Transformation of yeasts of the genus pichia

Claim 1

A process for transforming a host yeast strain of Pichia pastoris, which is defective in at least one biosynthetic pathway, said process comprising: (a) contacting the host yeast strain with a sulfhydryl group reducing agent; (b) contacting the product cells of step (a) with a cell wall degrading reagent under conditions suitable for the formation and maintenance of spheroplasts; (c) contacting the spheroplasts generated in step (b) with a hybrid plasmid which is capable of functioning as a selectable marker to complement said defect under conditions suitable for transformation; and (d) treating the product of step (c) under cell wall regenerating conditions wherein said conditions suitable for the formation of spheroplasts comprise: (i) 10-100 μg of cell wall degrading reagent per 10 mL of cell suspension; wherein said cell suspension is prepared by suspending exponentially growing cells in SCE buffer; (ii) maintained at 25-35° C.; (iii) for 15-60 minutes; and wherein said contacting conditions suitable for transformation comprise: (i) 2-10 volumes of CaCl 2 -polyethylene glycol solution per volume of spheroplast-containing suspension; (ii) maintained at 20-30° C.; (iii) for 5-30 minutes and where said cell wall regenerating conditions comprise: (i) adding transformed spheroplasts to regeneration agar, wherein said regeneration agar comprises: about 1 M sorbitol, about 0.1 M dextrose, about 7 g/L yeast nitrogen base, and about 3% agar; (ii) maintained at 25-35° C.; (iii) for about 3-10 days.