Molecular and functional characterization of Pichia pastoris MIG1 gene

The methylotrophic yeast Pichia pastoris is widely and successfully used as a host system to produce recombinant proteins for a variety of applications. The rising popularity of the system could be attributed to several characteristics, including the ability to perform many eukaryotic post-transcriptional modifications, and the capability of this yeast to produce heterologous proteins at high levels, either intra- or extracellularly. Being already the most productive expression system in protein expression within the other systems, Pichia pastoris is still being studied in order to enhance the efficiency of the system.In the P. pastoris expression system, the expression of foreign genes is usually driven by the promoter of the alcohol oxidase gene I (AOX1) which encodes the first enzyme in the methanol utilization pathway. Under a very strict control, the AOX1 promotor is highly induced by methanol (methanol induction) and repressed by glukoz and ethanol (catabolite repression). Although it is well known that the AOX1 promoter is regulated strictly by induction and catabolite repression mechanisms, the proteins involved in these regulation mechanisms have not been identified yet. So far only one gene product, Mxr1p, has been showed to be involved in the positive regulation (methanol induction) of the AOX1 promoter.In the scope of this project, the mechanism of catabolite repression of P. pastoris has been studied. For this purpose, the molecular and functional characterization of the MIG1 gene, which is determined to have the key role in catabolite repression mechanism for S. cerevisiae and other yeasts and encodes the protein Mig1 that is bound to DNA as repressor, were done. As a result of the functional analysis of single ( ? mig1, ? mig2) and double mutant ( ? mig1 ? mig2) P. pastoris strains, which have been derived by inactivation of MIG1 and homologous MIG2 genes, by building up their growth curves under repression (glukoz), derepression (glycerol) and induction (methanol) conditions and comparing the alcohol oxidase enzyme activities, it has been revealed that the MIG1 ve MIG2 genes have a role in catabolite repression mechanism of AOX1 gene. Although the inactivation of MIG1 and MIG2 genes has shown no phenotypic effect in growth of cells in glycerol and glukoz, a slight increase in growth speed of ? mig1 and ? mig1 ? mig2 strains and cell density at methanol has been observed. Regarding to AOX enzyme activity results, no activity has been identified at glukoz/methanol media, whereas the activities were found on ? mig2 ve ? mig1 ? mig2 strains at glycerol/methanol media at an approximate rate of 30% ve 50% respectively. Overall results suggested that the PpMig1 and PpMig2 proteins hold a place in negative regulation mechanism of AOX1 promotor.KEYWORDS: Pichia pastoris, catabolite repression, glukoz repression, Mig1, Mig2