Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-9012175-B2
Publication Date
2015-04-21
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 isolated Pichia pastoris alcohol oxidase 1 (AOX1) promoter sequences. The promoter comprises the wild type AOX1 promoter sequence as set forth in SEQ ID NO: 1, wherein nucleotides 170 to 235 are deleted.
The nucleotides 170 to 235 deletion causes increased expression activity compared to the wild-type promoter under methanol-induced conditions. The disclosed promoter variants are constructed to modify promoter behavior under methanol-induced and derepression conditions.
Beyond the 170 to 235 deletion, the disclosure refines AOX1 promoters by including mutations in transcription factor binding sites and by providing defined nucleotide-region mutations within SEQ ID NO: 1. Specific TFBS-related components are identified, including Rap1, Gcr1, Hsf, Adr1, Mat1MC, QA-1F, Hap1, Hap234, abaA, and Stre.
Claims Coverage
The independent claim set includes 4 inventive features focused on the mutant AOX1 promoter, its use in expression cassettes, recombinant expression, and isolation of super expression clones.
AOX1 promoter with nucleotides 170 to 235 deletion
An isolated Pichia pastoris alcohol oxidase 1 (AOX1) promoter comprising the wild type AOX1 promoter sequence as set forth in SEQ ID NO: 1, wherein nucleotides 170 to 235 are deleted, wherein the deletion causes increased expression activity compared to the wild-type promoter under methanol-induced conditions.
Mutant AOX1 promoter operably linked in single- or multi-copy expression cassette
An isolated cell wherein a mutant Pichia pastoris alcohol oxidase 1 (AOX1) promoter according to claim 1 is operably linked to one or more nucleic acids that encode a protein, peptide, or functional nucleic acid, wherein the one or more nucleic acids form a single- or multi-copy expression cassette.
Recombinant expression using mutant AOX1 promoter in a single- or multi-copy cassette
A method for expressing a recombinant protein, peptide, or functional nucleic acid in a cell by using at least one nucleic acid molecule with a mutant Pichia pastoris alcohol oxidase 1 (AOX1) promoter operably linked to a coding nucleic acid in a single- or multi-copy expression cassette, transforming said cell with the nucleic acid molecule, and culturing the transformed cell in a suitable culture medium to obtain expression.
Isolating super expression clones using a mutated methanol-inducible promoter and a VA protein or functional nucleic acid
A method isolating super expression clones by introducing an expression vector comprising a mutated methanol-inducible promoter operably linked to a nucleic acid encoding a VA protein or functional nucleic acid and a selective marker gene, growing the cells on selective medium comprising a non-repressing carbon source and methanol under inducing conditions, incubating, isolating colonies, and detecting super-expressing clones by measuring expression rate.
Across the independent claims, the core protection centers on an isolated mutant Pichia pastoris AOX1 promoter defined by a specific deletion (nucleotides 170 to 235 in SEQ ID NO: 1) that increases expression under methanol-induced conditions, and on using that mutant promoter within operably linked nucleic acids and single- or multi-copy expression cassettes to support recombinant expression and isolation of super expression clones.
Stated Advantages
Increased expression activity compared to the wild-type promoter under methanol-induced conditions.
Enables isolation of super expression clones by detecting super-expressing clones using expression rate.
Documented Applications
Recombinant expression of a protein, peptide, or functional nucleic acid in a cell using a mutant Pichia pastoris AOX1 promoter operably linked to coding nucleic acids in a single- or multi-copy expression cassette.
Isolation of super expression clones using an expression vector with a mutated methanol-inducible promoter, a VA protein or functional nucleic acid, and a selective marker gene, followed by colony isolation and detection by expression rate.
Interested in licensing this patent?