Development And Artificial Evolution Of Strong Promoters Of Bacillus Licheniformis

Bacillus licheniformis is an important industrial microorganism.It has the advantages of short fermentation cycle,good thermal stability,and strong protein secretion ability.It has been recognized as a biosafety GRAS strain.Bacillus licheniformis has very good industrial application prospects in terms of enzyme expression and metabolite synthesis.However,Bacillus licheniformis currently lacks standardized elements of synthetic biology.The existing promoters are mainly derived from model microorganisms such as Bacillus subtilis,but the effect of expressing heterologous genes is not satisfactory.In this study,a method for evaluating the constitutive expression strength of promoters in Bacillus licheniformis was established,and the activities of different Bacillus promoters were compared.The selected high-activity promoter was used to mediate the high-efficiency expression of maltotriose-producing amylase gene in Bacillus licheniformis,and the influence of different fermentation conditions on the expression of the promoter was investigated.The molecular analysis of the interaction between the upstream region and the RNA polymerase aCTD was completed,and the transformation of Bacillus promoter was further realized.The main contents and results of this study are as follows:（1）First,the promoter elements were predicted based on transcriptome data and then using green fluorescent protein egfp as the reporter gene,a method was established to evaluate the constitutive expression intensity of the promoter in Bacillus licheniformis.The expression system of Bacillus licheniformis were constructed,and the intensity of 11 Bacillus licheniformis promoters P_Hpall,P_glvA,P_spovG,P_acpp,P_ydzA,P43,P2,P_TrxA,P_DS,P_r2 and P_shuttle-09 were compared.The results showed that P2 was 28 times of the strong constitutive promoter Pshuttle-09 and 52 times of the natural strong promoter P43.（2）The expression plasmid harbouring the new promoter was transformed into Bacillus licheniformis to realize the secretion and expression of maltotriose-producing amylase.And the fermentation conditions were compared,the fermentation conditions of mixed carbon source maltodextrin 60 g·L^-1,glucose 10 g·L^-1 and 42℃ were more conducive to enzyme production,and the highest enzyme activity of genetically modified bacteria fermentation reached 578.47 U·mL^-1.（3）The factors affecting the initial transcription efficiency of prokaryotic promoter were investigated,and the molecular mechanism of RNA polymerase αCTD recognizing the upstream region of the promoter was analyzed.The expression and purification of RNA polymerase αCTD from Bacillus licheniformis were completed,and the specific binding of RNA polymerase αCTD with promoter was verified in vitro.The upstream base of promoter PTrxA-35 region was removed,and its core region was taken as the basic skeleton.The upstream region of-35 region of other promoters was replaced and connected to the skeleton of PTrxA to obtain the modified promoters.In addition,the fluorescence polarization of the modified promoters was verified in vitro and the fluorescence intensity was evaluated in vivo,the results showed that the modified promoter P2-TrxA had higher activity.The promoters P2,P_shuttle-09,and P_spovG were linked in tandem.The tandem promoters P_shuttle-09-P2,P_spovG-P2,and P_shuttle-09-P_spovG were successfully obtained.It is further proved that the interaction between RNA polymerase αCTD and the up elements of the promoter can provide basic theoretical guidance for the determination of promoter strength.