Expression constructs and methods of genetically engineering methylotrophic yeast
Inventors
Shankar, Smita • Hoyt, Martin Andrew
Assignees
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Publication Number
US-10689656-B2
Publication Date
2020-06-23
Expiration Date
Abstract
Methods and materials for genetically engineering methylotrophic yeast are provided.
Core Innovation
The invention provides an engineered methylotrophic yeast cell in which a recombinant nucleic acid molecule includes a first exogenous nucleic acid encoding a methanol expression regulator 1 (Mxr1) transcriptional activator operably linked to at least one methanol-inducible promoter element. The promoter element comprises a sequence to which the Mxr1 transcriptional activator binds. A second exogenous nucleic acid encodes a heterologous polypeptide and is operably linked to at least one methanol-inducible promoter element.
The disclosed system uses methanol-inducible promoters, including promoter elements such as AOX1, AOD1, MOX, MOD1, DHAS, FLD1, and PEX8, together with Mxr1, and further contemplates other activators such as Adr1 and Trm1/Trm2. The document describes a positive feedback arrangement in which AOX1-promoter transgenes are driven to enable high-level expression of transgenes, including heme-biosynthesis enzymes and soybean leghemoglobin (LegH).
The invention is described as enabling high-level expression of AOX1-promoter transgenes with reduced or no methanol induction once repressing carbon sources are depleted. The document further discloses methylotrophic yeast strains and recombinant nucleic acid configurations, including stable genomic integration or plasmid expression, and additional strain engineering for marker-free embodiments and hybrid promoter strains with constitutive LegH expression.
Claims Coverage
The partial content includes two independent claims (clm-00001 and clm-00026). Across these independent claims, the coverage centers on an Mxr1 transcriptional activator under methanol-inducible promoter control and the operable linkage of a heterologous polypeptide, or a defined globin/heme-biosynthesis set in the Pichia-specific case, to methanol-inducible promoter elements containing an Mxr1 binding sequence.
Mxr1 transcriptional activator under methanol-inducible promoter binding control
A methylotrophic yeast cell comprising a recombinant nucleic acid molecule wherein the recombinant nucleic acid molecule comprises a first exogenous nucleic acid encoding a methanol expression regulator 1 (Mxr1) transcriptional activator operably linked to at least one methanol-inducible promoter element comprising a sequence to which the Mxr1 transcriptional activator binds.
Heterologous polypeptide under methanol-inducible promoter element control
A methylotrophic yeast cell comprising a recombinant nucleic acid molecule wherein the recombinant nucleic acid molecule comprises a second exogenous nucleic acid encoding a heterologous polypeptide operably linked to at least one methanol-inducible promoter element.
Pichia-specific Mxr1 plus globin-family member PF00042 plus heme-biosynthesis polypeptides under methanol-inducible promoter control
A methylotrophic Pichia yeast cell comprising a recombinant nucleic acid molecule wherein the recombinant nucleic acid molecule comprises a nucleic acid encoding a Mxr1 transcriptional activator sequence from P. pastoris; a nucleic acid encoding a member of globin family PF00042; and a nucleic acid encoding at least one polypeptide involved in heme biosynthesis, wherein each nucleic acid is operably linked to a methanol-inducible promoter element and the methanol-inducible promoter element comprises at least one sequence to which a Mxr1 transcriptional activator binds.
Overall, claim coverage is directed to methylotrophic yeast cells engineered with an Mxr1 transcriptional activator and promoter elements containing an Mxr1 binding sequence, where a heterologous polypeptide is operably linked to methanol-inducible promoter elements; in the Pichia-specific independent claim, the construct additionally specifies a globin-family PF00042 member together with at least one heme-biosynthesis polypeptide, each under methanol-inducible promoter element control.
Stated Advantages
Enables high-level expression of AOX1-promoter transgenes.
Enables reduced or no methanol induction once repressing carbon sources are depleted.
Provides improved yields and increased performance in fermenters.
Documented Applications
Production of soybean leghemoglobin (LegH) with heme-loaded protein in methylotrophic yeast, including phenotypic red color and SDS gel confirmation described in the partial content.
Fermenter-based production/processing, with described increased performance in fermenters.
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