Engineered proline hydroxylase biocatalysts for hydroxylation of chemical compounds
Inventors
Nazor, Jovana • Osborne, Robert • Liang, Jack • Vroom, Jonathan • Zhang, Xiyun • Entwistle, David • Voladri, Rama • Garcia, Ravi David • Moore, Jeffrey C. • Grosser, Shane • Kosjek, Birgit • Truppo, Matthew
Assignees
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Publication Number
US-12286648-B2
Publication Date
2025-04-29
Expiration Date
Abstract
The present invention provides engineered proline hydroxylase polypeptides for the production of hydroxylated compounds, polynucleotides encoding the engineered proline hydroxylases, host cells capable of expressing the engineered proline hydroxylases, and methods of using the engineered proline hydroxylases to prepare compounds useful in the production of active pharmaceutical agents.
Core Innovation
The invention relates to engineered proline hydroxylase biocatalysts having proline hydroxylase activity and being defined relative to reference sequence SEQ ID NO:604. The engineered polypeptides have at least 90% sequence identity to SEQ ID NO:604 with one or more residue differences confined to selected residue positions, and the sequence frameworks include variants with 80–99% identity and residue-position sets that specify where substitutions may occur.
The engineered proline hydroxylase polypeptides are disclosed as improving activity, regioselectivity, substrate tolerance, and stability/expression. The invention further provides encoding polynucleotides, expression vectors, and host cells for expressing the engineered enzymes, together with general process concepts for expressing and optionally isolating the engineered enzymes.
The document describes catalytic transformation using the proline hydroxylase activity, including conversion of (S)-pipecolic acid to (2S,5S)-5-hydroxypipecolic acid. It also provides quantitative performance concepts including fold activity relative to a naturally occurring enzyme and diastereomeric excess metrics.
Claims Coverage
The independent claims cover an engineered proline hydroxylase polypeptide defined by sequence identity to SEQ ID NO:604 and constrained residue differences at specified positions, with functional conversion toward hydroxylated pipecolic acid products. Four inventive features are identified in the provided claim coverage.
Engineered proline hydroxylase polypeptide defined by sequence identity to SEQ ID NO:604
An engineered polypeptide having proline hydroxylase activity comprises an amino acid sequence having at least 90% sequence identity to reference sequence SEQ ID NO:604 and one or more residue differences compared to SEQ ID NO:604 at residue positions selected from the listed residue positions.
Specific conversion of (S)-pipecolic acid to (2S,5S)-5-hydroxypipecolic acid
The engineered polypeptide converts (S)-pipecolic acid into (2S,5S)-5-hydroxypipecolic acid.
Fold-activity threshold over a naturally occurring enzyme
The engineered polypeptide can convert (S)-pipecolic acid to (2S,5S)-5-hydroxypipecolic acid with at least the specified fold-increase in activity over the naturally occurring enzyme.
Diastereomeric excess selectivity based on (2S,5R)-5-hydroxypipecolic acid
The engineered polypeptide converts (S)-pipecolic acid to (2S,5S)-5-hydroxypipecolic acid with a diastereomeric excess of (2S,5R)-5-hydroxypipecolic acid greater than 90% through at least 99% or more.
Across the provided claim coverage, the inventive thrust is a sequence-defined engineered proline hydroxylase relative to SEQ ID NO:604, restricted to a listed set of permissible residue differences, paired with conversion of (S)-pipecolic acid to (2S,5S)-5-hydroxypipecolic acid and quantitative activity and selectivity thresholds.
Stated Advantages
Improved conversion of substrate compound (1) to hydroxylated product compound (2).
Higher fold activity versus reference enzymes/naturally occurring enzyme.
Improved regioselectivity, activity, specific activity, and thermostability.
Improved conversion and kinetics.
Improved diastereomeric excess/selectivity for hydroxylated stereochemical outputs.
Improved substrate tolerance.
Improved stability/expression.
Documented Applications
Engineered proline hydroxylase variants for conversion of (S)-pipecolic acid to specified hydroxylated products, including (2S,5S)-5-hydroxypipecolic acid, with performance metrics for activity and diastereomeric excess.
Catalytic hydroxylation transformation of (S)-pipecolic acid to (2S,5S)-5-hydroxypipecolic acid using engineered proline hydroxylase activity.
Use of engineered hydroxylases in immobilized forms on solid supports.
Use of polynucleotides, expression vectors, and host cells to express engineered hydroxylases.
Use of kit and array formats for engineered enzyme preparations.
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