Rational Design And Modification Optimization Of Hydroxylase And Glycosylase For Flavonoids

Flavonoids are a class of secondary metabolites with C₆-C₃-C₆skeleton,which have received widespread attention due to their anti-inflammatory,antibacterial,antioxidant,anti-tumor and other physiological activities.Post-modification represented by hydroxylation and glycosylation,can effectively improve the solubility and medicinal value of flavonoids.With the rapid development of computational biology and synthetic biology technology,the research on flavonoids hydroxylase and glycosyltransferase has become increasingly in-depth.This study successfully constructed an artificial P450 enzyme sequence with the function of catalyzing the 6-position hydroxylation of flavonoids by combining various methods such as biological information analysis,deep learning algorithms,and molecular biology experiments.Through the cloud platform of the plant glycosyltransferase database website that integrates data retrieval,structural prediction,virtual screening,and donor prediction,it achieved efficient prediction and rational transformation of glycosyltransferases’glycosyldonors.The main results are as follows:（1）In the construction process of artificial flavone-6-hydroxylase,using the CYP706X1 from the genome of Erigeron breviscapus as a template,combining the"three point fixation"catalytic pocket design principle of flavone-6-hydroxylase analyzed in previous work and the generation of an antagonistic network（GAN）model,an artificial P450 enzyme deep learning design route was developed to generate seven unnatural P450 sequences.Finally,by performing fluorescent protein expression in the Saccharomyces cerevisiae expression system,it was verified that all seven sequences can be folded normally.Further,through fermentation experiments and functional characterization,an unnatural P450 sequence with flavone-6-hydroxylase function was successfully obtained.At the same time,molecular dynamics simulation（MD）was used to conduct high-temperature simulation tests on the seven sequences,it was found that the binding of the other six sequences without flavone 6-hydroxylase function to the substrate apigenin was significantly weaker than the active sequence.（2）In the research process of glycosyltransferase,this study developed a tool for predicting glycosyltransferase based on substrate by analyzing the binding mechanism of characteristic glycosyltransferase with its substrate,and by analyzing the similarity of the binding substrate,catalytic pocket,and substrate binding affinity.Three glycosyltransferases were selected for validation.In addition,through comprehensive structural analysis of characteristic glycosyltransferases and their sugar donors,28key sites were mined,thereby developing a functional prediction tool for UDP sugar donors.Through rational design of the partial sites of three characteristic glycosyltransferases,namely Eb F7GAT,UGT88D7,and UGT88D8,under in vitro enzyme activity testing,different choices of sugar donors for the characteristic glycosyltransferases were obtained,achieving the diversity of sugar donors for glycosyltransferases.