Engineered proline hydroxylase polynucleotides and methods
The present disclosure 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.
1. An engineered polynucleotide encoding an engineered polypeptide comprising an amino acid sequence having at least 95% sequence identity to SEQ ID NO:2 and a residue difference as compared to the sequence of SEQ ID NO:2 of H166Q, wherein the engineered polypeptide has proline hydroxylase activity.
2. The engineered polynucleotide encoding the engineered polypeptide of claim 1 which further comprises residue differences at residue positions X2; X3; X4; X5; X9; X13; X25; X26; X29; X30; X36; X42; X52, X57; X58; X59; X66; X92; X95; X103; X112; X115; X116; X121; X131; X150; X151; X225; X230; and X271,
wherein the residue differences are selected from X2K; X2T; X3S; X4Q; X4L; X4E; X4S; X5I; X5L; X5M; X9I; X13T; X25R; X26T; X29A; X30V; X30P; X36T; X42E; X52P; X57T; X57A; X58A; X59G; X66Q;
X92V; X95M; X103L; X103Q; X112T; X112V; X115E; X115H; X115D; X115G; X115S; X115A; X116L; X121F; X131Y; X131F; X150S; X151S; X225L; X225Y; X225W; X230V; X271K; and X271R.
3. The engineered polynucleotide encoding the engineered polypeptide of claim 1 in which the amino acid sequence further comprises at least one residue difference or combination of residue differences selected from the group consisting of:
(a) X103L;
(b) X52P and X255Y;
(c) X4E/L/S and X115A;
(d) X25R and X58A;
(e) X29A;
(f) X115H/D/G and X121F;
(g) X3S and X103L;
(h) X103L and X131Y/F;
(i) X26T and X103L;
(j) X25R, X66Q, X92V and X115E;
(k) X25R, X66Q, X92V, X103L, and X115E; and
(l) X3S, X25R, X66Q, X92V, X103L, and X115E.
4. The engineered polynucleotide encoding the engineered polypeptide of claim 1 which further comprises one or more residue differences as compared to the sequence of SEQ ID NO: 2 at residue positions selected from the group consisting of: X17, X24, X26, X62, X88, X98, X114, X140, X151, X186, X188, and X205.
5. The engineered polynucleotide encoding the engineered polypeptide of claim 4 in which the residue differences at residue positions X17, X24, X26, X62, X88, X98, X114, X140, X151, X186, X188, and X205 are selected from X17V, X24R, X24S, X26R, X26W, X62Q, X88R, X98F, X98T, X114N, X140L, X151A, X151H, X186G, X188G, and X205V.
6. The engineered polynucleotide encoding the engineered polypeptide of claim 1 in which the polypeptide converts substrate compound (2), (2S)-piperidine-2-carboxylic acid,
to product compound (1), (2S,5S)-5-hydroxypiperidine-2-carboxylic acid,
under suitable reaction conditions.
7. The engineered polynucleotide encoding the engineered polypeptide of claim 6 in which the polypeptide converts substrate compound (2) to product compound (1) with at least 2 fold the activity of SEQ ID NO:2, and wherein the amino acid sequence further comprises one or more residue differences selected from the group consisting of: X3S; X4Q; X4L; X51; X5L; X24S; X25R; X30P; X66Q; X86S; X92V; X103L; X103Q; X113E; X115E; X150S; X151S; X225L; and X270E.
8. The engineered polynucleotide encoding the engineered polypeptide of claim 1 in which the polypeptide converts substrate compound (2) to product compound (1) in excess of product compound (la), (2S,3R)-3-hydroxypiperidine-2-carboxylic acid,
wherein the amino acid sequence comprises one or more residue differences selected from the group consisting of: X103L; X115E; and X131Y.
9. The engineered polynucleotide encoding the engineered polypeptide of claim 1 in which the polypeptide forms product compound (1),
in diastereomeric excess of product compound (1R), (2S,5R)-5-hydroxypiperidine-2-carboxylic acid,
10. An expression vector comprising the polynucleotide of claim 1 , and further comprising a control sequence.
11. A host cell comprising the polynucleotide of claim 1 , wherein the host cell is Escherichia coli.
12. A method of preparing an engineered polypeptide, comprising culturing the host cell of claim 11 conditions suitable for expression of the polypeptide, and the method further comprises a step of isolating the engineered polypeptide.