Indentification Of PpHXS1, A Hexose Sensor Homelogue, And Two Regulators PpMPP1 And PpTRM1 Related To Methanol Induction Of AOX1 Promoter In Pichia Pastoris

The methylotrophic yeast Pichia pastoris has been widely used and is now the second most used host for recombinant gene expression. In P. pastoris, the most commonly used regulatory sequence to control the expression of foreign proteins is the AOXI promoter, which controls the expression of the alcohol oxidase 1 (Aoxl). In this work, a P. pastoris mutant BN8 with impaired glucose repression of AOX1 promoter was screened, and the mutation in mutant BN8 was further identified, that the phenotype of BN8 was due to the mutation of PpHXSl. Two genes, PpMPPl and PpTRMl which are related to methanol induction of AOXl promoter were also identified.The mutant BN8 was screened by UV-induced mutant on a plate containing methanol and 0.02% 2-deoxyglucose. In P. pastoris mutant BN8, about 36%,30%and 25%of the alcohol oxidase 1 promoter activity of that induced by methanol in wild type, could be detected when induced by glucose, fructose and mannose respectively when using green fluorescent protein as a reporter. By analyzing the sequence of several predicted gene in BN8 mutant, it was found that one mutation occurred in a predicted ORF which is the homelogue of S. cerevisiae SNF3 and RGT2. The ORF was designated as PpHXS1. (Pichia pastoris hexose sensor).PpHXS1 played an important role in glucose repession. We also found that PpHXSl is not essential for hexose signaling in catabolite inactivation by pexophagy.A homelogue of P. angusta MPP1 designated PpMPPl and a homelogue of Candida boidinii TRM1 designated PpTRMl were also identified. APpmppl and APptrml can not grow in methanol medium. Activity of AOX1 promoter is significantly reduced in△Ppmppl and△Pptrml cells compared with WT.In this work, we report P. pastoris mutant with impaired glucose repression of alcohol oxidase 1 promoter. The mutant is promising strain for heterologous protein production using Paoxl y glucose instead of methanol induction. The identification of PpMPPl and PpTRM1 could help us understanding the mechanism of methanol induction of AOX1 promoter.