Establishment Of A Novel Counter-selectable Marker Suitable For Pichia Pastoris

The advantages of Pichia pastoris determine that it has great potential and commercial value in the field of research and production.However,the number of marker genes in P.pastoris that can be used for genetic engineering is limited.Sleeping Beauty(SB)transposon mutation technology is a mutation method that can be used to isolate and screen unknown genes by the random insertion of SB transposon,which can be used to mine new marker genes.In this study,a mutant library covering the whole genome of P.pastoris GS115 was successfully constructed with the SB transposon mutation system.A rapamycin sensitive gene was screened from this mutant library,and a counter-selectable marker was developed in P.pastoris.Mut375,a rapamycin-resistant mutant,was isolated from P.pastoris mutant library with10 ng/m L rapamycin.The genomic DNA of the mutant as a template,TAIL-PCR was used to obtain the flanking sequence of SB transposon.The insertion site of SB transposon was mapped,and the mutated gene of mut375 was identified as PAS?Chr2-2?0375(named kFPR1).In this study,kFPR1 gene was knocked out by homologous recombination technique,and it was found that P.pastoris had rapamycin resistance.The sensitivity of the strain to rapamycin was restored after the function of kFPR1 gene was supplemented to prove the correlation between kFPR1 gene and rapamycin resistance.Based on the characteristics of kFPR1 gene,a marker-free gene manipulation method suitable for P.pastoris was established.A marker unit Zeo R-kFPR1 was constructed in which a zeocin resistance gene(Zeo R)as the active-selectable marker and kFPR1 as the counterselectable marker,with direct repeats on both sides for marker recycling.The marker unit was integrated into P.pastoris genome by homologous recombination,introducing an activeselectable marker.Upon rapamycin medium,the Zeo R cassette was successfully recycled via homologous recombination between the directed repeats.The experimental results demonstrated the feasibility of kFPR1 as a counter-selectable marker for P.pastoris.By this method,we knocked in the EGFP expression cassette and knocked out the LYS1 gene in P.pastoris respectively,all without introducing unnecessary selection markers.In this study,a counter-selectable marker kFPR1 was established to achieve unmarked genetic modification suitable for P.pastoris,providing a new tool for unmarked genetic manipulation in P.pastoris.