The Function Of PpGUP1 In Glycerol And Methanol Metabolism

Pichia pastoris(P. pastoris) became one of the most attractive hosts for heterologous protein expression, basing on one promoter of alcohol oxidase I(PAOX1), which is the most efficient promoter involved in regulation of recombinant protein expression. PAOX1 is tightly repressed by the presence of glycerol in the culture medium; thus, glycerol must be exhausted before methanol can be taken up by P. pastoris and the expression of heterologous protein can be induced. In this study, the function of PpGUP1(Gene ID: 238029505) in glycerol and methanol metabolism were analysed. Different mutants were constructed by knockout, heterologous expression and overexpression. When culturing with different carbon sources, biomass and carbon metabolism were measured. And the transcriptional level of its gene-specific response to different carbons were also analysed, with the purpose to explore the mechanism of glycerol restrain PAOX1. Main results were described as follows:(1) One gup1-defective strain named GUP1Δ and a mutant with PpGUP1 overexpressed were constructed. After shakening in a baffled flask in BMGY and BMMGY medium for 72 h, the biomass and content of glycerol in fermentation broth were analyzed. The GUP1Δ strain had no significantly distinction in the glycerol metabolism level and the biomass with the wild type. When PpGUP1 was overexpressed, the biomass increased 5% and improved 8% glycerol utilized. Schizosaccharomyces pombe(S. pombe) cannot transport glycerol inside to utlize as a carbon source though with gup1, when heterogeneous expressed PpGUP1 in S. pombe, it cannot use glycerol either. By means of SDS sensitivity test analysis, this finding found that the permeability of GUP1Δ enhanced. The findings clarified that PpGUP1 just improved glycerol metabolism indirectly by intervening cell wall or plasma membrane saturation rather than acting as a glycerol transporter.(2)When culturing in BMMY, the biomass of GUP1Δ was approximately 30% less than the wild type, with the levels of methanol metabolism reduced, meanwhile, the activity of AOX1 loosed 50% of the wild type and increased 5% when Pp GUP1 was overexpressed. The transcription level of gup1, aox1 and mxr1 culturing in different carbon sources were aslo analyzed, when PpGUP1 was silenced, the transcription level of aox1 reduced significantly with mxr1 declining 50% weaker than before. Moreover, the transcription level of aox1 and mxr1 improved slightly while PpGUP1 was overexpressed. Consequently, PpGUP1 regulated aox1 by two possible mechanism, one was that PpGUP1 regulates the transcription of aox1 directly, the other was that PpGUP1 regulates the aox1 by affecting the transcription of mxr1 indirectly. As a result, mxr1 regulates aox1 at transcription lever was more rigorous.