Preliminary Studies On The Riboflavin Biosynthetic Pathway In Escherichia Coli As A Target For Bacterial Inhibitionby

The contamination of food-borne drug-resistant bacteria is one of the current problems facing food safety.It is important to study the mechanism of bacterial inhibition of bacteria,especially drug-resistant bacteria,and to explore effective inhibition targets for efficient inhibition of the growth of harmful microorganisms and prevention and control of bacterial contamination.The electron transport chain is an inherent property of bacteria,and riboflavin,as an endogenous electron shuttle in E.coli,has a key role in its growth process.In this paper,we characterized the changes of riboflavin metabolism in E.coli under different environments by electrochemical means,and combined with transcriptomic sequencing technology to explore the key regulatory genes of riboflavin metabolism-related pathways at the level of gene expression regulation,which is intended to provide ideas for exploring novel inhibitory targets and mechanisms.The specific results are as follows:(1)The effects of oxygen,osmotic pressure,pH and temperature on riboflavin metabolism in E.coli were investigated,and the riboflavin metabolic activity of the strain was characterized by calculating the amount of riboflavin metabolism per unit density of the organism.The main results were as follows:riboflavin metabolism was greater under aerobic conditions and growth metabolism was more vigorous;riboflavin metabolism may not have a regulatory mechanism in response to osmotic stress;the strain was unable to maintain normal activity and overall metabolic levels were low under extreme conditions such as pH=3,11 and 55℃;riboflavin secretion was higher than normal at pH=9 and 45℃,suggesting that the bacteria may regulate riboflavin metabolism under these conditions by The enhanced riboflavin metabolism regulates the electron transfer activity to achieve stress regulation under stress conditions.(2)Transcriptome screening of key genes regulating riboflavin metabolism.The gene expression of E.coli under 45℃ and pH9 treatment was analyzed by transcriptional sequencing technology.Functional annotation and bioinformatics analysis of differentially expressed genes revealed that ribA,ribD,ribE,and rpiA were significantly up-regulated at 45℃,and sdhA,sdhC,and sdhD were significantly upregulated at pH=9,and the products encoded by these genes were related to cofactor coenzyme The products encoded by these genes are related to key life processes such as cofactor-coenzyme binding,biological carbon skeleton synthesis,and energy metabolism.These genes may play an important regulatory role in response to the corresponding environmental stresses and have the potential to become repressive targets.(3)A strain defective in riboflavin metabolism was constructed by Red recombinant method.The defective strain required exogenous supplementation of riboflavin to grow,and the concentration of riboflavin in the culture medium was less than 250 μM,which affected the time to enter the exponential phase of the defective strain.The lower the concentration of riboflavin,the slower E.coli entered the exponential phase.This indicates that riboflavin exists as a growth factor in E.coli growth,and ribA in the biosynthetic pathway of riboflavin is a potential target for bacterial inhibition.