1-Deoxynojirimycin(DNJ)is an imino sugar piperidine alkaloid which is mainly found in mulberry leaves,commelina communis and certain microorganisms such as Bacillus and Streptomyces.Because DNJ has a certain inhibitory activity on a-glucosidase,it has potential application value in the treatment of diabetes,AIDS,tumor diseases and so on.Due to the extremely low DNJ content of plant source(0.1341 mg/g dry weight for mulberry leaves and 0.1125 mg/g dry weight for duck grass),it has become a bottleneck for activity evaluation,structure-activity relationship research and industrial application in vitro and in vivo.Therefore,microbial source DNJ gradually attractes the attention of researchers.At present,the microbial source DNJ stimulation method is concentrated on the optimization of strain selection and fermentation process,lacking of understanding of DNJ biosynthetic pathway has greatly restricted the research on regulation the yield of product based on synthetic pathway information.In addition,as a highly potent a-glucosidase inhibitor,DNJ can inhibit the activity of various a-glucosidases(disaccharidase,polysaccharase),and its clarified inhibition mechanism will help to synthesize and design efficient alpha-glucosidase inhibitor drugs according to DNJ structureStrepto,myces lavendulae UN-8 was a DNJ-producing bacterium isolated from natural habitats in the early stage of our group.In this paper,the physiological characteristics of DNJ-producing strain UN-8 under different carbon sources and concentrations were studied,and other properties of the strain were explored firstly;Secondly,the DNJ biosynthetic pathway was studied.Then,according to the clarified biosynthetic pathway,the strategies for targeted regulation DNJ synthesis were developed;Finally,the inhibition mechanism of DNJ on different a-glucosidases(disaccharidase maltase,polysaccharase glucoamylase)were revealed from the perspective of biophysics The main results were as follows(1)The physiological characteristics of UN-8 showed that the growth of UN-8 were inhibited by 100%and 70%,respectively,when the concentration of glucose or maltose in the solid media was 40 g/L,showing low glucose concentration and low maltose concentration tolerance.UN-8 could grow using agar as the sole carbon source.The agarase activity was 0.03 U/ml after shake flask fermentation in liquid medium.Its production was independent on the presence of NaCl,which was different from marine agar degradation microorganisms.UN-8 could be grown in a nitrogen-free medium,and the length of cloned nitrogen-fixing gene,nifU was 462 bp.The similarity of the translated amino acid sequence reached 98%,98%,and 97%with nitrogen-fixing protein NifU from Streptomyces sp.NRRL S-104,Streptomyces sp.NRRL F-4428 and Strepto,myces sp.PCS3 respectively,indicating that UN-8 had the capacity of potential biological nitrogen fixation.These physiological characteristics will provide a reference for the subsequent regulation of DNJ synthesis.(2)The DNJ biosynthetic pathway in UN-8 was elucidated by precursor feeding,'3C stable isotope labeling-nuclear magnetic resonance and ultra performance liquid chromatography-mass spectrometry.The results showed that glucose was the best precursor for DNJ synthesis.During the synthesis,the glucose carbon skeleton undergone C2/C6 cyclization reaction,besides,2-amino-2-deoxy-D-mannitol(ADM),nojirimycin(NJ)and NJ intermediates were isolated and identified.The synthetic route were as follows:glucose was converted into fructose-6-phosphate by glycolytic pathway firstly,then ADM was formed by amination,and further oxidized to NJ intermediate state.Subsequently,NJ intermediate state was cyclized into cyclic NJ by C2-N-C6,followed by dehydration and hydrogenation forming DNJ.In addition,experiments have found that higher concentrations of glucose(>5 g/L)were not conducive to DNJ synthesis,and then this phenomenon was explored.Results show that regulation of DNJ synthesis by glucose was not due to process of glucose phosphorylation,but higher concentration of glucose made the pH of the growth environment of cell growth was acidic during cell metabolism,and the intracellular ATP content was low,and finally the cells entered the decay phase prematurely,thereby inhibiting the synthesis of DNJ.(3)According to the elucidated DNJ biosynthetic pathway,DNJ synthesis was regulated by targeted strategies such as the addition of metabolic inhibitors,precursors and intermediate analogs.The results indicated that shikimate pathway inhibitor disodium edetate,mevalonate pathway inhibitor simvastatin,glycolytic pathway inhibitors sodium citrate and iodoacetic acid,precursor glucose and intermediate analog sorbose promoted DNJ synthesis.On this basis of this,the control strategy of DNJ synthesis was developed by orthogonal experiment:sorbose and sodium citrate were added to the initial medium under concentrations of 1 g/L and 5 g/L,respectively.After fermented for 20 hours and 26 hours,iodoacetic acid and glucose were added under concentrations of 50 mg/L and 7 g/L respectively.Under the strategy,the highest yield of DNJ reached 296.56 mg/L for 72 hours,which was 2.3 times higher than the previous period,indicating that this regulation strategy was effective.(4)The inhibition mechanism of DNJ on disaccharidase maltase derived from saccharomyces cerevisiae was revealed by enzyme inhibition kinetics and multispectral spectroscopy.The results showed that DNJ inhibited the activity of maltase in a dose-dependent manner,showing a mixed inhibition mechanism dominated by non-competitive inhibition.The fluorescence quenching mechanism of DNJ on maltase was dynamic quenching,which bound to the maltase active center through hydrogen bonding and hydrophobic interaction,inducing the change of maltase conformation,leading to the decrease of a-helix ratio,the increase of ?-sheet ratio and reduce hydraulic radius.In addition,the hydrogen atoms of the 2-OH,3-OH,4-OH,6-OH and-NH groups in the DNJ structure formed hydrogen bonds with the active center amino acid residues Asp68,Asp214,Asp68,Glu276 and Asp349 by molecular docking.The bond lengths were 4.6 A,1.9 A,6.0 A,1.9 A,and 1.8 A,respectively,thereby interfering with the binding of the substrate to the maltase and inhibiting the activity of the maltase(5)The inhibition mechanism of DNJ on the glucoamylase derived from Aspergillus niger was studied.The results showed that DNJ inhibited the activity of glucoamylase in a dose-dependent manner,and showed a competitive inhibition mechanism dominated by competitive inhibition.The fluorescence quenching mechanism of DNJ on glucoamylase was dynamic quenching.The binding forces were hydrophobic interaction and hydrogen bonding.The binding of DNJ caused the conformation of glucoamylase and the hydrophilicity around the tryptophan residue,decreasing of a-helix ratio,increasing of ?-sheet ratio and reducing hydraulic radius.Molecular docking model showed that DNJ bound to the glucoamylase catalytic domain and formed hydrogen bonds with conserved amino acid residues Arg78,Asp79,Glu203,Glu424,and surrounded by hydrophobic amino acids Trp76,Leu201,Trp202,Leu343,Met422,Gln425,Ala445.Molecular dynamics simulations showed that the DNJ-bound glucoamylase in catalytic domain amino acid residues had lower root mean square fluctuations than free glucoamylase,and the binding sites remained rigid during the simulation. |
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