Metabolic Engineering Of E.Coli For The Production Of ?-aminoadipic Acid

Metabolic engineering has made it promising to increase the yield,titer and productivity in bio-based production of chemicals,materials of interest,and identification of innovative targets for drug encountering in the treatment of disease.The ?-aminoadipic acid also known as homoglutamic acid is a non-proteinogenic amino acid is a six-carbon analog of glutamic and aspartic acid.It is a potent prognosticator of prostate cancer,biomarker for diabetes,a pharmacological tool as an effective inhibitor of L-glutamate uptake and a hypothetically precarious step in the recycling of neurotransmitter glutamate.It has also a wide range of applications in the fields of cosmetics,medicine,etc.,and plays an important role in the synthesis of intermediates of certain chemicals,such as sweeteners and chelating agents,In the present situation homoglutamic acid can be synthesized through chemical routes which are high costly,low yield,not suitable for industrial use and even problems of contaminations in the process.Our work demonstrates the efforts of de-novo biosynthesis of homoglutamate through a platform strain of Escherichia coli.First,we constructed an E.coli strain harbouring pathway converting 2-ketoadipate to homoglutamate.Then we carried out molecular docking studies for glutamate dehydrogenase GDH enzyme and analyzed their binding affinity through docking simulations which revealed that the ?-Ketoadipic acid has a promising interaction to GDH enzyme.Furthermore the ?-Ketoadipic acid ligand showed an important interaction with the crucial moieties in the substrate binding pocket.We have replaced Methionine codon 101 by arginine 101,the alanine 325 by lysine 325 and replaced aspartate 347 codon by arginine 347 Our study revealed that there are two hydrogen bonding observed from the surrounding residues including Ser351(2.81 A)and Asn326(2.44 A)with the 2-Ketoadipic acid.Also,there are four hydrophobic contacts Met101,Arg324,Ala325 and Asp347 have been reported from the wild type GDH enzyme binding pocket which result with high stability and enhanced catalytic activity of the enzyme.The mutant M10lR has an enhanced activity in conversion of 2-ketoadipic acid into a-aminoadipic acid with 99.625mg/L as compared to the A325K with 78.574 mg/LIn the second project,we addressed antimicrobial analysis for the materials based hybrid to developed procedure for decreasing toxicity to the normal cells and enhance the antimicrobial efficacy.We have used medicinal plant extract as a reducing agent to kill the multidrug resistant MDR Gram-negative E.Coli base on Zinc Oxide(ZnO)and graphene oxide(GO)nanosheet ZnO@GO mediated nanoeomposite synergistieally enhanced its efficiency against MDR Gram-negative bacteria.From the TEM images,results show successfully synthesized trigonal small sizes ZnO on the surface of GO nanosheets.However,the DLS and C potential studies indicate that in an aqueous medium the average ZnO size and surface capped charge comes from plant extract of ZnO@GO are 30-40 nm and+20 mV,respectively.The spectroscopic characterization such as UV-visible and FTIR analyses reveals that in the synthesized ZnO@GO nanocomposite ZnO successfully well dispersed decorated on the surface of GO nanosheets.The as-synthesized ZnO@GO was used against MDR gram-negative pathogenesis(E-coli)and shows excellent antibacterial activity and killing 95%toxic bacteria within 5 hours.ZnO@GO having very biocompatible and shows significant antibacterial activities.The highly antibacterial activity is due to synthesized positive ZnO@GO produce electrostatic interaction with the cell membrane of E.coli.Hence nanocomposites subsequently enter the cytoplasm through damaging of the cell membrane of bacteria,as a results production of ROS into the cytoplasm led to metabolism imbalance in the cell.Moreover,the cell membrane damage of gram-negative bacteria verified through ? potential and propidium iodide(PI)study.From ? potential test,the cell membrane lost and disturbs their negative charge due to the interaction of ZnO@GO.Furthermore,the PI analysis also shows successfully bacteria cell membrane disruptor.Thus,the understanding of obtained results in depth will reduce and develop a new way to solve the existing world challenge problem in the field of medical treatment.