The conceptual basis for the use of the AOX1 promoter in the P. pastoris expression system stems from the observation that the alcohol oxidase enzyme encoded by AOX1 is required specifically and only for methanol metabolism of the yeast and is present at substantial levels only when cells are grown on methanol (Veenhuis et al., 1983; Egli et al., 1980). Biochemical studies have shown that methanol utilization utilizes a novel metabolic pathway involving several unique enzymes (Veenhuis et al., 1983). The enzyme alcohol oxidase (AOX) catalyzes the first step in the methanol utilization pathway, the oxidation of methanol to formaldehyde and hydrogen peroxide (see Fig.). AOX is sequestered within the peroxisome along with catalase, which degrades hydrogen peroxide to oxygen and water. A portion of the formaldehyde generated by AOX leaves the peroxisome and is further oxidized to formate and carbon dioxide by two cytoplasmic dehydrogenases, reactions that are a source of energy for cells growing on methanol.
The remaining formaldehyde is assimilated to form cellular constituents by a cyclic pathway that starts with the condensation of formaldehyde with xylulose 5-monophosphate, a reaction catalyzed by a third peroxisomal enzyme dihydroxyacetone synthase (DHAS). The products of this reaction, glyceraldehyde 3-phosphate and dihydroxyacetone, leave the peroxisome and enter a cytoplasmic pathway that regenerates xylulose 5-monophosphate and, for every three cycles, one net molecule of glyceraldehyde 3-phosphate. Two of the methanol pathway enzymes, AOX and DHAS, are present at high levels in cells grown on methanol but are not detectable in cells grown on most other carbon sources (e.g., glucose, glycerol, or ethanol). In cells fed methanol at growth-limiting rates in fermenter cultures, AOX levels are dramatically induced, constituting >30% of total soluble protein (Couderc and Baratti, 1980; Roggenkamp et al., 1984).Figure. Methanol-metabolism in yeast. 1. Alcohol oxidase; 2. Catalase; 3. Formate dehydrogenase; 4. Formaldehyde dehydrogenase; 5. Dihydroxyacetone synthase; 6. Dyhydroxyacetone kinase; 7. Fructose 1,6-bisphosphate aldolase; 8, FBP phosphatase.