Biocatalysts and methods for hydroxylation of chemical compounds
Inventors
Vroom, Jonathan • Ortega Guerra, Alberto • Liang, Jack • Cope, Gregory A. • Dellas, Nikki • Prier, Christopher Keltie
Assignees
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Publication Number
US-12344865-B2
Publication Date
2025-07-01
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 and engineered polypeptides having proline hydroxylase activity. The engineered polypeptides comprise an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 4, or to specified alternative reference sequences, and further comprise one or more amino acid substitutions at defined residue positions.
The disclosed variants are based on sequence identity to reference proteins including SEQ ID NO: 562, SEQ ID NO: 598, and SEQ ID NO: 630, with explicit residue substitution sets relative to those reference sequences. The engineered polypeptides and sequence variants are described as improved relative to a wild-type enzyme for converting L-proline to trans-3-hydroxyproline.
The invention further includes polynucleotides encoding the engineered proline hydroxylase variants, including codon optimized nucleic acids for E. coli, expression vectors, host cells, and enzyme preparations. Additional embodiments include engineered polypeptide fragments with functional activity, variants with additional residue differences and deletions, insertions, or substitutions, and downstream use formats that include immobilization on solid supports and kit or array formats.
Claims Coverage
The provided claim coverage centers on two independent claims: one directed to an engineered proline hydroxylase polypeptide and one directed to a polynucleotide encoding the engineered polypeptide. The inventive features are defined by sequence identity to SEQ ID NO: 4, and in some embodiments alternative SEQ ID sequences, together with one or more amino acid substitutions restricted to enumerated residue positions, with dependent claims adding higher identity thresholds or substitution restrictions.
Engineered proline hydroxylase polypeptide defined by sequence identity and selected substitutions
An engineered polypeptide having proline hydroxylase activity, comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 4, and further comprising one or more amino acid substitutions as compared to SEQ ID NO: 4 at positions selected from the group consisting of 194, 123, 21, 28, 58/247, 65, 80, 85, 95, 98, 117, 120, 159, 185, 199, 200, 233, 237, 243, 250, 268, 281, 282, 287, 289, 307, 324, 326, 327, 330, 338, 343, 346, and 348.
Engineered polypeptide variants with additional identity constraints and restricted substitutions
The engineered polypeptide having at least 85% or at least 87% sequence identity to the amino acid sequence of SEQ ID NO: 4, together with one or more amino acid substitutions at positions selected from specified groups, including the substitution at position 194 to 194L for at least one variant.
Engineered polypeptide variants based on alternative reference sequences
The engineered polypeptide having at least 80% sequence identity to at least one amino acid sequence selected from the specified set of SEQ ID NOs.
Polynucleotide encoding the engineered proline hydroxylase polypeptide
A polynucleotide that includes a nucleotide sequence encoding the engineered polypeptide.
Across the independent and dependent claims, coverage focuses on engineered proline hydroxylase polypeptides defined by sequence identity to SEQ ID NO: 4 and/or alternative SEQ ID sequences, together with one or more amino acid substitutions restricted to specified residue positions. The claim set also includes a polynucleotide encoding the engineered polypeptide.
Stated Advantages
Enhanced enzymatic conversion of L-proline to trans-3-hydroxyproline.
Enhanced regioselectivity, including trans-3 versus trans-4 hydroxyproline selectivity.
Enhanced specific activity.
Enhanced thermostability.
Improved conversion kinetics, including fold-activity thresholds and conversion performance metrics.
Improved substrate tolerance.
High trans-3 isomer excess is referenced as an outcome.
Host-expression enhancement, including expression in E. coli.
Documented Applications
Hydroxylating L-proline to trans-3-hydroxyproline using engineered proline hydroxylase biocatalysts, including variants of ANO11243.
Polynucleotide, expression vector, and host-cell embodiments, including E. coli codon optimization and E. coli host cell example, for production of engineered proline hydroxylase enzymes.
Preparing and isolating engineered enzymes.
Immobilization on solid supports and use in kit or array formats.
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