Functional Analysis Of An Alternative Sigma Factor,?~E,in Corynebacterium Glutamicum

?-Factor is an important component of RNA polymerase,which can identify the consensus sequence in the promoter,and binds with the core enzyme of RNA polymerase,thus initiates gene transcription.Gene expression regulation through?factor is one of the important manners for bacteria to regulate its physiological metabolic activities.Therefore,investigating the function and regulatory mechanism of?factor contributes to perfecting the gene regulatory network of microorganisms,thus facilitating the formulation of strain optimizing strategy in industrial production.Corynebacterium glutamicum is an important industrial microorganism,whose genome contains seven RNA polymerase?subunit coding genes.Apart from?~A,the remaining six?factors belong to the alternative?factors.This project aimed to identify the basic function of the less-known?~E.The main research results were shown below.(1)Changes in the transcription levels of the 6 alternative?factors except for sigM after various stress treatments were systemically identified,which suggested that the expression quantities of sigB and sigE would be markedly improved after treated with 10%ethanol.(2)The knockout and covering strains of sigE,sigB were constructed,which discovered that the knockout strains of sigE and sigB shared a lot of similar properties,including reduced total biomass,bacterial aggregation and precipitation,and improved sensitivity to ethanol stress.(3)The expression levels of sigB in the sigE over-expression and knockout strains were determined,which indicated that sigE could up-regulate sigB expression at transcription level.Afterwards,it was identified through Ch IP experiment that?~E could directly bind with the sigB promoter.(4)The TSPs of sigE-cseE were identified.As was shown in our results,the TSP of sigE corresponded to the first nucleotide of the initiation codon ATG annotated in NCBI,while cseE,the anti-factor coding gene of?~E,had an independent TSP,which was regulated by?~E.(5)It was also discovered that?~E and?~H shared some functional similarities,and the role of?~E in ethanol stress was also explored.(6)Our findings suggested that ethanol could promote the expression of enhanced green fluorescent protein(EGFP)when it was utilized by the bacteria,and a self-induced promoter was constructed accordingly based on such phenomenon.At the same time,this project also preliminarily explored whether ethanol addition was of certain industrial application value in terms of costs and effects.Moreover,this project had also conducted plenty of methodological discussion based on sufficiently summarizing domestic and foreign studies,with an aim to provide certain reference for related studies on?factor in the future.