Polynucleotides encoding engineered ketoreductase polypeptides
Inventors
Voladri, Rama • Gooding, Owen • Jenne, Stephan • Mundorff, Emily
Assignees
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Publication Number
US-8455230-B2
Publication Date
2013-06-04
Expiration Date
Abstract
The present disclosure provides engineered ketoreductase enzymes having improved properties as compared to a naturally occurring wild-type ketoreductase enzyme including the capability of stereospecifically reducing (R)-2-methylpentanal to (R)-2-methylpentanol. Also provided are polynucleotides encoding the engineered ketoreductase enzymes, host cells capable of expressing the engineered ketoreductase enzymes, and methods of using the engineered ketoreductase enzymes to produce (R)-2-methylpentanol and related compounds.
Core Innovation
The patent describes engineered ketoreductase (KRED) polypeptide variants defined relative to reference sequences, including polypeptides comprising residues 90-211 and optionally residues 1-89. The variants are specified by residue substitutions at numbered positions, including residues corresponding to X82, X145, X153, X147, and X223, with allowed conservative differences and defined amino-acid identity ranges, including identity thresholds in the range of 85%-99%.
Further residue feature options and additional improvements are described through enumerated substitutions relative to wild-type, including options for conservative mutations and deletions that maintain ketoreductase activity. The engineered polypeptides can also be provided as fusion polypeptides, and the document addresses polypeptides including non-encoded or non-standard amino acids.
The document also describes polynucleotides encoding the engineered KRED polypeptides, including codon optimization and multiple nucleotide sequences corresponding to amino-acid sequence variants. The polynucleotides are further characterized by strict hybridization conditions and by excluding certain sequences, including exclusion of SEQ ID NO:15 and SEQ ID NO:41.
As a use case, the patent describes an enantiospecific reduction of (R)-2-alkyl aldehydes, including mixtures, to chiral (R)-2-alkyl alcohols, including formation of (R)-2-methylpentanol from 2-methylpentanal, with stereomeric excess exceeding 98% and in some instances exceeding 99%. The document further discusses use of cofactor/cofactor-regeneration systems, including GDH/glucose, FDH/formate, and sADH/isopropanol.
Claims Coverage
The identified independent-claim family is directed to isolated or recombinant polynucleotides encoding engineered ketoreductase polypeptides. The independent claim requires a minimum identity to a reference sequence (SEQ ID NO:4) and fixes the residue corresponding to X153, with dependent claims adding additional positional residue constraints and optional construct features such as expression vectors and production/recovery steps.
Isolated or recombinant polynucleotide encoding a ketoreductase with at least 90% identity to SEQ ID NO: 4 and X153=glutamine
An isolated or recombinant polynucleotide encoding a ketoreductase polypeptide comprising an amino acid sequence having at least 90% identity to a reference sequence of SEQ ID NO: 4, wherein the residue corresponding to X153 is glutamine.
Additional residue identity constraints at X145
The polynucleotide encodes a ketoreductase polypeptide whose amino acid sequence has serine at the residue corresponding to X145.
Positional residue constraints across multiple numbered positions with option for other differences
The polynucleotide encodes a ketoreductase polypeptide amino acid sequence including specified residue identities at positions X72, X82, X96, X104, X117, X147, X157, X177, X178, X202, X223, and/or X236, with optional additional differences at other amino acid residues relative to a reference sequence.
Expression vector with operably linked control sequences
An expression vector where the polynucleotide from claim 1 is operably linked to control sequences that direct gene expression in a host cell.
Expressing the polynucleotide in a host cell and recovering the ketoreductase polypeptide
A method prepares a ketoreductase polypeptide by expressing a polynucleotide in a host cell and recovering the resulting polypeptide from the host cell or culture medium.
Across the identified independent-claim family, the core coverage focuses on polynucleotides that encode ketoreductase polypeptides with at least 90% identity to SEQ ID NO:4 while specifying the X153 glutamine residue, with dependent embodiments further fixing additional residue identities at numbered positions. The claims also extend to expression vector embodiments and to a method centered on expression in a host cell and recovery of the ketoreductase polypeptide.
Stated Advantages
Increased activity.
Enhanced stereospecificity, including high stereomeric excess.
Improved enantiospecificity, via reported E-values.
Improved thermostability.
Enantiospecific reduction of (R)-2-alkyl aldehydes, including mixtures, to chiral (R)-2-alkyl alcohols.
Stereomeric excess of the product exceeding 98%, and in some instances exceeding 99%.
Use of cofactor/cofactor-regeneration systems, including GDH/glucose, FDH/formate, and sADH/isopropanol, in the described reduction process.
Documented Applications
Production or resolution of (R)-2-methylpentanol from (R)-2-methylpentanal, including stereospecific reduction from racemic mixtures where the (S) enantiomer is left unreacted.
Encoding and expression of engineered ketoreductase polypeptides for achieving stereospecific reduction, using polynucleotides and host cells.
Enantiospecific reduction of (R)-2-alkyl aldehydes, including mixtures, to chiral (R)-2-alkyl alcohols, including (R)-2-methylpentanol from 2-methylpentanal.
Use of cofactor regeneration systems, including GDH/glucose, FDH/formate, and sADH/isopropanol, in the enantiospecific reduction context described in the document.
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