Patent ID: 7479389

Claim:
A method for alternately inactivating two or more biosynthetic pathways in a P. pastoris host, comprising the steps of: (a) inactivating a first gene from a first biosynthetic pathway in the P. pastoris host, wherein said first gene is involved in the synthesis of a first amino acid or a nucleotide selected from the group consisting of adenine, arginine, histidine, methionine and uracil, by deleting or disrupting the first gene with a first nucleic acid encoding a first selectable marker, thereby rendering the P. pastoris host auxotrophic for the first amino acid or nucleotide whose synthesis the first gene is involved in; and (b) inactivating a second gene from a second biosynthetic pathway in the P. pastoris host from step (a), wherein said second gene is involved in the synthesis of a second amino acid or a nucleotide selected from the group consisting of adenine, arginine, histidine, methionine, and uracil, by deleting or disrupting the second gene with a second nucleic acid, wherein said second nucleic acid is the gene that was inactivated in step (a), wherein the second gene is not involved in the synthesis of the first nucleotide or amino acid whose synthesis the first gene of step (a) is involved in, thereby rendering the P. pastoris host auxotrophic for the second amino acid or nucleotide whose synthesis the second gene is involved in, and no longer auxotrophic for the first amino acid or nucleotide of step (a).