Characterization and reconstitution of enzymes and pathways in the biosynthetic production of nitroaromatic compounds

Nitroaromatic;p-aminobenzoate N-oxygenase(AurF);Mangotoxin;N-oxygenase;Nitro natural products

Chanco, Emmanuel ; Zhao ; Huimin

关键词: Nitroaromatic;    p-aminobenzoate N-oxygenase(AurF);    Mangotoxin;    N-oxygenase;    Nitro natural products;   

Others  :  https://www.ideals.illinois.edu/bitstream/handle/2142/42125/Emmanuel_Chanco.pdf?sequence=1&isAllowed=y

美国|英语

来源: The Illinois Digital Environment for Access to Learning and Scholarship

【 摘 要 】

Nitro-containing compounds are an important class of chemicals with high value in industry and medicine. Several of these compounds are produced naturally in many different species; however key issues in their discovery and biosynthesis are problems which are under active research. The prototypical enzyme for N-oxygenation of arylamines is the protein AurF from Streptomyces thioletus. In this thesis, further characterization of this enzyme is performed for protein engineering. The activity of the enzyme was increased more than 600-fold from previous literature results by both optimized pH conditions and by using the chemical reductants PMS/NADH. Alanine scanning and substrate specificity studies were performed to identify key residues and mechanistic information of the protein. The electron transfer mechanism of AurF was also studied, resulting in better understanding of the kinetics of each important step in the reaction mechanism.Finally, an in vivo screening method for directed evolution was developed and validated. Screening work was performed to find mutants with activity towards meta-aminobenzoic acid, however positive hits are unconfirmed. Based on the bioinformatics searches for AurF analogs, the Mangotoxin gene cluster from Pseudomonas syringae was identified as possibly making a nitroaromatic product. Isolation and identification of this cluster’s gene product was attempted based on heterologous expression of the cluster in E.coli, and fermentation studies in the native host. To that end, the gene cluster was recloned in a variety of constructs using several methods such as DNA assembler, Golden Gate and Gibson assembly. Expression of the gene cluster’s proteins was observed in E.coli, while feeding studies confirmed the production of nitroaromatic compounds from the cluster. A product detection method based on reduction/diazotization was developed in order to find the putative gene product.