Analysis And Modification Of Key Enzyme Genes Of Neomycin Biosynthetic Pathway

Neomycin is a classical of aminoglycoside antibiotics with broad-spectrum antibacterial properties,which are widely used in medicine and agriculture and have high economic benefits.In the previous work,we found that the structural genes neo N and neo E have important effects on the production of neomycin by Streptomyces fradiae in the biosynthetic pathway of neomycin.NeoN encoded by neo N,catalyzes the final step of neomycin biosynthesis from neomycin C to produce the highly efficient and less toxic neomycin B with higher economic value.Neo E encoded by neo E,a zinc-containing dehydrogenase in the synthesis of neomycin parent ring 2-DOS,and its in situ overexpression resulted in a large increase in neomycin potency.Neither NeoN nor Neo E has been characterized so far.Based on the importance of NeoN,this study mainly explores the sequence and structural characteristics of NeoN by means of bioinformatics and molecular biology.The key sites of NeoN were also analyzed and modified.A preliminary exploration of the sequence and structure of Neo E was also conducted.The main research is summarized as follows.（1）Amino acid sequence analysis of NeoNThe sequence of NeoN protein was analyzed using online software,the results showed that NeoN is a stable hydrophilic intracellular protein.Bioinformatics analysis showed that it belongs to the superfamily of free radical-dependent SAM enzymes.Each member in this family possessing the conserved sequence Cxxx Cxx C.Amino acids 26～33 are the sites where iron-sulfur clusters are formed and bound to SAM.（2）Tertiary structure modeling and molecular docking of NeoNHomology modeling of NeoN amino acid sequences revealed a low homology of 32%for the existing template sequences in its protein database.Therefore,the NeoN Tertiary structure obtained by Alpha Fold calculation is used in this study.Since the catalytic reaction of NeoN is dependent on SAM,the whole molecular docking work is divided into two steps.First,the NeoN-SAM complex structure was obtained by docking the SAM molecule with the NeoN protein and comparing the NeoN conformational changes before and after the entry of the SAM molecule.Second,the NeoN-SAM complex structure was docked with the obtained NeoN-SAM molecule to analyze the key sites of action.Overlapping comparison of the NeoN and NeoN-SAM complex structures revealed that the binding of SAM prompted an induced fit in the substrate binding pocket of NeoN.Three regions in NeoN underwent significant structural changes（L174～F177,E278～S282,D226～N230）.The originalβ-sheet layer folds outward and the loop area increases;and the pocket changes from an acidic environment to a neutral environment.The change may have increased the internal space of the catalytic pocket to allow the entry and reaction of the neomycin C molecule.The binding of neomycin C to NeoN is mainly dependent on hydrogen bonding and salt bridges,and Thr38,Gly24,Asp68,Asp23,Gln236,Arg243,Asp248,Tyr250 can form hydrogen bonding interaction.Among them,Asp68 contributes three hydrogen bonds and salt-bridging forces,while Thr38 can form three hydrogen bonds.The docking results also showed that the catalytic pocket of NeoN is very narrow,and four amino acids,Glu34,Lys36,Ser251,and Val252,located at the entrance of the binding pocket,caused a bulge on the protein surface,which may prevent the entry and reaction of neomycin C molecules.（3）Optimization of NeoN heterologous expression conditionsThe conditions for NeoN heterologous expression were optimized,and the plasmid p ET-28a（+）-NeoN and strain BL21-p N were successfully constructed for the expression of the target protein.Under the conditions of the presence of iron-sulfur-derived cysteine and ferrous salts,maximum expression of the target protein was achieved in the supernatant of crushed cells with the addition of the inducer IPTG at a concentration of 0.4mmol/L,an induction time of 18 h,an induction temperature of 28°C and a starting OD₆₀₀ value of 0.6.（4）Site-directed mutagenesis and result analysisBased on the results of the docking analysis,mutations were performed on Thr38,Asp68,Glu34,Lys36,Ser251,and Val252.The experimental results showed that the enzyme activities of mutant enzymes NeoN ^D68Aand NeoN ^T38A decreased by 27.2%and 60.4%,respectively.The hydrophobicity of both threonine at position 38 and aspartic acid at position 68 was found to increase significantly after mutation to alanine by software analysis.Docking analysis showed that the original hydrogen-bonding and salt-bridging forces at the mutation site were lost.Among the mutants NeoN^E34A,NeoN^K36A,NeoN^S251A,and NeoN^V252A constructed to increase the catalytic pocket,the surface area of the catalytic pocket increased by 128（?）²for NeoN^K36A,the volume of the catalytic pocket increased by 51（?）3 for NeoN^V252A,and the volume and surface area of the catalytic pocket increased significantly for NeoN^S251A.The enzyme activity was measured and no activity was detected in mutant NeoN^E34A,while the enzyme activities of NeoN^K36A,NeoN^S251A and NeoN^V252A were 87.5%,66.9%and 104.2%of those before the mutation,respectively.（5）Transcriptional analysis of genes associated with neo E overexpression strain SF-neo E revealed that the transcript level of neo E was significantly up-regulated by 2.1-fold at 48 h compared with that of SF-p PR containing a null plasmid,and neo H was up-regulated to a small extent at different time periods.（6）Bioinformatics analysis showed that Neo E is a stable hydrophobic protein.The N-terminal catalytic structural domain of alcohol dehydrogenase（ADH）at amino acid sequences 24～129 aa and the Rossnman folded structural domain used to bind the coenzyme NAD（P）⁺at 171～298 aa.A highly conserved structure of GXGXXG exists at its sequence 168～173 aa,a binding site for NAD（P）⁺,possessing a binding pocket for phosphate formation from an alanine and an arginine.