Selection And Modification Of The Inorganic Nitrogen Source Signal Switch For The Auto-Inducible Aspergillus Heterologous Expression System

Heterologous expression is an important way of life science;it is also an important method for industrial protein production.The promising heterologous expression host Aspergillus fungi,as the eukaryotic microorganism,contain both protein modification ability of post-translation and the advantages of low operating costs.Aspergillus nidulans stands out from other aspergillus species by its characteristic of fast growth,high recombination efficiency and clear genetic and metabolic background.Although Aspergillus nidulans have the potential to be an excellent host for the expression of heterologous proteins,its expression patterns and exploited promoters are limited.The ultimate goal of this project is to develop Aspergillus nidulans as a protein expression host,which utilize cheap inorganic nitrogen source nitrate/ammonium salt as inducer and inhibitor,to realize the valuable innovation in expression host and expression pattern.As an important part of a large project,this paper mainly focused on the characteristics of key genes in the nitrate assimilation pathway of Aspergillus nidulans,from which the promoter for heterologous expression was found.The promoter could be highly induced by nitrate and significantly inhibited by ammonium salt,and be further optimized could make it more suitable for protein expression.Research results are as follows:Using the reporter gene uidA(encoding GUS)fused with promoter,the promoter activity of Aspergillus nidulans nitrate assimilation genes nrtA,niaD and niiA could be compared.These three key genes respectively encode for nitrate transporter,nitrate reducer and nitrite reducer,which can be induced by nitrate and be inhibited by ammonium.We found that the promoter of nitrate transporter encoding gene nrtA have the better promoter strength and higher rigor,which was selected as the heterologous expression switch.It is the first time to analyze the function of the transcription factor AreA and NirA in vitro and in vivo,which regulated the activity of nrtA promoter.In addition,we found that the effective length of nrtA promoter was 1477 bp,including five important transcription factors binding sites,such as N1/N2,N3,A4,A5 and A6.Finally,according to the location of transcription factor binding sites and core regions of nrtA promoter,the promoter sequence was modified,and the optimized promoter induced activity was increased by twice,with no significant change in repressed rigor.Thus,an auto-inducible promoter with higher strength and strict transcription regulation was obtained,which could respond to the signal of mixed inorganic nitrogen source in the environment.This promoter can be a switch for Aspergillus nidulans to build an auto-inducible eukaryotic heterologous expression.