Transcriptional Regulatory Mechanism Of AOX1 Promoter In Pichia Pastoris

Pichia pastoris has been one of the most widely used host to express heterologous proteins. P. pastoris AOX1 promoter (PAOX1) is the most widely used promoter as its specific characteristics, i.e., powerful, strictly repressed by glucose, glycerol, ethanol, and strongly induced by methanol. It is well known that the regulation of Paoxi is a two step processes: repression/derepression and methanol induction. However, the detailed methanisms in these processes are still in infancy. Moreover, the activation of Paoxi is severely depended on methanol, which has many drawbacks in large-scale fermentation industry, as methanol is toxic, flammable, and explosive. The use of methanol in fermentation also restricts its application. Thus, it is meaningful to ecucidate regulatory mechanism of Paoxi to improve the productivity of rencent Pichia expression system and develop novel methanol-free Pichia expression system. To clarify transcriptional regulatory mechanism of Paoxi, we studied the detailed regulatory mechanism of Paoxi by Mitl, Prm1, and Nrg1. Moreover, the regulatory profile of PAOX1 by three activators, Mitl, Prm1, Mxrl, and three repressors, Mig1, Mig2, Nrgl, has been studied. We elucidated the methanol signal transduction in these activators and proposed a putative regulatory model of PAOX1 regulating in glucose, glycerol, and methanol. Finally, two outstanding methanol-free Pichia expression systems in which PAOX1 could be induced by glycerol have been developed by rational reconstruction of the PAOX1 regulatory network. They hold great potential for industrial use for production of recombinant proteins.Firstly, Mitl and Prml were both zinc finger proteins with conserved Zn2(Ⅱ)Cys6 zinc fingers at their N terminus. Mitl and Prml were involved in methanol metabolism as the growth of Amit1 and Aprm1 was totally missing in methanol but normal in glucose, glycerol, ethanol, sorbitol, and oleate. Mitl and Prml were responsible for the activation of genes in methanol utilization (AOX1, DAS1, DAS2, and FLD). Distinct to Mitl, Prml also specificially activated the expression of FDH. In addition, Mitl and Prml did not participate in peroxisome proliferation and transport of peroxisomal proteins. The regulation of PAOX1 by Mitl and Prml occurred at the transcriptional level. Chromatin immunoprecipitation (ChIP) assay revealed that Mitl and Prml directly bound to PAOX1 and the binding ability increased from weak binding or no binding to strong binding when cells were response to glucose, glycerol, and methanol, sequentially. Besides, Mitl and Prml also bound to promoters of other genes that they tightly regulated, respectively.When H. polymorpha Mppl was complemented to △mit1, Mppl not only recovered the gowth of Amitl in methanol, but also released the strict repression of PAOX1 by glycerol. The difference of Mitl and Mppl in glycerol repression of PAOX1 was caused by their structural dissimilarity. The UR3, RR1, and RR3 domains of Mitl repressed PAOX1 in glycerol while the RR2 domain was inverse. In addition, Mitl and Prml were nuclear localized in glucose, glycerol, and methanol. The UR2 and TM1 domians were responsible for the nuclear localization of Mitl and Prml, respectively. Moreover, the ZF and UR2 domains of Mit1 and ZF, TM1, FT, and UR1 domains of Prm1 were indispensable for the function of Mit1 and Prm1, while the RR2, UR3, and RR3 domains of Mit1 and CC1, TM2, and CC2 domains of Prml were dispensable for the function of Mit1 and Prm1.Homology alignment revealed Nrg1, a Cys2His2 zinc finger protein, was a repressor of PAOX1. Nrgl was nuclear localized in glycose, glycerol, and methanol, and involved in the repression of Paoxi by glucose and glycerol. Nrg1 might compete the binding sites of Mxr1 and Prm1, as it bound to PAOX1 at five sites that included two binding sites of Mxr1 and one sites of Prml. Besides, there was no protein-protein interaction between repressors (Mig1 and Nrg1) and activators (Mit1 and Prm1). We revealed that repressors Mig1, Mig2, and Nrg1 repressed the expression of PAOX1 by repressing Mit1 expression and/or direct bound to PAOX1. There was also no protein-protein interaction between activators (Mit, Prm1, and Mxr1), as methanol signal transferred in cascade among these activators. Specificially, Mxr1 was mainly responsible for the derepression of PAOX1 while Prm1 and Mit1 participated in the methanol-specific activation of PAOX1. Methanol signal prioritily transmitted to Prm1, which responded to methanol signal rapidly by inducing its self-expression, and then induced the strong expression of Mit1. Large amount of Mit1 accumulated intracellular then induced the expression of PAOX1. Moreover, Mitl could represse the expression of Prm1 as a feedback loop, which is meaningful for cells to utilize their intracellular resources economically. Collectively, we proposed a regulatory model of PAOX1 in glycose, glycerol, and methanol.Based on the regulatory model of PAOX1, we constructed two Pichia strains by deleting repressors and overexpessing activators. Specificially, the PAOX1 activities of △mig1△mig2△nrg1-Mit1 strain, in which Mitl was overexpressed by PGAP, came up to 77% in glycerol and 126% in methanol compared to WT in methanol. Another strain △mig1△mig2△nrg1-AMit1 has been obtained by substituting PGAP with PAOX1. By this means, a positive feedback loop was constructed to improve Mit1 expression as well as automatically keep Mit1 in balance. The △mig1Amig2△nrg1-AMit1 could express GFP in glycerol up to 107% of WT in methanol. These two strains would be promising hosts for heterologous proteins expression in industry.