The Analysis And Optimization Of Rhamnose-Inducible Promoter P_LRA3 In Pichia Pastoris

The methylotrophic yeast Pichia pastoris is one of the most widely used eukaryotic expression hosts for heterologous expression of various recombinant proteins.Currently,P_GAP and P_AOX1,which can express recombinant proteins efficiently,are the most frequently used promoters,however some disadvantages of the two promoters seriously decreased the application in Pichia pastoris expression system.In detail,P_GAP,which is constitutive promoter,leads to uncontrollable fermentation process and is unsuitable to expression of toxic proteins.Additionally,P_AOX1,which is induced by toxic and flammable methanol,is not feasible to express food-grade recombinant proteins.Therefore,it is necessary to develop some more inducible promoters for Pichia pastoris expression system.In a previous study,five genes,LRA1-LRA4 and the regulatory gene rhaR,associated with rhamnose metabolism in P.pastoris were predicted.The rhamnose-inducible promoter P_LRA3 from LRA3,was disclosed to achieve high expression of recombinant proteins in P.pastoris.And more indeep studies were needed to investigate P_LRA3.In this study,it was found that rhaR was involved in rhamnose metabolism.RhaR encoded by rhaR could bind to P_LRA3,and the binding site was further revealed.Key sites in P_LRA3 were reveal by CR-RT-PCR method combined with bioinformatics analysis.Finally,P_LRA3RA3 mutants with improved transcriptional activity were acquired via random mutation.The main results are as follows:1.rhaR was deleted by markerless gene deletion system,developing the rhaR-deleted strain P.pastoris GS115/?rhaR.Subsequently,rhaR-complemented strain P.pastoris GS115/rhaRC was constructed by introduction of rhaR into P.pastoris GS115/?rhaR.It was found that P.pastori GS115/?rhaR could not grow normally on medium with rhamnose as the sole carbon source,and P.pastoris GS115/rhaRC grew normally.Therefore,it indicated that rhaR was involved in rhamnose metabolism in P.pastoris.2.The conserved domain of transcriptional factor RhaR,Zn?II?₂Cys₆ domain,was analyzed.This domain was expressed and purified as GST-tagged proteins in Escherichia coli.Then the binding of RhaR to P_LRA3 was verified by EMSA,and the binding site,5?-TAAACTGAAA-3?,was disclosed by DNase I footprinting.3.It was found that the transcription initiation site of P_LRA3 was located 22 nucleotides upstream of translation start site of LRA3 via CR-RT-PCR method,simutaneously,core promoter,TATA Box and cis-acting elements of P_LRA3 were predicted.4.To improve transcriptional activity of P_LRA3,the error-prone PCR was performed to construct a mutation library of P_LRA3,and finally two mutants with transcriptional activity improved by 25%were isolated.