Expression constructs and methods of genetically engineering methylotrophic yeast

A method of producing a heme-containing protein, the method comprising:

• fermenting a recombinant Pichia pastoris cell lacking a recombinant nucleic acid encoding an antibiotic resistance gene in a fermentation broth,
• wherein the recombinant Pichia pastoris cell comprises:
• one or more stably integrated recombinant nucleic acids encoding one or more heme biosynthesis polypeptides selected from ALA synthase, ALA dehydratase, porphobilinogen deaminase, uroporphyrinogen III synthase, uroporphyrinogen III decarboxylase, coproporphyrinogen oxidase, protoporphyrinogen III oxidase, and ferrochelatase,
• a stably integrated recombinant nucleic acid encoding the heme-containing protein operably linked to a first methanol-inducible promoter, and
• a recombinant nucleic acid encoding a transcriptional activator.

A method of producing a heme-containing protein, the method comprising:

• fermenting a recombinant Pichia pastoris cell lacking a recombinant nucleic acid encoding an antibiotic resistance gene in a fermentation broth,
• wherein the recombinant Pichia pastoris cell comprises:
• a stably integrated recombinant nucleic acid encoding ALA synthase, operably linked to a first methanol-inducible promoter,
• a stably integrated recombinant nucleic acid encoding ALA dehydratase, operably linked to a second methanol-inducible promoter,
• a stably integrated recombinant nucleic acid encoding porphobilinogen deaminase, operably linked to a third methanol-inducible promoter,
• a stably integrated recombinant nucleic acid encoding uroporphyrinogen III synthase, operably linked to a fourth methanol-inducible promoter,
• a stably integrated recombinant nucleic acid encoding uroporphyrinogen III decarboxylase, operably linked to a fifth methanol-inducible promoter,
• a stably integrated recombinant nucleic acid encoding coproporphyrinogen oxidase, operably linked to a sixth methanol-inducible promoter,
• a stably integrated recombinant nucleic acid encoding protoporphyrinogen III oxidase, operably linked to a seventh methanol-inducible promoter,
• a stably integrated recombinant nucleic acid encoding ferrochelatase, operably linked to a eighth methanol-inducible promoter,
• two or more copies of a stably integrated recombinant nucleic acid encoding the heme-containing protein, operably linked to a ninth methanol-inducible promoter, and
• a recombinant nucleic acid encoding a transcriptional activator operably linked to a constitutive promoter or a tenth methanol-inducible promoter,
• wherein each of the first through ninth methanol-inducible promoters comprises a sequence to which the transcriptional activator binds.