Roles Of Pichia Pastoris HXT And MIG Genes In Catabolite Repression Of AOX1 Promoter

In Pichia pastoris system, the expression of foreign genes is usually driven by the outstanding promoter of the alcohol oxidase I gene (AOX1) which encodes the first enzyme in the methanol utilization pathway. The AOX1 promoter (PAOX1) is transcribed only in response to methanol and repressed by other carbon sources, such as glucose, fructose or glycerol. In this work, the identification and characterization of two hexose transporter homologs (PpHxtl and PpHxt2) and two catabolite repressor (PpMig1 and PpMig2) in P. pastoris are described.Firstly, PpHxtl and PpHxt2 were identified. The predicted 12 transmembrane regions, the comparison of the sequence with various hexose transporters from other organisms as well as functional complementation of an S. cerevisiae hxt null mutant unable to take up hexose showed that the two proteins are members of hexose transporters. Both PpHXT genes were transcriptionally regulated by glucose. Full transcription of PpHXTl was induced by high level glucose, whereas the transcription of PpHXT2 was relatively lower and was fully induced by low level glucose. Notably, it was found that the deletion of PpHXTl, but not PpHXT2, led to the induced expression of heterologous protein GFP based on AOX1 promoter in response to glucose or fructose. These observations suggest that PpHxt1 is involved in AOX1 repression directly rather than just as a sugar carrier. However, PpHxt2 plays an important role in glycolysis-dependent fermentative growth since PpHxt2 is essential for growth on glucose or fructose when respiration is inhibited by antimycin A. We also elucidated that a sharp dropping of sugar-induced expression level of GFP-SKL in Ahxtl at a later growth phase was mainly caused by pexophagy, a degradation pathway in methylotrophic yeast.Based on degenerate PCR and genome walking method, two catabolite repressor genes were cloned in P. patoris. Both PpMIG genes encoded the proteins homologous to the DNA-binding repressor Migl from S. cerevisiae (ScMigl), which was a C2H2 zinc finger protein and could bind to the promoters of many genes repressed by glucose.Δmig1 mutant was constructed by the gene replacement method using the zeocin resistance gene Sh ble as a marker, andΔmig2 mutant was constructed as same as Amigl except using the G418 resistance gene KAN as a marker. TheΔmig1Δmig2 double-deletion mutant was isolated via genetic crossing method. Remarkablely, it was found that glycerol could induce the expression of heterologous proteins based on AOX1 promoter inΔmig1Δmig2 mutant.In this work, genes involved in in catabolite repression of AOX1 promoter were identified, and mutants with altered catabolite regulation of the PAOX were obtained via gene deletion, which might be promising the expression of recombinant proteins in a methanol-free medium.