Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-9279129-B2
Publication Date
2016-03-08
Expiration Date
Abstract
A mutant Pichia pastoris alcohol oxidase 1 (AOX1) promoter of the wild type Pichia pastoris AOX1 promoter (SEQ ID No. 1) comprising at least one mutation selected from the group consisting of: a) a transcription factor binding site (TFBS), b) nucleotides 170 to 235 (−784 to −719), nucleotides 170 to 191 (−784 to −763), nucleotides 192 to 213 (−762 to −741), nucleotides 192 to 210 (−762 to −744), nucleotides 207 to 209 (−747 to −745), nucleotides 214 to 235 (−740 to −719), nucleotides 304 to 350 (−650 to −604), nucleotides 364 to 393 (−590 to −561), nucleotides 434 to 508 (−520 to −446), nucleotides 509 to 551 (−445 to −403), nucleotides 552 to 560 (−402 to −394), nucleotides 585 to 617 (−369 to −337), nucleotides 621 to 660 (−333 to −294), nucleotides 625 to 683 (−329 to −271), nucleotides 736 to 741 (−218 to −213), nucleotides 737 to 738 (−217 to −216), nucleotides 726 to 755 (−228 to −199), nucleotides 784 to 800 (−170 to −154) or nucleotides 823 to 861 (−131 to −93) of Seq ID No. 1, and combinations thereof.
Core Innovation
The invention relates to an isolated mutant Pichia pastoris alcohol oxidase 1 (AOX1) promoter of a wild type Pichia pastoris AOX1 promoter comprising SEQ ID NO: 1 for expression under derepression conditions. The promoter is defined by deletion of nucleotides 694 to 723 of SEQ ID NO: 1, with the mutant promoter producing expression behavior described as altered glucose repression/derepression and methanol induction behavior.
The document further describes mutant AOX1 promoter variants in which additional defined mutations are introduced in transcription factor binding sites and within specific nucleotide ranges of SEQ ID NO: 1. TFBS regions are emphasized, including Hap1, Hsf, Hap234, abaA, Stre, Rap1, Adr1, Mat1MC, and QA-1F, together with further sequence changes within defined nucleotide ranges, resulting in surprisingly enhanced derepression and methanol-induced expression.
The effectiveness of the mutant promoter is reported as depending on promoter copy number, where multicopy strains show substantially enhanced expression relative to single-copy strains. The document also highlights key promoter regions and specific nucleotide position ranges, including 694 to 723 and additional ranges including 729 to 763, 736 to 741, 737 to 738, 726 to 755, and 784 to 800, as producing derepression and methanol-induced expression outcomes in engineered expression cassettes using operably linked nucleic acids.
Claims Coverage
The independent claims cover four inventive features: a defined mutant AOX1 promoter variant for derepression conditions, nucleic acids containing the mutant promoter in single- or multi-copy expression cassettes, recombinant expression workflows using the construct in cells, and kit formats including a vector and cell configured for AOX1-promoter-controlled expression.
Mutant AOX1 promoter for expression under derepression conditions with defined deletion
An isolated mutant Pichia pastoris alcohol oxidase 1 (AOX1) promoter comprising SEQ ID NO: 1 for expression under derepression conditions, wherein nucleotides 694 to 723 of SEQ ID NO: 1 are deleted.
Nucleic acid with mutant AOX1 promoter in single- or multi-copy expression cassettes
A nucleic acid molecule containing at least one mutant Pichia pastoris alcohol oxidase 1 (AOX1) promoter operably linked to at least one nucleic acid encoding a protein or functional nucleic acid to form a single- or multi-copy expression cassette.
Recombinant expression workflow using a nucleic acid or vector carrying the mutant construct
A method expresses a recombinant protein, peptide, or functional nucleic acid in a cell by providing a nucleic acid molecule or a vector carrying it, transforming the cell, culturing the transformed cell, expressing the target molecule, and isolating it.
Kit including vector and cell configured to express a selected protein under AOX1 promoter control
A kit that includes a vector and a cell configured to express a selected protein under control of the AOX1 promoter of the vector.
Overall, the claims tie a defined mutant AOX1 promoter deletion to derepression-condition expression, extend to promoter refinements and nucleic acid constructs in single- or multi-copy expression cassettes, and further cover recombinant expression in cells and kit formats providing the vector together with an expression-configured cell.
Stated Advantages
Substantially enhanced expression in multicopy strains.
Surprisingly enhanced derepression and methanol-induced expression.
Documented Applications
Operably linked nucleic acids for expression of a protein or functional nucleic acid in engineered cells or hosts using the mutant AOX1 promoter under derepression conditions.
Recombinant expression use cases where a recombinant protein, peptide, or functional nucleic acid is expressed in a cell and the expressed material is isolated.
Reporter and characterization use cases involving promoter variants assessed with reporter constructs including GFP-Zeo and other reporter enzymes.
Kit use cases that include a vector and a cell configured to express a selected protein under the AOX1 promoter.
Interested in licensing this patent?