Expression constructs and methods of genetically engineering methylotrophic yeast
Inventors
Shankar, Smita • Hoyt, Martin Andrew
Assignees
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Publication Number
US-12084667-B2
Publication Date
2024-09-10
Expiration Date
Abstract
Methods and materials for genetically engineering methylotrophic yeast are provided.
Core Innovation
The invention relates to genetically engineered methylotrophic yeast, including recombinant Pichia pastoris cells, for producing a heme-containing protein. The disclosed method includes fermenting a recombinant Pichia pastoris cell lacking a recombinant nucleic acid encoding an antibiotic resistance gene in a fermentation broth, and inducing expression of the heme-containing protein with methanol. The recombinant Pichia pastoris cell includes stably integrated recombinant nucleic acids encoding heme biosynthesis polypeptides and a heme-containing protein operably linked to a methanol-inducible promoter, together with a recombinant nucleic acid encoding a transcriptional activator.
A central aspect is the integration of methanol-pathway transcriptional regulation with heme biosynthesis, using methanol-inducible promoter control and a transcriptional activator. In the described construct logic, stably integrated heme biosynthesis enzymes are provided to support production of heme, while a heme-containing protein nucleic acid is operably linked to a methanol-inducible promoter to enable methanol-responsive expression. The transcriptional activator is linked to constitutive or methanol-inducible promoter contexts and is defined by binding to sequences present in the methanol-inducible promoters.
The disclosed embodiments additionally include promoter architecture and gene copy architecture features. Separate heme biosynthesis polypeptides are each operably linked to methanol-inducible promoters in a multi-promoter arrangement, and two or more copies of a stably integrated heme-containing protein nucleic acid are provided with a designated methanol-inducible promoter. The framework includes specific sets of heme biosynthesis polypeptides, including ALA synthase, ALA dehydratase, porphobilinogen deaminase, uroporphyrinogen III synthase, uroporphyrinogen III decarboxylase, coproporphyrinogen oxidase, protoporphyrinogen III oxidase, and ferrochelatase, and supports selection of named transcriptional activators and methanol-inducible promoter choices.
Claims Coverage
The partial content provides two independent claims. Across these claims, the inventive features cover methanol-induced heme-containing protein production in recombinant Pichia pastoris, stably integrated heme biosynthesis polypeptides, a methanol-inducible heme-containing protein expression cassette, and a transcriptional activator arranged to regulate methanol-inducible promoters, with additional promoter and copy-number constraints in the second independent claim.
Methanol-induced heme-containing protein production with integrated heme biosynthesis and transcriptional activator
Fermenting a recombinant Pichia pastoris cell lacking a recombinant nucleic acid encoding an antibiotic resistance gene in a fermentation broth, inducing expression of the heme-containing protein with methanol, 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.
Multi-promoter heme pathway integration with multiple copies of heme-containing protein and activator-bound methanol-inducible promoters
Fermenting a recombinant Pichia pastoris cell lacking a recombinant nucleic acid encoding an antibiotic resistance gene in a fermentation broth, inducing expression of the heme-containing protein with methanol, 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 an 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.
Across the two independent claims, the coverage is centered on recombinant Pichia pastoris fermentation followed by methanol-induced expression of a heme-containing protein, enabled by stably integrated heme biosynthesis polypeptides under methanol-inducible promoter control and a transcriptional activator that regulates methanol-inducible promoter sequences. The second independent claim further specifies a multi-step promoter integration of the heme pathway genes, provides multiple copies of the heme-containing protein nucleic acid, and requires that the relevant methanol-inducible promoters contain sequences bound by the transcriptional activator.
Stated Advantages
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Documented Applications
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