Inactivation of alcohol oxidase genes (AOX1 and AOX2) in Pichia pastoris and utilization of the AOX-defective strain in recombinant protein production

Pichia pastoris is an expression system for the production of recombinant proteins needed by the biotechnology and pharmaceutical industries and by academic researchers. Like bacteria, P. pastoris grows rapidly on inexpensive media containing methanol, glucose, glycerol or ethanol as a sole carbon source. Furthermore, like humans, it is also a eukaryotic organism with a subcellular environment more conducive to the folding of eukaryotic proteins and with an ability to perform post-translational modifications such as proteolytic processing, disulfide bridge formation and glycosylation.The genome of P. pastoris contains two copies of the alcohol oxidase gene. The AOX1 gene is responsible for 85% of alcohol oxidase regulation within the cell while AOX2 gene is responsible for %15. Therefore if is the AOX1 promoter which is more widely used in recombinant protein production. P. pastoris has three different phenotypes depending on methanol utilization ability. Mut+ strain is indistinguishable from wild-type methanol utilizing strain. Muts (AOX1 gene is defective) and Mut- (AOX1 and AOX2 genes are defective) strains can be better than wild-type in recombinant protein production. In addition, these strains require less methanol for induction of expression. Less methanol requirement is an advantage of Mut- strains in large scale fermentation.This research consists of two parts. In the first part the objective was the inactivation of AOX genes (AOX1 and AOX2) in P. pastoris. AOX1 and AOX2 were inactivated by inserting ADE1 and HIS4 genes, respectively. ADE1 and HIS4 genes were also selection markers. As a result of this part, P. pastoris MK321 (his4::PHIS4, aox1::ADE1), MK431 (ade1::PADE1 aox2::HIS4) and MK500 (aox1::ADE1 aox2::HIS4) strains have been developred.In the second part, recombinant protein production of these strains was studied. Therefore, model protein EGFP gene was transformed into the strains by using pPICZ?A as a vector for extracellular expression. Commercial strains P. pastoris X33 and KM71H which are mostly-used in recombinant protein production were also transformed with EGFP, simultaneously. Single copy gene containing clones were selected from protein producing X33, KM71H, MK321, MK431 and MK500 strains using Southern Blot method and protein production of these strains were compared.Results showed that P. pastoris MK500 is capable of producing higher specific yield of protein (FU/OD) compared to P.pastoris X33 which is the most commonly used in recombinant protein production. Extracellular production of EGFP have been confirmed using SDS-PAGE and Western Blot methods.As a result, P. pastoris MK500 which is newly developed host system is unable to metabolize methanol so requires methanol only as inducer. Consequently, this strain allows to carry out large scale protein production with small amounts of methanol.