Identification And Transformation Of The Key Sites Of The Strong Promoter P_CAT1 Of Pichia Pastoris

Pichia pastoris is one of the important industrial microorganisms for heterologous protein expression.It has advantages including suitable for high-density fermentation and post-translational modification.The important goal of studying the heterologous protein expression system is to increase the yield of heterologous proteins,and screening for strong promoters can increase the transcription level of heterologous genes,thereby effectively increasing the yield of heterologous proteins.P_AOX1 is currently the most widely used Pichia pastoris promoter.It has strong activity,but methanol needs to be used to induce P_AOX1.Methanol is flammable,explosive,and toxic.It has a certain security risk for industrial storage and transportation of methanol.P_CAT1 is a promoter of peroxidase in the methanol utilization pathway of Pichia pastoris.Its methanol-inducing activity is comparable to P_AOX1,and P_CAT1 activity is higher than P_AOX1 when expressing certain enzymes;meanwhile,P_CAT1 is repressed by high concentrations of glucose and derepressed by low concentration of glucose.P_CAT1 can be used to express transcription factors related to methanol induction,and P_AOX1-based non-methanol-utilizing Pichia highly expressing strains can be constructed.However,as of now,there is a lack of research on P_CAT1.In this study,we modified the core promoter region of P_CAT1,randomly mutated the P_CAT1sequence,and engineered the transcription factor binding site of P_CAT1 to screen for more active P_CAT1 variants and analyze the functional elements affecting P_CAT1 activity.The main research contents and results are as follows:?1?Transformation of P_CAT1 core startup areaBy truncating the P_CAT1-1000 sequence and characterizing the activity of P_CAT1 variants,the P_CAT1 core promoter region was determined;the P_CAT1 core promoter was divided into five parts,and each core promoter regions was knocked out from P_CAT1-1000 respectively to determine the most influential P_CAT1 activity.Analysis of the transcription factor binding sites?TFBSs?of P_CAT1-1000,using the online prediction software Mat Inspector,found that the core promoter region that most affects P_CAT1 activity only has the binding site of the factor HAP1.01.Duplication of the binding site of HAP1.01 lead to the activity of the constructed P_CAT1 variant significantly improve,and its methanol-inducing activity was increased by 16%.?2?Random mutation of P_CAT1 sequenceMutation was randomly introduced into the P_CAT1 sequence by EP-PCR,and the activity of the P_CAT1 variant was characterized by EGFP.The P_CAT1 promoter library with an activity range of 26%to 138%was obtained.The-118 bp base of P_CAT1 mutating from A to G can increase the methanol-induced activity of the P_CAT1 promoter.Through the analysis of P_CAT1promoter variants by Mat Inspector,it was found that this mutation lead to F$YMCM binding site decrease and F$CSRE and F$YGAL binding sites increase in P_CAT1.?3?Modification of the transcription factor binding sites?TFBSs?of P_CAT1According to Mat Inspector's analysis of TFBSs of P_CAT1-1000,several TFBSs of P_CAT1 were duplicated or deleted,and it was found that the duplicating HAP1.05 binding site can effectively improve the activity of P_CAT1.Combined with the results of the transformation of the core promoter region of P_CAT1,duplicating the binding sites of HAP1.05 and HAP1.01 at the same time on P_CAT1-1000,the activity of the P_CAT1 variant was greatly improved.Among them,under the conditions of methanol induction,CALB was expressed with this P_CAT1 variant,and the Candida antarctica lipase B?CALB?yield phase was obtained 77%improvement over the control.