Biocatalysts and methods for hydroxylation of compounds

Abstract

To provide biocatalysts and methods for hydroxylation of compounds.SOLUTION: 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. The engineered enzymes with one or more improved properties have one or more residue differences as compared to the naturally occurring proline hydroxylase, where the residue differences occur at residue positions affecting one or more of the foregoing enzyme properties.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 940,647, filed November 26, 2019, which is incorporated by reference in its entirety for all purposes.

[0002] Technical Field The present invention relates to biocatalysts for the hydroxylation of compounds.

[0003] Reference to a sequence listing, table or computer program An official copy of the Sequence Listing is being submitted contemporaneously with the present specification as a text file in ASCII format via EFS-Web with the file name "CX2-193WO1_ST25.txt," a creation date of November 17, 2020, and a size of 1.39 megabytes. The Sequence Listing filed via EFS-Web is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] background Due to the constrained conformation of proline, proline derivatives bearing functional groups on the ring carbons are useful building blocks for the synthesis of pharmaceutical compounds. One such derivative, hydroxylated proline, is used in the synthesis of carbapenem antibiotics (see, e.g., Altamura et al., J. Med., Chem. 38(21):4244-56

[1995] ), angiotensin-converting enzyme inhibitors, and protease inhibitors (see, e.g., Chen et al., J. Med., Chem. 38(21):4244-56

[1995] ). al., J. Org. Chem., 67(8):2730-3

[2002] ; Chen et al., 2006, J. Med. Chem. 49(3):995-1005), nucleic acid analogs (see, e.g., Efimov et al., Nucleic Acids Res., 34(8):2247-2257

[2006] ), isoprenyltransferase inhibitors (O'Connell et al., Chem. Pharm. Bull., 48(5):740-742

[2000] ) and drug library construction (Vergnon et al., J. Comb. Chem., 6(1):91-8

[2004] ; and Remuzon, Tetrahedron 52:13803-13835

[1996] ).

[0005] Hydroxyproline can be obtained from natural sources, such as plant materials and collagen hydrolysates. Hydroxyproline can be obtained from the starting materials, allyl bromide and diethylacetamidomalonic acid (Kyun Lee et al., Bull. Chem. Soc. Japan, 46:2924

[1973] ), D-glutamic acid (Eguchi et al., Bull. Chem. Soc. Japan, 47:1704-08

[1974] ), glyoxal and oxaloacetic acid (Ramaswamy et al., J. Org. Chem., 42(21):3440-3443

[1977] ), and α-alanine (Sinha et al., Proc. ECSOC-4, The Fourth International Electronic Conference on Synthetic Organic Chemistry, ISBN It can also be chemically synthesized from hydroxybenzoates such as hydroxybenzoates (3-906980-05-7

[2000] ).

[0006] Isolation from natural sources is limited by raw material availability, requires purification from significant background contaminants, and lacks certain desired diastereomers. Chemical synthesis methods require complex steps, can be difficult to scale up to industrial levels, and may require additional purification steps due to the formation of multiple hydroxylated products.

[0007] Another approach to preparing hydroxylated proline uses proline hydroxylase, a 2-oxoglutarate-dependent dioxygenase that utilizes 2-oxoglutarate (α-ketoglutarate) and O as cosubstrates and ferrous iron as a cofactor (see, e.g., Klein et al., Adv. Synth. Catal., 353:1375-1383

[2011] ; U.S. Patent No. 5,364,775; and Shibasaki et al., Appl. Environ. Microbiol., 65(9):4028-4031

[1999] ). Unlike prolyl hydroxylases, which specifically recognize peptidylproline in procollagen and related peptides, proline hydroxylases can convert free proline to hydroxyproline. Several microbial enzymes that produce cis-3-, cis-4-, or trans-4-hydroxyproline are known in the art (see, e.g., U.S. Pat. Nos. 5,962,292, 5,963,254, and 5,854,040; WO2009139365; and EP2290065), and enzymes that produce trans-3-hydroxyproline have been identified in fungal extracts. Many of the proline hydroxylases are found in bacteria and fungi, where they are involved in the biosynthesis of peptide antibiotics.

[0008] No naturally occurring proline hydroxylases selective for trans-3-hydroxyproline are known in the art. GloF, a fungal proline hydroxylase from Glarea lozoyensis, produces trans-3-hydroxyproline as the minor isomer along with the major isomer trans-4-hydroxyproline (Petersen et al., Appl. Microbiol. Biotechnol. 2003, 62, 263; Houwaart et al., ChemBioChem 2014, 15, 2365). Another fungal prolyl hydroxylase, HtyE, from Emericella rugulosa NRRL 11440, which shares approximately 64% sequence identity with GloF, was reported as part of the echinocandin B biosynthetic gene cluster (Cacho et al., J. Am. Chem. Soc. 2012, 134, 16781). HtyE was also found to produce trans-3-hydroxyproline as a minor isomer along with the major isomer trans-4-hydroxyproline. Recently, a gene cluster containing three hydroxylase genes was identified in the fungus sp. 11243 (Matsui et al., J. Biosci. Bioeng. 2017, Feb; 123(2): 147-153), and one gene was subsequently identified as having homology to HtyE. Although whole recombinant cells expressing cloned prolyl hydroxylase are more suitable for large-scale industrial processes, the use of whole cells limits the variability of reaction conditions, such as high substrate concentrations; restricts the types of substrates that can be used to those that are permeable to the cells; and results in undesirable by-products that must be separated from the final product. In addition, the use of rich growth media prepared from protein hydrolysates contains free proline, which can be a competitive inhibitor when substrates other than proline are targeted, so in vivo systems may require a defined growth medium that is neither optimal nor cost-effective. Alternative methods are needed for synthesizing hydroxylated forms of proline and proline analogs and other compounds that can be easily scaled up and can produce substantially pure isomeric products. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] U.S. Patent No. 5,364,775 [Patent Document 2] U.S. Patent No. 5,962,292 [Patent Document 3] U.S. Patent No. 5,963,254 [Patent Document 4] U.S. Patent No. 5,854,040 [Non-patent literature]

[0010] [Non-Patent Document 1] Cacho et al., J. Am. Chem. Soc. (2012) 134, 16781 [Non-patent document 2] Matsui et al., J. Biosci. Bioeng. (2017) 123(2):147~153 Summary of the Invention [Means for solving the problem]

[0011] Summary of the Invention The present invention provides engineered proline hydroxylase biocatalysts, polynucleotides encoding the biocatalysts, methods for their preparation, and processes for preparing hydroxylated compounds using such engineered biocatalysts. The proline hydroxylases of the present invention have been engineered to have one or more improved properties compared to the naturally occurring proline hydroxylase of ANO11243 from fungal species No. 11243 (SEQ ID NO: 2 with an N-terminal His tag). The improved biocatalytic properties of the engineered proline hydroxylases include, among others, activity, substrate tolerance, stereoselectivity, regioselectivity, and thermostability. The engineered proline hydroxylases have also been found to hydroxylate various substrate compounds, including the hydroxylation of L-proline to trans-3-hydroxyproline using alpha-ketoglutarate as a cosubstrate. In some embodiments, the process is carried out in the presence of oxygen (i.e., air) and iron (i.e., Fe(II)). An engineered enzyme having one or more improved properties has one or more residue differences compared to naturally occurring prolyl hydroxylases, the residue differences being at residue positions that affect one or more of the aforementioned enzyme properties.

[0012] Thus, in one aspect, the invention provides engineered polypeptides having proline hydroxylase activity, wherein the polypeptide comprises an amino acid sequence having at least about 80% identity to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630. In some embodiments, the invention provides engineered polypeptides having proline hydroxylase activity, wherein the polypeptide comprises an amino acid sequence set forth in an even-numbered sequence within the range of SEQ ID NOs: 6 to 658. The detailed description below provides guidance on the selection of residue differences that can be used in the preparation of engineered proline hydroxylases with desired improved biocatalytic properties.

[0013] The present invention provides engineered polypeptides with prolyl hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4. In some embodiments, the present invention provides engineered polypeptides with proline hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4 and one or more residue differences compared to SEQ ID NO:4 at residue positions selected from 21, 28, 58 / 247, 65, 80, 85, 95, 98, 117, 120, 159, 185, 194, 199, 200, 233, 237, 243, 250, 268, 281, 282, 287, 289, 307, 324, 326, 327, 330, 338, 343, 346 and 348. In some embodiments, the present invention provides engineered polypeptides with proline hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4 and one or more residue differences compared to SEQ ID NO:4 at residue positions selected from 21, 28, 45, 65, 95, 112, 117, 139, 177, 185, 199, 233, 243, 250, 281, 282, 287, 289, 307, 324, 326, 327, 335, 338, 343 and 346.In some embodiments, the present invention provides engineered polypeptides with proline hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4 and one or more residue differences compared to SEQ ID NO:4 at residue positions selected from 48 / 66 / 189 / 194, 48 / 66 / 194 and 66 / 82 / 85 / 135 / 189 / 194 / 267. In some embodiments, the present invention provides engineered polypeptides with proline hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4 and one or more residue differences compared to SEQ ID NO:4 at residue positions selected from 20 / 56 / 76 / 168 / 169 / 296, 20 / 56 / 232 / 294, 20 / 119 / 294 / 296, 56 / 76 / 119 / 124 / 147 / 232, 56 / 76 / 294, 76 / 168 / 232 / 294, 76 / 294 / 296, 76 / 296, 147 and 232. In some embodiments, the engineered polypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one of the even-numbered sequences in SEQ ID NOs: 4-658.

[0014] The present invention provides engineered polypeptides with proline hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 116. In some embodiments, the present invention provides engineered polypeptides with proline hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 116 and one or more residue differences compared to SEQ ID NO: 116 at residue positions selected from 123, 189, 195, 233 and 296. In some embodiments, the present invention provides amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 116, as well as 20 / 21 / 56, 20 / 21 / 56 / 76 / 95 / 232 / 294 / 307 / 335, 20 / 21 / 56 / 76 / 147 / 225 / 232 / 233 / 281 / 294 / 296 / 307 / 335, 20 / 21 / 56 / 95 / 147 / 281 / 294 / 307, 20 / 21 / 56 / 281 / 307, 20 / 21 / 76 / 232 / 243, 20 / 21 / 95 / 232 / 307, 20 / 21 / 95 / 281 / 294 / 296 , 20 / 21 / 147 / 189 / 233 / 243 / 281 / 307, 20 / 56, 20 / 56 / 76 / 95 / 281 / 307, 20 / 56 / 76 / 147 / 294 / 296 / 307, 20 / 56 / 95 / 147 / 294, 20 / 56 / 281, 20 / 76, 20 / 76 / 95 / 281 / 294 / 296, 20 / 76 / 95 / 281 / 296 / 30 7, 20 / 76 / 233 / 294 / 307, 20 / 76 / 243 / 281 / 294, 21 / 76 / 147 / 233 / 294 / 307, 21 / 76 / 147 / 243 / 296 / 307 / 335, 21 / 95 / 185 / 189 / 232 / 281 / 296, 21 / 95 / 233 / 243 / 281 / 296, 21 / 95 / 294 / 296 / 307 / 335,21 / 95 / 307, 21 / 281 / 307, 29 / 76 / 281, 56 / 76 / 95 / 232 / 243 / 281, 56 / 76 / 147 / 281 / 307, 56 / 76 / 243 / 294, 56 / 76 / 281 / 294, 56 / 76 / 296, 56 / 76 / 307, 56 / 95 / 147 / 307 / 335 / 348, 56 / 95 / 232 / 233 / 281 / 294 / 307, 56 / 95 / 243 / 281, 56 / 147 / 281, 56 / 232 / 243 / 281, 56 / 232 / 281, 56 / 232 / 281 / 294 / 296, 56 / 233 / 281 / 294 / 296, 56 / 281 / 307, 76 / 95 / 232 / 243 / 281 / 307, 76 / 95 / 243 / 281 / 307 / 335, 76 / 95 / 294 / 307, 76 / 147, 76 / 147 / 233 / 243 / 294, 76 / 147 / 233 / 281 / 294 / 307, 76 / 147 / 243 / 294 / 296 / 307 / 335, 76 / 147 / 281 / 307, 76 / 189 / 296, 76 / 232 / 2 33 / 243 / 294 / 296 / 307, 76 / 281, 76 / 281 / 294, 76 / 294 / 296, 95 / 120, 95 / 147 / 335, 95 / 232 / 243 / 281 / 294 / 307, 95 / 232 / 281 / 294 / 296, 95 / 281 / 294 / 296, 95 / 335, 147, 147 / 225 / 232 / 243 / 281 / 296 / 307 / 335, 147 / 233 / 243 / 281 / 307, 147 / 233 / 281 / 307 / 335, 147 / 243 / 281 , 147 / 307, 232 / 233 / 281 / 294 / 296 / 307, 232 / 281, 232 / 284 / 307, 233 / 243 / 281 / 296 / 307 / 335, 233 / 281 / 296 / 307, 243 / 281 / 294 / 296, 281, 281 / 294, 281 / 307, 307 and 335. In some embodiments, the present invention provides an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 116,and one or more residue differences compared to SEQ ID NO: 116 at residue positions selected from 21 / 76 / 147 / 243 / 296 / 307 / 335, 56 / 76 / 147 / 281 / 307, and 95 / 147 / 335. In some embodiments, the engineered polypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one of the even-numbered sequences in SEQ ID NOs: 4-658.

[0015] The present invention provides engineered polypeptides with proline hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:162. In some embodiments, the present invention provides amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 162, as well as amino acid sequences having at least 2 / 85 / 123 / 237, 28 / 115 / 117 / 120 / 123 / 268 / 270 / 343 / 346 / 348, 45 / 123 / 326, 65 / 117 / 120 / 123 / 34 The present invention provides engineered polypeptides having proline hydroxylase activity, comprising one or more residue differences compared to SEQ ID NO: 162 at residue positions selected from: 3 / 346, 85 / 123 / 281 / 282, 114 / 115 / 117 / 120 / 123 / 268 / 271 / 313 / 326 / 343 / 346, 123 / 139 / 233 / 237 / 281 / 282 / 289 / 324 / 326 and 123 / 199 / 200 / 247 / 250 / 338. In some embodiments, the engineered polypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one of the even-numbered sequences in SEQ ID NOs: 6-658.

[0016] The present invention provides engineered polypeptides with prolyl hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:322. In some embodiments, the invention provides engineered polypeptides with proline hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 322 and one or more residue differences compared to SEQ ID NO: 322 at residue positions selected from 26, 54, 61, 129, 132, 149, 156, 175, 189, 201, 209, 228, 236, 248, 262, 272, 277, 291 and 345. In some embodiments, the invention provides engineered polypeptides with proline hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 322 and one or more residue differences compared to SEQ ID NO: 322 at residue positions selected from 25, 43, 54, 58, 61, 79, 129, 132, 143, 156, 163, 175, 179, 201, 209, 236, 248, 278, 291, 345 and 347.In some embodiments, the present invention provides amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 322, as well as ... Provided are engineered polypeptides having prolyl hydroxylase activity, comprising one or more residue differences compared to SEQ ID NO: 322 at residue positions selected from 135 / 208 / 281 / 282 / 289, 85 / 117 / 120 / 270 / 281 / 289, 85 / 117 / 135 / 139 / 208, and 117 / 120 / 208 / 270 / 324 / 343 / 346. In some embodiments, the engineered polypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one of the even-numbered sequences in SEQ ID NOs: 6-658.

[0017] The present invention provides engineered polypeptides with prolyl hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:412. In some embodiments, the invention provides engineered polypeptides with proline hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:412 and one or more residue differences compared to SEQ ID NO:412 at residue positions selected from 47, 48, 56 / 118, 85, 95, 95 / 289, 113, 118, 118 / 247, 154, 162, 162 / 204, 164, 164 / 198 / 271, 168, 169, 187, 195, 243, 271, 275, 281, 314, 330 and 342. In some embodiments, the invention provides engineered polypeptides with proline hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:412 and one or more residue differences compared to SEQ ID NO:412 at residue positions selected from 25 / 129 / 163 / 236 / 262 / 345 / 347, 120 / 156 / 175 / 179 / 201, 129 / 189 / 236 / 262 / 277 / 278, 129 / 236 / 262, 156 / 175 / 179 / 228 and 162. In some embodiments, the engineered polypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one of the even-numbered sequences in SEQ ID NOs: 6-658.

[0018] The present invention provides engineered polypeptides with prolyl hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:492. In some embodiments, the invention provides engineered polypeptides with proline hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:492 and one or more residue differences compared to SEQ ID NO:492 at residue positions selected from 15, 17, 28, 29, 65, 135, 167, 177, 199, 208, 228, 235, 287, 294, 307 and 343. In some embodiments, the invention provides engineered polypeptides with prolyl hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:492 and one or more residue differences compared to SEQ ID NO:492 at residue positions selected from 85 / 187 / 281 / 347, 85 / 187 / 347, 118 / 120 / 162 / 175 / 179 / 330, 118 / 120 / 162 / 175 / 330, 162 / 175 / 179 / 330, 175 / 228 / 330, 195 / 347 and 278 / 314 / 347. In some embodiments, the engineered polypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one of the even-numbered sequences in SEQ ID NOs: 6-658.

[0019] The present invention provides engineered polypeptides with prolyl hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:562. In some embodiments, the invention provides engineered polypeptides with proline hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:562 and one or more residue differences compared to SEQ ID NO:562 at residue positions selected from 15, 40, 43, 44, 59, 79, 82, 149, 164, 179, 345 and 347. In some embodiments, the present invention provides amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 562, as well as 29 / 85 / 177 / 208 / 228 / 347, 29 / 85 / 208 / 228 / 343 / 347, 29 / 177 / 195 / 228 / 343 , 29 / 208 / 228 / 278 / 294 / 347, 56 / 195 / 278, 85 / 187 / 205 / 208 / 278, 113 / 177 / 187 / 195 / 208 / 278 / 294 / 343 / 347, and 177 / 205 / 208 / 228. In some embodiments, the engineered polypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one of the even-numbered sequences in SEQ ID NOs: 6-658.

[0020] The present invention provides engineered polypeptides with proline hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 598. In some embodiments, the present invention provides engineered polypeptides with proline hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 598 and one or more residue differences compared to SEQ ID NO: 598 at residue positions selected from 47, 162, 209, 219, 227 and 342. In some embodiments, the present invention provides amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 598, as well as 17 / 44 / 179 / 195 / 250 / 313 / 345, 17 / 44 / 199 / 313, 43 / 44 / 195 / 199, 44 / 149 / 164 / 171 / 187, 44 / 179 / 195 / 19 The present invention provides engineered polypeptides having proline hydroxylase activity, comprising one or more residue differences compared to SEQ ID NO:598 at residue positions selected from: 9, 44 / 179 / 195 / 199 / 345, 79 / 163 / 164 / 171 / 187 / 201 / 286 / 288, 82 / 163 / 164, 82 / 163 / 164 / 171 / 187 / 201 / 203 / 208 / 286 / 288 / 320, 149 / 164 / 171 / 288 and 187 / 286. In some embodiments, the engineered polypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one of the even-numbered sequences in SEQ ID NOs: 6-658.

[0021] The present invention provides engineered polypeptides with prolyl hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:630. In some embodiments, the invention provides engineered polypeptides with prolyl hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 630 and one or more residue differences compared to SEQ ID NO: 630 at residue positions selected from 82 / 164 / 171 / 203 / 208, 135 / 163 / 164 / 201 / 203 / 208, 162, 162 / 219 / 236, 162 / 219 / 313 / 338, 162 / 236 / 342, 162 / 313 / 342 and 164 / 171 / 201 / 203 / 282. In some embodiments, the engineered polypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one of the even-numbered sequences in SEQ ID NOs: 6-658.

[0022] The present invention also provides engineered polypeptides having proline hydroxylase activity capable of converting L-proline to trans-3-hydroxyproline. In some embodiments, the engineered polypeptides are capable of converting L-proline to trans-3-hydroxyproline with an activity that is at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more than that of the naturally occurring enzyme. In some further embodiments, the engineered polypeptides are capable of converting L-proline to trans-3-hydroxyproline with an isomeric excess of trans-3-hydroxyproline of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0023] The present invention also provides polynucleotides encoding engineered polypeptides having prolyl hydroxylase activity. In some embodiments, the polynucleotides comprise nucleic acid sequences optimized for expression in E. coli.

[0024] The present invention further provides an expression vector comprising a polynucleotide encoding an engineered polypeptide having prolyl hydroxylase activity. In some embodiments, the expression vector comprises at least one regulatory sequence.

[0025] The present invention also provides host cells comprising a polynucleotide encoding an engineered polypeptide having prolyl hydroxylase activity. In some embodiments, the host cell is E. coli.

[0026] The present invention further provides methods for preparing an engineered polypeptide having prolyl hydroxylase activity, the method comprising culturing a host cell comprising an expression vector comprising at least one polynucleotide encoding an engineered polypeptide having prolyl hydroxylase activity under conditions suitable for expression of the polypeptide(s). In some embodiments, the method further comprises isolating the engineered polypeptide(s). In certain embodiments, for example, the following items are provided: (Item 1) and / or 630. Engineered polynucleotides having proline hydroxylase activity comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence of SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630, and one or more residue differences compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630. peptide. (Item 2) 2. The engineered polypeptide of item 1, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence, SEQ ID NO:4, and one or more residue differences compared to SEQ ID NO:4 at residue positions selected from 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. (Item 3) 2. The engineered polypeptide of item 1, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence, SEQ ID NO:4, and one or more residue differences compared to SEQ ID NO:4 at residue positions selected from 21, 28, 45, 65, 95, 112, 117, 139, 177, 185, 199, 233, 243, 250, 281, 282, 287, 289, 307, 324, 326, 327, 335, 338, 343, and 346. (Item 4) 2. The engineered polypeptide of item 1, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence, SEQ ID NO:4, and one or more residue differences compared to SEQ ID NO:4 at residue positions selected from 48 / 66 / 189 / 194, 48 / 66 / 194 and 66 / 82 / 85 / 135 / 189 / 194 / 267. (Item 5) 2. The engineered polypeptide of item 1, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence, SEQ ID NO:4, and one or more residue differences compared to SEQ ID NO:4 at residue positions selected from 20 / 56 / 76 / 168 / 169 / 296, 20 / 56 / 232 / 294, 20 / 119 / 294 / 296, 56 / 76 / 119 / 124 / 147 / 232, 56 / 76 / 294, 76 / 168 / 232 / 294, 76 / 294 / 296, 76 / 296, 147 and 232. (Item 6) 2. The engineered polypeptide of item 1, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence, SEQ ID NO: 116, and one or more residue differences compared to SEQ ID NO: 116 at residue positions selected from 123, 189, 195, 233 and 296. (Item 7) Amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence, SEQ ID NO: 116, as well as 20 / 21 / 56, 20 / 21 / 56 / 76 / 95 / 232 / 294 / 307 / 335, 20 / 21 / 56 / 76 / 147 / 225 / 232 / 2 33 / 281 / 294 / 296 / 307 / 335, 20 / 21 / 56 / 95 / 147 / 281 / 294 / 307, 20 / 21 / 56 / 281 / 307, 20 / 21 / 76 / 232 / 243, 20 / 21 / 95 / 232 / 307, 20 / 21 / 95 / 281 / 294 / 296, 20 / 21 / 147 / 189 / 233 / 243 / 281 / 307, 20 / 56, 20 / 56 / 76 / 95 / 281 / 307, 2 0 / 56 / 76 / 147 / 294 / 296 / 307、20 / 56 / 95 / 147 / 294、20 / 56 / 281、20 / 76、20 / 76 / 95 / 281 / 294 / 296、20 / 76 / 95 / 281 / 296 / 307、20 / 76 / 233 / 294 / 307、20 / 76 / 243 / 281 / 294、21 / 76 / 147 / 233 / 294 / 307、21 / 76 / 147 / 243 / 296 / 307 / 335、21 / 95 / 185 / 189 / 232 / 281 / 296、21 / 95 / 233 / 243 / 281 / 296、21 / 95 / 294 / 296 / 307 / 335、21 / 95 / 307、21 / 281 / 307、29 / 76 / 281、56 / 76 / 95 / 232 / 243 / 281、56 / 76 / 147 / 281 / 307、56 / 76 / 243 / 294、56 / 76 / 281 / 294、56 / 76 / 296、56 / 76 / 307、56 / 95 / 147 / 307 / 335 / 348、56 / 95 / 232 / 233 / 281 / 294 / 307、56 / 95 / 243 / 281、56 / 147 / 281、56 / 232 / 243 / 281、56 / 232 / 281、56 / 232 / 281 / 294 / 296、56 / 233 / 281 / 294 / 296、56 / 281 / 307、76 / 95 / 232 / 243 / 281 / 307、76 / 95 / 243 / 281 / 307 / 335、76 / 95 / 294 / 307、76 / 147、76 / 147 / 233 / 243 / 294、76 / 147 / 233 / 281 / 294 / 307、76 / 147 / 243 / 294 / 296 / 307 / 335、76 / 147 / 281 / 307、76 / 189 / 296、76 / 232 / 233 / 243 / 294 / 296 / 307、76 / 281、76 / 281 / 294、76 / 294 / 296、95 / 120、95 / 147 / 335、95 / 232 / 243 / 281 / 294 / 307、95 / 232 / 281 / 294 / 296、95 / 281 / 294 / 296、95 / 335、147、147 / 225 / 232 / 243 / 281 / 296 / 307 / 335、147 / 233 / 243 / 281 / 307、147 / 233 / 281 / 307 / 335、147 / 243 / 281、147 / 307、232 / 233 / 281 / 294 / 296 / 307、232 / 281、232 / 284 / 307、233 / 243 / 281 / 296 / 307 / 335、233 / 281 / 296 / 307、2. The engineered polypeptide of item 1, comprising one or more residue differences compared to SEQ ID NO: 116 at residue positions selected from 243 / 281 / 294 / 296, 281, 281 / 294, 281 / 307, 307, and 335. (Item 8) 2. The engineered polypeptide of item 1, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence, SEQ ID NO: 116, and one or more residue differences compared to SEQ ID NO: 116 at residue positions selected from 21 / 76 / 147 / 243 / 296 / 307 / 335, 56 / 76 / 147 / 281 / 307 and 95 / 147 / 335. (Item 9) Amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence, SEQ ID NO: 162, as well as 2 / 85 / 123 / 237, 28 / 115 / 117 / 120 / 123 / 268 / 270 / 343 / 346 / 348, 45 / 123 / 326, 65 / 117 / 120 / 123 2. The engineered polypeptide of item 1, comprising one or more residue differences compared to SEQ ID NO: 162 at residue positions selected from: 123 / 139 / 233 / 237 / 281 / 282 / 289 / 324 / 326, 123 / 199 / 200 / 247 / 250 / 338, 85 / 123 / 281 / 282, 114 / 115 / 117 / 120 / 123 / 268 / 271 / 313 / 326 / 343 / 346, 123 / 139 / 233 / 237 / 281 / 282 / 289 / 324 / 326, and 123 / 199 / 200 / 247 / 250 / 338. (Item 10) Amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence, SEQ ID NO: 322, as well as 26, 54 2. The engineered polypeptide of item 1, comprising one or more residue differences compared to SEQ ID NO: 322 at residue positions selected from: 61, 129, 132, 149, 156, 175, 189, 201, 209, 228, 236, 248, 262, 272, 277, 291 and 345. (Item 11) 2. The engineered polypeptide of item 1, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence, SEQ ID NO: 322, and one or more residue differences compared to SEQ ID NO: 322 at residue positions selected from 25, 43, 54, 58, 61, 79, 129, 132, 143, 156, 163, 175, 179, 201, 209, 236, 248, 278, 291, 345 and 347. (Item 12) Amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence, SEQ ID NO: 322, as well as 85 / 117 / 120 / 135 / 208 / 270 / 324 / 343 / 346, ... 2. The engineered polypeptide of item 1, comprising one or more residue differences compared to SEQ ID NO: 322 at residue positions selected from 20 / 135 / 208 / 281 / 282 / 289, 85 / 117 / 120 / 270 / 281 / 289, 85 / 117 / 135 / 139 / 208 and 117 / 120 / 208 / 270 / 324 / 343 / 346. (Item 13) 2. The engineered polypeptide of item 1, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence, SEQ ID NO: 412, and one or more residue differences compared to SEQ ID NO: 412 at residue positions selected from 47, 48, 56 / 118, 85, 95, 95 / 289, 113, 118, 118 / 247, 154, 162, 162 / 204, 164, 164 / 198 / 271, 168, 169, 187, 195, 243, 271, 275, 281, 314, 330 and 342. (Item 14) 2. The engineered polypeptide of item 1, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence, SEQ ID NO: 412, and one or more residue differences compared to SEQ ID NO: 412 at residue positions selected from 25 / 129 / 163 / 236 / 262 / 345 / 347, 120 / 156 / 175 / 179 / 201, 129 / 189 / 236 / 262 / 277 / 278, 129 / 236 / 262, 156 / 175 / 179 / 228 and 162. (Item 15) 2. The engineered polypeptide of item 1, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence, SEQ ID NO: 492, and one or more residue differences compared to SEQ ID NO: 492 at residue positions selected from 15, 17, 28, 29, 65, 135, 167, 177, 199, 208, 228, 235, 287, 294, 307, and 343. (Item 16) Amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence, SEQ ID NO: 492, as well as 85 / 18 2. The engineered polypeptide of item 1, comprising one or more residue differences compared to SEQ ID NO: 492 at residue positions selected from: 7 / 281 / 347, 85 / 187 / 347, 118 / 120 / 162 / 175 / 179 / 330, 118 / 120 / 162 / 175 / 330, 162 / 175 / 179 / 330, 175 / 228 / 330, 195 / 347 and 278 / 314 / 347. (Item 17) 2. The engineered polypeptide of item 1, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence, SEQ ID NO: 562, and one or more residue differences compared to SEQ ID NO: 562 at residue positions selected from 15, 40, 43, 44, 59, 79, 82, 149, 164, 179, 345, and 347. (Item 18) Amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence, SEQ ID NO: 562, as well as 29 / 85 / 177 / 208 / 228 / 347, 29 / 85 / 208 / 228 / 343 / 347, 29 / 177 / 195 / 228 / 2. The engineered polypeptide of item 1, comprising one or more residue differences compared to SEQ ID NO: 562 at residue positions selected from: 343, 29 / 208 / 228 / 278 / 294 / 347, 56 / 195 / 278, 85 / 187 / 205 / 208 / 278, 113 / 177 / 187 / 195 / 208 / 278 / 294 / 343 / 347 and 177 / 205 / 208 / 228. (Item 19) 2. The engineered polypeptide of item 1, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence, SEQ ID NO: 598, and one or more residue differences compared to SEQ ID NO: 598 at residue positions selected from 47, 162, 209, 219, 227 and 342. (Item 20) Amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to the reference sequence, SEQ ID NO: 598, as well as 17 / 44 / 179 / 195 / 250 / 313 / 345, 17 / 44 / 199 / 313, 43 / 44 / 195 / 199, 44 / 149 / 164 / 171 / 187, 44 / 179 / 195 201 / 286 / 288, 82 / 163 / 164, 82 / 163 / 164 / 171 / 187 / 201 / 203 / 208 / 286 / 288 / 320, 149 / 164 / 171 / 288 and 187 / 286. (Item 21) 2. The engineered polypeptide of item 1, comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence, SEQ ID NO: 630, and one or more residue differences compared to SEQ ID NO: 630 at residue positions selected from 82 / 164 / 171 / 203 / 208, 135 / 163 / 164 / 201 / 203 / 208, 162, 162 / 219 / 236, 162 / 219 / 313 / 338, 162 / 236 / 342, 162 / 313 / 342 and 164 / 171 / 201 / 203 / 282. (Item 22) 2. The engineered polypeptide of item 1, having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one of the even-numbered sequences in SEQ ID NOs: 6 to 658. (Item 23) 2. The engineered polypeptide according to item 1, which is capable of converting L-proline to trans-3-hydroxyproline. (Item 24) 24. The engineered polypeptide of claim 23, which is capable of converting L-proline to trans-3-hydroxyproline with an activity that is at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more greater than the activity of the naturally occurring enzyme. (Item 25) 24. The engineered polypeptide of claim 23, which is capable of converting L-proline to trans-3-hydroxyproline in a diastereomeric excess of trans-3-hydroxyproline of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. (Item 26) 26. A polynucleotide encoding the engineered polypeptide according to any one of items 1 to 25. (Item 27) 23. A polynucleotide encoding the engineered polypeptide of item 22. (Item 28) 25. A polynucleotide encoding the engineered polypeptide of item 24. (Item 29) 29. The polynucleotide according to any one of Items 26 to 28, comprising a nucleic acid sequence optimized for expression in E. coli. (Item 30) 30. An expression vector comprising the polynucleotide according to any one of items 26 to 29, and optionally further comprising at least one regulatory sequence. (Item 31) 31. The expression vector of item 30, comprising an engineered polypeptide of SEQ ID NO: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630. (Item 32) 30. A host cell comprising the polynucleotide according to any one of items 26 to 29. (Item 33) A host cell comprising the expression vector of item 30 or 31. (Item 34) 34. The host cell according to item 32 or 33, which is E. coli. (Item 35) 35. A method for preparing an engineered polypeptide, comprising culturing a host cell according to any of items 32 to 34 under conditions suitable for expression of said polypeptide. (Item 36) 36. The method of claim 35, further comprising isolating the engineered polypeptide. DETAILED DESCRIPTION OF THE INVENTION

[0027] Description of the Invention Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry, and nucleic acid chemistry described below are those well known and commonly employed in the art. Such techniques are well known and are described in numerous textbooks and reference works familiar to those skilled in the art. Standard techniques, or modifications thereof, are used for chemical synthesis and chemical analysis. All patents, patent applications, articles, and publications mentioned herein, both above and below, are hereby expressly incorporated by reference.

[0028] Although any suitable methods and materials similar or equivalent to those described herein will find use in the practice of the present invention, some methods and materials are described herein.It should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described, as these may vary depending on the context in which they are used by those skilled in the art.Therefore, the terms defined immediately below are more fully explained by referring to the present invention as a whole.

[0029] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention.

[0030] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0031] Numerical ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to include every narrower numerical range that falls within that broader numerical range, as if such narrower numerical ranges were all expressly written herein. Every maximum (or minimum) numerical limit disclosed herein is also intended to include every numerical lower (or upper) limit, as if such lower (or upper) numerical limits were expressly written herein.

[0032] Abbreviation The abbreviations used for the genetically encoded amino acids are conventional and are as follows: [Table 1]

[0033] When a three-letter abbreviation is used, an amino acid is referred to as having an α-carbon (C α) can be in either the L- or D-configuration about the α-carbon. For example, "Ala" designates alanine without specifying the configuration about the α-carbon, while "D-Ala" and "L-Ala" designate D-alanine and L-alanine, respectively. When single-letter abbreviations are used, an uppercase letter designates an amino acid in the L-configuration about the α-carbon, and a lowercase letter designates an amino acid in the D-configuration about the α-carbon. For example, "A" designates L-alanine and "a" designates D-alanine. When a polypeptide sequence is presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequence is presented in the amino (N) to carboxy (C) direction, according to common convention.

[0034] The abbreviations used for genetically encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless otherwise depicted, abbreviated nucleosides can be either ribonucleosides or 2'-deoxyribonucleosides. Nucleosides can be designated as either ribonucleosides or 2'-deoxyribonucleosides, either individually or in aggregate. When nucleic acid sequences are presented as a string of single-letter abbreviations, the sequences are presented in the 5' to 3' direction, according to common convention, and the phosphate is not indicated.

[0035] definition With respect to the present invention, technical and scientific terms used in the description herein shall have the meanings commonly understood by those of ordinary skill in the art, unless otherwise specified. Accordingly, the following terms are intended to have the following meanings:

[0036] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes more than one polypeptide.

[0037] Similarly, the terms "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting. Thus, as used herein, the term "comprising" and its cognates are used in their inclusive sense (i.e., equivalent to the term "including" and its corresponding cognates).

[0038] It is further understood that where the descriptions of various embodiments use the term "comprising," those skilled in the art will understand that in some specific instances, the embodiments may instead be described using the words "consisting essentially of" or "consisting of."

[0039] The term "about" refers to an acceptable error for a particular value. In some instances, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value range. In some instances, "about" means within 1, 2, 3, or 4 standard deviations of a given value.

[0040] The "EC" numbers refer to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) enzyme nomenclature. The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.

[0041] "ATCC" is the American Type Culture Collection. The National Institute of Genetics and Biotechnology (NIH) refers to the National Institute of Genetics and Biotechnology (NIB), a biorepository collection of genetic material, including genes and strains.

[0042] "NCBI" refers to the National Center for Biological Information and the sequence databases it hosts.

[0043] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to polymers of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation). Included within this definition are D- and L-amino acids and mixtures of D- and L-amino acids, and polymers comprising D- and L-amino acids and mixtures of D- and L-amino acids.

[0044] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes.

[0045] As used herein, "polynucleotide" and "nucleic acid" refer to two or more nucleosides covalently linked together. A polynucleotide may be composed entirely of ribonucleotides (i.e., RNA), entirely of 2' deoxyribonucleotides (i.e., DNA), or a mixture composed of ribo- and 2' deoxyribonucleotides. The nucleosides will typically be linked together via standard phosphodiester linkages, although a polynucleotide may contain one or more non-standard linkages. A polynucleotide may be single-stranded or double-stranded, or may contain both single- and double-stranded regions. Furthermore, a polynucleotide will typically be composed of naturally occurring coding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), but may contain one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleobases are nucleobases that encode an amino acid sequence.

[0046] "Coding sequence" refers to a portion of a nucleic acid (eg, a gene) that codes for the amino acid sequence of a protein.

[0047] "Proline hydroxylase" refers to a polypeptide having the enzymatic ability to convert free proline to hydroxyproline in the presence of the co-substrates α-ketoglutarate and dioxygen, as described below: [ka]

[0048] It should be understood that proline hydroxylases are not limited to the aforementioned reaction with proline, but can also hydroxylate other substrates or produce various isomers of hydroxyproline, such as trans-3-hydroxyproline. As used herein, proline hydroxylase includes naturally occurring (wild-type) proline hydroxylases as well as non-naturally occurring engineered polypeptides produced by human engineering. In some embodiments, the proline hydroxylase variants of the present invention can convert L-proline to trans-3-hydroxyproline, as shown in Scheme 1 below: [ka]

[0049] A "co-substrate" for proline hydroxylase refers to α-ketoglutarate and co-substrate analogs that can replace α-ketoglutarate in the hydroxylation of proline and proline substrate analogs. Co-substrate analogs include, by way of example and not limitation, 2-oxoadipate (e.g., Majamaa et al., Biochem. J., 229:127-133 (1985).

[0050] As used herein, "wild-type" and "naturally occurring" refer to forms found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and has not been intentionally modified by human manipulation.

[0051] "Recombinant" or "engineered" or "non-naturally occurring," when used in reference to a cell, nucleic acid, or polypeptide, refers to a material that has been modified in a way that would not otherwise occur in nature, or a material that corresponds to the natural or native form of the material. In some embodiments, the cell, nucleic acid, or polypeptide is identical to a naturally occurring cell, nucleic acid, or polypeptide, but is produced or derived from synthetic materials and / or by manipulation using recombinant techniques. Non-limiting examples include, among others, recombinant cells that express genes not found within the native (non-recombinant) form of the cell, or that express native genes that are otherwise expressed at different levels.

[0052] The term "percent (%) sequence identity" is used herein to refer to the comparison between polynucleotides or polypeptides, and is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence due to the optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue exists in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue exists in both sequences, or the number of positions where the nucleic acid base or amino acid residue is aligned with gaps, to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will recognize that there are many established algorithms available for aligning two sequences. Optimal alignment of sequences for comparison can be performed by any suitable method, including, but not limited to, the Smith and Waterman local homology algorithm (Smith and Waterman, Adv. Appl. Math., 2:482

[1981] ), the Needleman and Wunsch homology alignment algorithm (Needleman and Wunsch, J. Mol. Biol., 48:443

[1970] ), the Pearson and Lipman similarity search method (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444

[1988] ), computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or visual inspection, as known in the art.Examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include, but are not limited to, the BLAST and BLAST 2.0 algorithms described by Altschul et al. (See, Altschul et al., J. Mol. Biol., 215: 403-410

[1990] ; and Altschul et al., Nucl. Acids Res., 3389-3402

[1977] , respectively.) Software for performing BLAST analyses is available from the National Center for Biotechnology Information (NCBI). The algorithm is publicly available via the Center for Biotechnology Information website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that, when aligned with words of the same length in a database sequence, match or meet a positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using the parameters M (giving a score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0) for nucleotide sequences. For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of word hits in each direction is stopped when: the cumulative alignment score falls by an amount X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

[1989] . Exemplary sequence alignments and determination of percent sequence identity can be performed using the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI) using the default parameters provided.

[0053] A "reference sequence" refers to a defined sequence used as a basis for sequence and / or activity comparison. A reference sequence can be a subset of a larger sequence, such as a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length, or the full length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides can each contain (1) sequences that are similar between the two sequences (i.e., portions of the complete sequence) and (2) additional sequences that diverge between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, which can have one or more changes in the primary sequence.

[0054] As used herein, a "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues, where a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and the portion of the sequence in the comparison window can include 20 percent or less additions or deletions (i.e., gaps) compared to the reference sequence (no additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, including windows of 30, 40, 50, 100, or longer, as appropriate.

[0055] "Corresponding to," "with reference to," or "compared to," when used in the context of numbering a given amino acid or polynucleotide sequence, refers to the numbering of residues in a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, residue numbers or residue positions in a given polymer are specified with respect to the reference sequence, rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as that of an engineered prolyl hydroxylase, can be aligned with a reference sequence by introducing gaps to optimize residue matches between the two sequences. In such cases, despite the presence of gaps, the numbering of residues in a given amino acid or polynucleotide sequence can be done with respect to the reference sequence to which it is aligned.

[0056] "Substantial identity" refers to a polynucleotide or polypeptide sequence having at least 80 percent sequence identity, at least 85 percent identity, at least 89-95 percent sequence identity, or more usually at least 99 percent sequence identity over a comparison window of at least 20 residue positions, frequently over a window of at least 30-50 residues, relative to a reference sequence, where the percentage of sequence identity is calculated by comparing the reference sequence to a sequence containing deletions or additions totaling 20 percent or less of the reference sequence over the comparison window. In some specific embodiments, as applied to polypeptides, the term "substantial identity" means that two polypeptide sequences share at least 80 percent sequence identity, preferably at least 89 percent sequence identity or at least 95 percent sequence identity or more (e.g., 99 percent sequence identity), when optimally aligned, such as by programs GAP or BESTFIT using default gap weights. In some embodiments, non-identical residue positions in the compared sequences differ by conservative amino acid substitutions.

[0057] As used herein, "amino acid difference" and "residue difference" refer to the difference in an amino acid residue at a position in a polypeptide sequence compared to the amino acid residue at the corresponding position in a reference sequence. The position of an amino acid difference is generally referred to herein as "Xn," where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "a residue difference at position X93 compared to SEQ ID NO:4" refers to the difference in the amino acid residue at the polypeptide position corresponding to position 93 of SEQ ID NO:4. Thus, if a reference polypeptide of SEQ ID NO:4 has a serine at position 93, then "a residue difference at position X93 compared to SEQ ID NO:4" refers to an amino acid substitution of any residue other than serine at the polypeptide position corresponding to position 93 of SEQ ID NO:4. In most examples herein, a specific amino acid residue difference at a position is designated as "XnY," where "Xn" designates the corresponding position as described above, and "Y" is the single-letter identifier of the amino acid found in the engineered polypeptide (i.e., the residue that differs from the residue in the reference polypeptide). In some instances (e.g., in Tables 4.1, 4.2, 4.3, 4.4, 5.1, 5.2, 5.3, 6.1, 7.1, 7.2, 7.3, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, 11.2, and / or 12.1), the invention also provides specific amino acid differences, designated by the conventional notation "AnB," where A is the single-letter identifier of the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and B is the single-letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some instances, the polypeptides of the invention contain one or more amino acid residue differences compared to the reference sequence, as indicated by a list of designated positions at which the residue difference compared to the reference sequence occurs. In some embodiments, when more than one amino acid can be used at a specific residue position in the polypeptide, the various amino acid residues that can be used are separated by a " / " (e.g., X307H / X307P or X307H / P). A slash can also be used to indicate multiple substitutions within a given variant (ie, more than one substitution is present in a given sequence, such as in a combinatorial variant).In some embodiments, the invention includes engineered polypeptide sequences that contain one or more amino acid differences, including conservative or non-conservative amino acid substitutions. In some additional embodiments, the invention provides engineered polypeptide sequences that contain both conservative and non-conservative amino acid substitutions.

[0058] As used herein, " conservative amino acid substitution " refers to the substitution of a residue with a different residue that has a similar side chain, and thus typically includes the substitution of an amino acid in a polypeptide with an amino acid within the same or similar defined class of amino acids.By way of example and not limitation, in some embodiments, an amino acid with an aliphatic side chain is substituted with another aliphatic amino acid (for example, alanine, valine, leucine and isoleucine); an amino acid with a hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (for example, serine and threonine); an amino acid with an aromatic side chain is substituted with another amino acid with an aromatic side chain (for example, phenylalanine, tyrosine, tryptophan and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basic side chain (for example, lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (for example, aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid is substituted with another hydrophobic or hydrophilic amino acid, respectively.

[0059] As used herein, a "non-conservative substitution" refers to the replacement of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions can use amino acids between defined groups rather than within them, and affect (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0060] As used herein, "deletion" refers to a modification to a polypeptide by the removal of one or more amino acids from a reference polypeptide. Deletions can include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids comprising the reference enzyme, or up to 20% of the total number of amino acids, while retaining the enzymatic activity and / or improved properties of the engineered prolyl hydroxylase enzyme. Deletions can be directed to internal and / or terminal portions of the polypeptide. In various embodiments, deletions can include contiguous segments or can be discontinuous.

[0061] As used herein, "insertion" refers to a modification to a polypeptide by the addition of one or more amino acids from a reference polypeptide. Insertions can be made in the internal portion of the polypeptide or at the carboxy or amino terminus. As used herein, insertions include fusion proteins, as known in the art. Insertions can be contiguous segments of amino acids or can be separated by one or more amino acids in a naturally occurring polypeptide.

[0062] A "functional fragment" or "biologically active fragment," as used interchangeably herein, refers to a polypeptide that has amino- and / or carboxy-terminal deletion(s) and / or internal deletions, but where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full-length engineered prolyl hydroxylase of the invention), and which retains substantially all of the activity of the full-length polypeptide.

[0063] As used herein, an "isolated polypeptide" refers to a polypeptide that has been substantially separated from other contaminants that naturally accompany it (e.g., proteins, lipids, and polynucleotides). The term encompasses a polypeptide that has been removed or purified from its naturally occurring environment or expression system (e.g., within a host cell or by in vitro synthesis). Recombinant proline hydroxylase polypeptides can be present intracellularly, in cell culture medium, or prepared in various forms, such as a lysate or isolated preparation. Thus, in some embodiments, a recombinant proline hydroxylase polypeptide can be an isolated polypeptide.

[0064] As used herein, "substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e., more abundant than any other individual macromolecular species in the composition, on a molar or weight basis); generally, a composition is substantially purified when the target species constitutes at least about 50 percent of the macromolecular species present, on a molar or weight basis. However, in some embodiments, a composition comprising proline hydroxylase contains a proline hydroxylase that is less than 50% pure (e.g., about 10%, about 20%, about 30%, about 40%, or about 50%). Generally, a substantially pure proline hydroxylase composition will constitute about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of the total macromolecular species present in the composition, on a molar or weight basis. In some embodiments, the target species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods), and the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant prolyl hydroxylase polypeptide is a substantially pure polypeptide composition.

[0065] As used herein, "improved enzymatic properties" refers to at least one improved property of an enzyme. In some embodiments, the present invention provides engineered proline hydroxylase polypeptides that exhibit an improvement in any enzymatic property compared to a reference proline hydroxylase polypeptide and / or a wild-type proline hydroxylase polypeptide and / or another engineered proline hydroxylase polypeptide. Thus, the level of "improvement" can be determined and compared among various proline hydroxylase enzymes, including wild-type as well as engineered proline hydroxylases. Improved properties include, but are not limited to, properties such as increased protein expression, increased thermoactivity, increased thermostability, increased pH activity, increased stability, increased enzymatic activity, increased substrate specificity or affinity, increased specific activity, increased resistance to substrate or end-product inhibition, increased chemical stability, improved chemoselectivity, improved solvent stability, increased tolerance to acidic pH, increased tolerance to basic pH, increased resistance to proteolytic activity (i.e., decreased susceptibility to proteolysis), reduced aggregation, increased solubility, and an altered temperature profile.

[0066] As used herein, "increased enzymatic activity" and "enhanced catalytic activity" refer to improved properties of an engineered proline hydroxylase polypeptide, which can be expressed as an increase in specific activity (e.g., product produced / time / weight protein) or an increase in percent conversion of substrate to product (e.g., percent conversion of product from a starting amount of substrate in a specified period of time using a specified amount of proline hydroxylase) compared to a reference proline hydroxylase enzyme. Exemplary methods for determining enzymatic activity are provided in the Examples. m , V max or k catAny property related to enzyme activity can be affected, including classical enzyme properties, and such changes can result in increased enzyme activity. Improvements in enzyme activity can be as much as about 1.1-fold the enzyme activity of the corresponding wild-type enzyme, to 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or more higher enzyme activity than the naturally occurring proline hydroxylase or another engineered proline hydroxylase from which the proline hydroxylase polypeptide is derived.

[0067] As used herein, "conversion" refers to the enzymatic conversion (or biotransformation) of a substrate(s) to the corresponding product(s). "Percent conversion" refers to the percent of a substrate that is converted to a product within a period of time under specified conditions. Thus, the "enzyme activity" or "activity" of a proline hydroxylase polypeptide can be expressed as the "percent conversion" of substrate to product over a particular period of time.

[0068] An enzyme with "generalist properties" (or "generalist enzyme") refers to an enzyme that exhibits improved activity against a broad range of substrates compared to the parent sequence. A generalist enzyme does not necessarily demonstrate improved activity against all possible substrates. In some embodiments, the present invention provides prolyl hydroxylase variants with generalist properties in that they demonstrate similar or improved activity against a broad range of sterically and electronically diverse substrates compared to the parent gene. In addition, the generalist enzymes provided herein have been engineered to be improved across a broad range of diverse API-like molecules, resulting in increased production of metabolites / products.

[0069] The term "stringent hybridization conditions" is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those skilled in the art, hybrid stability is determined by the melting temperature (T m) Generally, hybrid stability is a function of ionic strength, temperature, G / C content, and the presence of chaotropic agents. m The value can be calculated using known methods for predicting melting temperatures (e.g., Baldino et al., Meth. Enzymol., 168:761-777

[1989] ; Bolton et al., Proc. Natl. Acad. Sci. USA 48:1390

[1962] ; Bresslauer et al., Proc. Natl. Acad. Sci. USA 83:8893-8897

[1986] ;Freier et al., Proc. Natl. Acad. Sci. USA 83:9373-9377

[1986] ;Kierzek et al., Biochem., 25:7840-7846

[1986] ;Rychlik et al., Nucl. 18:6409-6412

[1990] (misprint, Nucl. Acids Res., 19:698

[1991] );Sambrook et al., supra);Suggs et al., 1981, in Developmental Biology Using Purified Genes, Brown (See, e.g., J. Med. et al. [eds.], pp. 683-693, Academic Press, Cambridge, MA

[1981] ; and Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227-259

[1991] .) In some embodiments, the polynucleotide encodes a polypeptide disclosed herein and hybridizes under defined conditions, such as moderately stringent or highly stringent conditions, to the complement of a sequence encoding an engineered prolyl hydroxylase enzyme of the invention.

[0070] "Hybridization stringency" refers to hybridization conditions, such as washing conditions, in nucleic acid hybridization. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washing at varying but higher stringency. The term "moderately stringent hybridization" refers to conditions that allow a target DNA to bind to a complementary nucleic acid having about 60% identity to the target DNA, preferably about 75% identity, about 85% identity, or more than about 90% identity to the target polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS at 42°C. "High stringency hybridization" generally ... a hybridization that is performed under solution conditions for a defined polynucleotide sequence, such as a thermal melting temperature T m High stringency conditions refer to conditions at or below about 10°C. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only nucleic acid sequences that form stable hybrids at 0.018M NaCl at 65°C (i.e., if a hybrid is not stable at 0.018M NaCl at 65°C, it will not be stable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridization at 42°C in conditions equivalent to 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS, followed by washing in 0.1x SSPE and 0.1% SDS at 65°C. Another high stringency condition is hybridization in 5x SSC containing 0.1% (w:v) SDS at 65°C, followed by washing in 0.1x SSC containing 0.1% SDS at 65°C. Other highly stringent hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.

[0071] "Codon-optimized" refers to changing the codons of a polynucleotide encoding a protein to those preferentially used in a particular organism, so that the encoded protein is efficiently expressed in the target organism. Although the genetic code is degenerate in that most amino acids are represented by several codons called "synonyms" or "synonymous" codons, it is well known that codon usage by a particular organism is non-random and biased toward certain codon triplets. This codon usage bias can be higher for a given gene, for genes of common function or ancestral origin, for highly expressed proteins versus low copy number proteins, and for the total protein-coding region of an organism's genome. In some embodiments, a polynucleotide encoding a prolyl hydroxylase enzyme may be codon-optimized for optimal production in the host organism selected for expression.

[0072] "Preferred, optimal, high codon usage bias codons" refer interchangeably to codons that are used more frequently in protein-coding regions than other codons that encode the same amino acid. Preferred codons can be determined with respect to a single gene, a set of genes of common function or origin, codon usage in highly expressed genes, codon frequency in the total protein-coding regions of an entire organism, codon frequency in the total protein-coding regions of related organisms, or a combination thereof. Codons whose frequency increases with the level of gene expression are typically optimal codons for expression. Various methods are known for determining codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in a particular organism, including, for example, cluster analysis or correspondence analysis, and multivariate analysis using the effective number of codons used in a gene (see, e.g., GCG CodonPreference, Genetics Computer Group Wisconsin Package; CodonW, Peden, University of Nottingham; McInerney, Bioinform., 14:372-73).

[1998] ;Stenico et al., Nucl. Acids Res., 222437-46

[1994] ;Wright, Gene 87:23-29

[1990] Codon usage tables are available for many different organisms (e.g., Wada et al., Nucl. Acids Res., 20:2111-2118

[1992] ; Nakamura et al., Nucl. Acids Res., 28:292

[2000] ; Duret, et al., (See, supra; Henaut and Danchin, in Escherichia coli and Salmonella, Neidhardt, et al. (eds.), ASM Press, Washington DC, pp. 2047-2066

[1996] ). Data sources for obtaining codon usage can rely on any available nucleotide sequence capable of encoding a protein. These data sets include nucleic acid sequences that are actually known to encode expressed proteins (e.g., complete protein coding sequences - CDS), expressed sequence tags (ESTS), or predicted coding regions of genomic sequences (see, e.g., Mount, Bioinformatics: Sequence and Genome Analysis, Chapter 8, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

[2001] ; Uberbacher, Meth. Enzymol., 266:259-281

[1996] ; and Tiwari et al., Comput. Appl. Biosci., 13:263-270

[1997] ).

[0073] The term "control sequences," as used herein, includes all components necessary or advantageous for expression of a polynucleotide and / or polypeptide of the present invention. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter sequence, signal peptide sequence, initiation sequence, and transcription terminator. At a minimum, control sequences include a promoter, and transcription and translation stop signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites to facilitate ligation of the control sequences with the coding region of the nucleic acid sequence encoding the polypeptide.

[0074] "Operably linked" is defined herein as a configuration in which a control sequence is suitably positioned (i.e., in a functional relationship) relative to a polynucleotide of interest such that the control sequence directs or regulates expression of the polynucleotide and / or polypeptide of interest.

[0075] A "promoter sequence" refers to a nucleic acid sequence recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences that mediate the expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence that exhibits transcriptional activity in the host cell of choice, including mutant promoters, truncated promoters, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular polypeptides that are either homologous or heterologous to the host cell.

[0076] "Suitable reaction conditions" refer to the conditions in an enzyme conversion reaction solution (e.g., enzyme load, substrate load, temperature, pH, buffer, co-solvent ranges, etc.) under which a proline hydroxylase polypeptide of the invention can convert a substrate into a desired product compound. Some exemplary "suitable reaction conditions" are provided herein.

[0077] As used herein, "load" as in "compound load" or "enzyme load" refers to the concentration or amount of a component in the reaction mixture at the start of the reaction.

[0078] As used herein, "substrate" in the context of an enzymatic conversion reaction process refers to a compound or molecule that is acted upon by a prolyl hydroxylase polypeptide.

[0079] As used herein, "product" in the context of an enzymatic conversion process refers to a compound or molecule that results from the action of a proline hydroxylase polypeptide on a substrate.

[0080] As used herein, the term "culturing" refers to growing a population of microbial cells under any suitable conditions (e.g., using liquid, gel, or solid media).

[0081] Recombinant polypeptides can be produced using any suitable method known in the art. A gene encoding a wild-type polypeptide of interest can be cloned into a vector such as a plasmid and expressed in a desired host, such as E. coli. Variants of recombinant polypeptides can be generated by various methods known in the art. Indeed, there are a wide variety of different mutagenesis techniques known to those skilled in the art. In addition, mutagenesis kits are available from many commercial molecular biology suppliers. Methods are available for making specific substitutions at defined amino acids (site-directed), specific or random mutations in localized regions of a gene (position-directed), or random mutagenesis throughout a gene (e.g., saturation mutagenesis). Numerous suitable methods for generating enzyme variants are known to those skilled in the art, including, but not limited to, site-directed mutagenesis of single- or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or any other suitable method known in the art. Non-limiting examples of methods used for DNA and protein engineering are provided in the following patents: U.S. Patent No. 6,117,679; U.S. Patent No. 6,420,175; U.S. Patent No. 6,376,246; U.S. Patent No. 6,586,182; U.S. Patent No. 7,747,391; U.S. Patent No. 7,747,393; U.S. Patent No. 7,783,428; and U.S. Patent No. 8,383,346. After variants are produced, they can be screened for any desired properties (e.g., high or increased activity, or low or decreased activity, increased thermal activity, increased thermal stability, and / or acidic pH stability, etc.). In some embodiments, "recombinant proline hydroxylase polypeptides" (also referred to herein as "engineered proline hydroxylase polypeptides," "variant proline hydroxylase enzymes," and "proline hydroxylase variants") find use.

[0082] As used herein, a "vector" is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, the vector is an expression vector operably linked to a suitable control sequence that can cause the expression of a polypeptide encoded by the DNA sequence in a suitable host. In some embodiments, an "expression vector" has a promoter sequence operably linked to a DNA sequence (e.g., a transgene) to drive expression in a host cell, and in some embodiments, also includes a transcription terminator sequence.

[0083] As used herein, the term "expression" includes any step involved in producing a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from the cell.

[0084] As used herein, the term "produce" refers to the production of a protein and / or other compound by a cell. The term is intended to encompass any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses the secretion of a polypeptide from the cell.

[0085] As used herein, an amino acid or nucleotide sequence (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) is "heterologous" to another sequence to which it is operably linked if the two sequences are not associated in nature. For example, a "heterologous polynucleotide" is any polynucleotide introduced into a host cell by laboratory techniques, including a polynucleotide that is removed from a host cell, subjected to laboratory manipulation, and reintroduced into the host cell.

[0086] As used herein, the terms "host cell" and "host strain" refer to a suitable host for an expression vector containing DNA (e.g., a polynucleotide encoding a prolyl hydroxylase variant) provided herein. In some embodiments, a host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA techniques as known in the art.

[0087] The term "analog" refers to a polypeptide having greater than 70% sequence identity but less than 100% sequence identity (e.g., greater than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) with a reference polypeptide. In some embodiments, an analog refers to a polypeptide that contains one or more non-naturally occurring amino acid residues, including, but not limited to, homoarginine, ornithine, and norvaline, along with naturally occurring amino acids. In some embodiments, an analog also includes one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues.

[0088] The term "effective amount" means an amount sufficient to produce a desired result. One of ordinary skill in the art can determine an effective amount by using routine experimentation.

[0089] The terms "isolated" and "purified" are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that is removed from at least one other component with which it is naturally associated. The term "purified" does not require absolute purity; rather, it is intended as a relative definition.

[0090] "Stereoselectivity" refers to the preferential formation of one stereoisomer over another in a chemical or enzymatic reaction. Stereoselectivity can be partial, in which case the formation of one stereoisomer is favored over the other, or complete, in which case only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity is referred to as enantioselectivity, which is the fraction (typically reported as a percentage) of one enantiomer over the sum of the two. This is instead generally reported in the art as enantiomeric excess (ee), which is calculated therefrom (typically as a percentage) according to the formula [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer]. When the stereoisomers are diastereoisomers, the stereoselectivity is referred to as diastereoselectivity, which is the fraction (typically reported as a percentage) of one diastereomer in a mixture of two diastereomers, and is generally reported instead as diastereomeric excess (d). Enantiomeric excess and diastereomeric excess are types of stereomeric excess.

[0091] "Highly stereoselective" refers to a chemical or enzymatic reaction capable of converting a substrate (e.g., L-proline) to its corresponding hydroxylated product (e.g., trans-3-hydroxyproline) in at least about 85% stereoisomeric excess.

[0092] "Regioselectivity" or "regioselective reaction" refers to a reaction in which bond making or breaking occurs preferentially in one direction over all other possible directions. The reaction can be completely (100%) regioselective, where discrimination is complete; substantially regioselective (at least 75%); or partially regioselective (x%, where the percentage is set depending on the reaction of interest), where the product of the reaction at one site predominates over the product of the reaction at another site, e.g., preferential formation of a product compound (i.e., trans-3-hydroxyproline over the undesired product trans-4-hydroxyproline).

[0093] "Selective" or "selectivity," as defined above, can refer to either stereoselectivity or regioselectivity, or can refer to both stereoselectivity and regioselectivity.

[0094] "Isomeric excess" refers to a percentage calculated according to the formula [major isomer - minor isomer] / [major isomer + minor isomer]. This percentage represents the preferential formation of one isomer over another in a chemical or enzymatic reaction. Enantiomeric excess is a form of isomeric excess.

[0095] As used herein, "thermostable" refers to a proline hydroxylase polypeptide that maintains similar activity (e.g., greater than 60% to 80%) after exposure to elevated temperatures (e.g., 40 to 80°C) for a period of time (e.g., 0.5 to 24 hours) compared to a wild-type enzyme exposed to the same elevated temperature.

[0096] As used herein, "solvent stable" refers to a proline hydroxylase polypeptide that maintains similar activity (e.g., greater than 60% to 80%) after exposure to varying concentrations (e.g., 5-99%) of a solvent (e.g., ethanol, isopropyl alcohol, dimethyl sulfoxide [DMSO], tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.) for a period of time (e.g., 0.5-24 h) compared to a wild-type enzyme exposed to the same solvent at the same concentration.

[0097] As used herein, "thermo- and solvent stable" refers to a proline hydroxylase polypeptide that is both thermo- and solvent-stable.

[0098] As used herein, a "reducing agent" is defined as Fe +3 Fe +2 An exemplary reducing agent is ascorbic acid, which is generally in the form of L-ascorbic acid.

[0099] "Alkyl" refers to a saturated hydrocarbon group of 1 to 18 (inclusive) carbon atoms, more preferably 1 to 8 (inclusive) carbon atoms, and most preferably 1 to 6 (inclusive) carbon atoms, which is either straight-chained or branched. Alkyl groups having a designated number of carbon atoms are indicated in parentheses (e.g., (C1-C6) alkyl refers to an alkyl of 1 to 6 carbon atoms).

[0100] "Alkenyl" refers to a hydrocarbon group of 2 to 12 carbon atoms (inclusive), either straight or branched, containing at least one double bond, but optionally containing more than one double bond.

[0101] "Alkynyl" refers to a hydrocarbon group of 2 to 12 carbon atoms (inclusive), either straight-chained or branched, containing at least one triple bond, but optionally containing more than one triple bond, and optionally containing one or more double-bonded moieties as well.

[0102] "Alkylene" refers to a straight- or branched-chain divalent hydrocarbon radical having 1 to 18 (inclusive) carbon atoms, more preferably 1 to 8 (inclusive) carbon atoms, and most preferably 1 to 6 (inclusive) carbon atoms, optionally substituted with one or more suitable substituents. Exemplary "alkylene" groups include, but are not limited to, methylene, ethylene, propylene, butylene, etc.

[0103] "Alkenylene" refers to a straight- or branched-chain divalent hydrocarbon radical having 2 to 12 (inclusive) carbon atoms and one or more carbon-carbon double bonds, more preferably 2 to 8 (inclusive) carbon atoms, and most preferably 2 to 6 (inclusive) carbon atoms, optionally substituted with one or more suitable substituents.

[0104] "Heteroalkyl," "heteroalkenyl," and "heteroalkynyl" refer to alkyl, alkenyl, and alkynyl, respectively, as defined herein, in which one or more of the carbon atoms are each independently replaced with the same or different heteroatom or heteroatom group. The heteroatoms and / or heteroatom groups that can replace the carbon atoms include -O-, -S-, -SO-, -NR γ -, -PH-, -S(O)-, -S(O)2-, -S(O)NR γ -, -S(O)NR γ -, etc., including combinations thereof, where each R γ are independently selected from hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl, and heteroaryl.

[0105] "Aryl" refers to an unsaturated aromatic carbocyclic group of 6 to 12 carbon atoms (inclusive) having a single ring (e.g., phenyl) or multiple condensed rings (e.g., naphthyl or anthryl). Exemplary aryls include phenyl, pyridyl, naphthyl, and the like.

[0106] "Arylalkyl" refers to an aryl-substituted alkyl (i.e., an aryl-alkyl group) preferably having from 1 to 6 (inclusive) carbon atoms in the alkyl moiety and from 6 to 12 (inclusive) carbon atoms in the aryl moiety. Such arylalkyl groups are exemplified by benzyl, phenethyl, and the like.

[0107] "Aryloxy" is -OR λ group (in the formula, R λ is an optionally substituted aryl group).

[0108] "Cycloalkyl" refers to cyclic alkyl groups of 3 to 12 carbon atoms (inclusive) having a single cyclic ring or multiple condensed rings, which may be optionally substituted with 1 to 3 alkyl groups. Exemplary cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, and other single ring structures, or multiple ring structures, including bridged ring systems, such as adamantyl.

[0109] "Cycloalkylalkyl" refers to cycloalkyl-substituted alkyl (i.e., cycloalkyl-alkyl groups) preferably having from 1 to 6 (inclusive) carbon atoms inclusively in the alkyl moiety and from 3 to 12 (inclusive) carbon atoms inclusively in the cycloalkyl moiety. Such cycloalkylalkyl groups are exemplified by cyclopropylmethyl, cyclohexylethyl, and the like.

[0110] "Amino" refers to the group -NH2. Substituted amino refers to the group -NHR η , N.R. η Rη and N.R. η R η R η (In the formula, each R η are independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfanyl, sulfinyl, sulfonyl, etc. Exemplary amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylysulfonylamino, furanyl-oxy-sulfamino, etc.

[0111] "Aminoalkyl" refers to an alkyl group in which one or more of the hydrogen atoms has been replaced by one or more amino groups, including substituted amino groups.

[0112] "Aminocarbonyl" refers to -C(O)NH2. Substituted aminocarbonyl is -C(O)NR η R η (Wherein, an amino group NR η R η refers to a compound having a structure as defined herein.

[0113] "Oxy" refers to the divalent group --O--, which can have a variety of substituents to form different oxy groups, including ethers and esters.

[0114] "Alkoxy" or "alkyloxy" refers to the group -OR ζ (In the formula, R ζ is used interchangeably herein to refer to an alkyl group, optionally including a substituted alkyl group.

[0115] "Carboxy" refers to --COOH.

[0116] "Carbonyl" refers to -C(O)- which can have a variety of substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters, and ketones.

[0117] "Carboxyalkyl" refers to an alkyl in which one or more of the hydrogen atoms has been replaced by one or more carboxy groups.

[0118] "Aminocarbonylalkyl" refers to an alkyl substituted with an aminocarbonyl group, as defined herein.

[0119] "Halogen" or "halo" refers to fluoro, chloro, bromo and iodo.

[0120] "Haloalkyl" refers to an alkyl group in which one or more hydrogen atoms have been replaced with halogen. Thus, the term "haloalkyl" is meant to include monohaloalkyl, dihaloalkyl, trihaloalkyl, etc., up to perhaloalkyl. For example, the expression "(C1-C2)haloalkyl" includes 1-fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1 trifluoroethyl, perfluoroethyl, etc.

[0121] "Hydroxy" refers to --OH.

[0122] "Hydroxyalkyl" refers to an alkyl group in which one or more of the hydrogen atoms has been replaced by one or more hydroxy groups.

[0123] A "thiol" or "sulfanyl" refers to -SH. A substituted thiol or sulfanyl is -SR η (In the formula, R η is an alkyl, aryl or other suitable substituent).

[0124] "Alkylthio" is -SR ζ(In the formula, R ζ is an optionally substituted alkyl. Typical alkylthio groups include, but are not limited to, methylthio, ethylthio, n-propylthio, and the like.

[0125] "Alkylthioalkyl" refers to an alkylthio group, -SR ζ (In the formula, R ζ refers to alkyl substituted with (wherein is an optionally substituted alkyl).

[0126] "Sulfonyl" refers to -SO2-. Substituted sulfonyl is -SO2-R η (In the formula, R η is an alkyl, aryl or other suitable substituent).

[0127] "Alkylsulfonyl" is -SO2-R ζ (In the formula, R ζ is an optionally substituted alkyl. Typical alkylsulfonyl groups include, but are not limited to, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, and the like.

[0128] "Alkylsulfonylalkyl" refers to an alkylsulfonyl group, -SO2-R ζ (In the formula, R ζ refers to alkyl substituted with (wherein is an optionally substituted alkyl).

[0129] "Heteroaryl" refers to an aromatic heterocyclic group of 1 to 10 (inclusive) carbon atoms and 1 to 4 (inclusive) heteroatoms selected from oxygen, nitrogen, and sulfur within the ring. Such heteroaryl groups can have a single ring (e.g., pyridyl or furyl) or multiple condensed rings (e.g., indolizinyl or benzothienyl).

[0130] "Heteroarylalkyl" refers to heteroaryl-substituted alkyl (i.e., heteroaryl-alkyl) groups preferably having from 1 to 6 (inclusive) carbon atoms in the alkyl moiety and from 5 to 12 (inclusive) ring atoms in the heteroaryl moiety. Such heteroarylalkyl groups are exemplified by pyridylmethyl and the like.

[0131] "Heterocycle," "heterocyclic," and interchangeably, "heterocycloalkyl" refer to saturated or unsaturated groups having a single ring or multiple condensed rings of 2 to 10 (inclusive) carbon ring atoms and 1 to 4 (inclusive) hetero ring atoms selected from nitrogen, sulfur, or oxygen within the ring. Such heterocyclic groups can have a single ring (e.g., piperidinyl or tetrahydrofuryl) or multiple condensed rings (e.g., indolinyl, dihydrobenzofuran, or quinuclidinyl). Examples of heterocycles include, but are not limited to, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, pyrrolidine, indoline, and the like.

[0132] "Heterocycloalkylalkyl" refers to an alkyl substituted with a heterocycloalkyl (i.e., a heterocycloalkyl-alkyl group) preferably having from 1 to 6 (inclusive) carbon atoms inclusively ...

[0133] "Membered ring" is meant to encompass any cyclic structure. The number preceding the term "membered" indicates the number of skeletal atoms that make up the ring. Thus, for example, cyclohexyl, pyridine, pyran, and thiopyran are six-membered rings, and cyclopentyl, pyrrole, furan, and thiophene are five-membered rings.

[0134] "Fused bicyclic ring," as used herein, refers to both unsubstituted and substituted carbocyclic and / or heterocyclic ring moieties having 5 to 8 atoms in each ring, wherein the rings have two common atoms.

[0135] Unless otherwise specified, positions occupied by hydrogen in the aforementioned groups are substituted with hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluoro, chloro, bromo, iodo, halo, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxy, alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonamido, substituted sulfonamido, cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo, doximo), hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocycle, (heterocycle)oxy, and (heterocycle)alkyl; preferred heteroatoms are oxygen, nitrogen, and sulfur. It is understood that when open valences exist in these substituents, they may be further substituted with alkyl, cycloalkyl, aryl, heteroaryl and / or heterocyclic groups; when these open valences exist on carbon, they may be further substituted with halogens and with oxygen, nitrogen or sulfur bonded substituents; and when multiple such open valences exist, these groups may be linked to form rings by direct bond formation or by bond formation to new heteroatoms, preferably oxygen, nitrogen or sulfur.It is further understood that the above-described substitutions can be made provided that replacing hydrogen with a substituent does not introduce unacceptable instability into the molecules of the invention and is otherwise chemically reasonable.

[0136] "Optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes cases where the event or circumstance occurs and cases where it does not occur. Those of skill in the art will understand that for any molecule described as containing one or more optional substituents, only sterically practical and / or synthetically feasible compounds are intended to be included. "Optionally substituted" refers to any subsequent modifier in a term or series of chemical groups. For example, in the term "optionally substituted arylalkyl," the "alkyl" and "aryl" portions of the molecule can be substituted or unsubstituted, and for the series "optionally substituted alkyl, cycloalkyl, aryl, and heteroaryl," the alkyl, cycloalkyl, aryl, and heteroaryl groups can be substituted or unsubstituted, independently of the others.

[0137] Engineered proline hydroxylase polypeptides The present invention provides polypeptides having prolyl hydroxylase activity, polynucleotides encoding the polypeptides, methods for preparing the polypeptides, and methods for using the polypeptides. It should be understood that where a description refers to a polypeptide, a description may also be given of the polynucleotide encoding the polypeptide.

[0138] Proline hydroxylase belongs to the class of dioxygenase enzymes that catalyze the hydroxylation of proline in the presence of alpha-ketoglutarate and oxygen (O2). Alpha-ketoglutarate is stoichiometrically decarboxylated during hydroxylation, with one atom of the O2 molecule being incorporated into succinate and the other atom being incorporated into the hydroxyl group formed on the proline residue. As noted above, proline hydroxylase is distinguished from prolyl hydroxylase by its ability to hydroxylate free proline.

[0139] Several types of proline hydroxylases have been identified based on the major diastereomeric products formed in the enzymatic reaction: cis-3-proline hydroxylase (cis-P3H), cis-4-proline hydroxylase (cis-P4H), trans-3-proline hydroxylase (trans-P3H), and trans-4-proline hydroxylase (trans-P4H). The cis-P3H enzyme was identified in Streptomyces sp. TH1, Streptomyces canus, and Bacillus sp. TH2 and TH3 (Mori et al., Appl. Environ. Microbiol., 62(6):1903-1907

[1996] ). Cis-P4H enzymes have been identified in Lotus corniculatus rhizobia, Mesorhibozium loti, Sinorhizobium meliloti, and Medicago sativa rhizobia (Hara and Kino, Biochem. Biophys. Res. Commun., 379(4):882-6

[2009] ; U.S. Patent Application Publication No. 2011 / 0091942). Trans-P4H has been identified in Dactylosporangium sp., Amycolatopsis sp., Streptomyces griseoviridus, Streptomyces sp., Glarea lozoyensis, and Emericella rugulosa NRRL 11440 (Shibasaki et al., Appl. Environ. Microbiol., 65(9):4028-31

[1999] ; Petersen et al., Appl. Microbiol. Biotechnol., 62(2-3):263-7

[2003] ; Mori et al., Appl. Environ. Microbiol., 62:1903-1907

[1996] ; Lawrence et al., Biochem. J., 313:185-191

[1996] ; and EP0641862; Cacho et al., J. Am. Chem. Soc. 2012, 134, 16781).

[0140] Recently, a gene cluster containing three hydroxylase genes was identified in fungal species (sp.) 11243 (Matsui et al., J. Biosci. Bioeng. 2017, Feb; 123(2): 147-153). One of these genes was subsequently identified as a proline hydroxylase and characterized as a trans-selective proline hydroxylase. The proline hydroxylase from fungal species 11243 (designated ANO11243 or ANO) converts free proline to both trans-4-hydroxyproline and trans-3-hydroxyproline, with a slight enrichment for the trans-3-hydroxyproline isomer. However, the naturally occurring ANO11243 proline hydroxylase lacks properties that would be useful in large-scale industrial processes, including low specific activity, low thermostability, and low selectivity for the desired trans-3-hydroxyproline isomer.

[0141] Described herein are engineered proline hydroxylases that overcome the deficiencies of wild-type proline hydroxylase from fungal species No. 11243. The engineered proline hydroxylase polypeptide, derived from the wild-type enzyme ANO from fungal species No. 11243, can efficiently convert L-proline to trans-3-hydroxyproline. The present invention identifies amino acid residue positions and corresponding mutations in the proline hydroxylase polypeptide sequence that improve enzymatic properties compared to the naturally occurring enzyme, including, inter alia, activity, stability, expression, regioselectivity, and stereoselectivity. In particular, the present invention provides engineered polypeptides that can efficiently convert L-proline to trans-3-hydroxyproline in the presence of a co-substrate (e.g., alpha-ketoglutarate) under suitable reaction conditions (e.g., in the presence of oxygen and Fe(II)) (as illustrated in Scheme 1 above).

[0142] In some embodiments, the engineered proline hydroxylase polypeptides exhibit increased activity in the hydroxylation of L-proline to trans-3-hydroxyproline with the same amount of enzyme in a defined time compared to the polypeptide of SEQ ID NO: 4. In some embodiments, the engineered proline hydroxylase polypeptides have at least about 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more greater activity under suitable reaction conditions compared to the polypeptides represented by SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630.

[0143] In some embodiments, the engineered proline hydroxylase polypeptide has increased regioselectivity compared to wild-type proline hydroxylases. Specifically, naturally occurring enzymes convert proline primarily, if not exclusively, to trans-3-hydroxyproline. In some embodiments, the engineered proline hydroxylase polypeptide herein can selectively form trans-3-hydroxyproline over trans-4-hydroxyproline. In some embodiments, the engineered polypeptide can selectively form trans-3-hydroxyproline over trans-4-hydroxyproline, and the ratio of the formed trans-3-hydroxyproline to the compound trans-4-hydroxyproline under suitable reaction conditions is at least 1.5, 2, 3, 4, 5, 10, 15, 20, 25, 30, or more.

[0144] In some embodiments, the engineered proline hydroxylase polypeptide is capable of converting L-proline to trans-3-hydroxyproline under suitable reaction conditions with a percent conversion of at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% at a substrate loading concentration of at least about 10 g / L, about 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, about 70 g / L, about 100 g / L, about 125 g / L, about 150 g / L, about 175 g / L, or about 200 g / L or more, in a reaction time of about 120 hours or less, 72 hours or less, about 48 hours or less, about 36 hours or less, or about 24 hours or less.

[0145] Suitable reaction conditions under which the engineered polypeptides with the above-described improved properties carry out hydroxylation reactions can be determined with respect to conditions including the concentration or amount of polypeptide, substrate, co-substrate, transition metal cofactor, reducing agent, buffer, co-solvent, pH, temperature and reaction time, and / or conditions with the polypeptide immobilized on a solid support, as further described below and in the Examples.

[0146] In some embodiments, exemplary engineered polypeptides with proline hydroxylase activity, particularly those with improved properties in converting L-proline to trans-3-hydroxyproline, comprise amino acid sequences having one or more residue differences compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 at the residue positions indicated in Tables 4.1, 4.2, 4.3, 4.4, 5.1, 5.2, 5.3, 6.1, 7.1, 7.2, 7.3, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, 11.2 and / or 12.1.

[0147] Structural and functional information for exemplary non-naturally occurring (or engineered) proline hydroxylase polypeptides of the present invention is based on the conversion of L-proline to trans-3-hydroxyproline, the results of which are shown in Tables 4.1, 4.2, 4.3, 4.4, 5.1, 5.2, 5.3, 6.1, 7.1, 7.2, 7.3, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, 11.2, and / or 12.1 below. Odd-numbered sequence identifiers (i.e., SEQ ID NOs) refer to nucleotide sequences encoding the amino acid sequences provided by even-numbered SEQ ID NOs. Exemplary sequences are provided in the electronic sequence listing file accompanying this application, which is hereby incorporated by reference. Amino acid residue differences are based on comparison to the reference sequences of SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598, and / or 630. The naturally occurring amino acid sequence of proline hydroxylase ANO from fungal species no. 11243 is provided herein as SEQ ID NO:2 (the corresponding polynucleotide sequence, as provided herein, is SEQ ID NO:1). The activity of each engineered polypeptide relative to the reference polypeptides of SEQ ID NOs:4, 116, 162, 322, 412, 492, 562, 598, and / or 630 was determined as the conversion of substrate as described in the Examples herein. In some embodiments, shake flask powder (SFP) or downstream processing (DSP) powder assays are used as secondary screens to evaluate the properties of engineered proline hydroxylases, the results of which are provided in Tables 4.1, 4.2, 4.3, 4.4, 5.1, 5.2, 5.3, 6.1, 7.1, 7.2, 7.3, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, 11.2, and / or 12.1. The SFP format provides a more purified powder preparation of the engineered polypeptide and can contain up to about 30% of the engineered polypeptide total protein. Because the DSP preparation can contain up to about 80% of the engineered proline hydroxylase total protein, the DSP preparation can provide an even more purified form of the engineered polypeptide.

[0148] In some embodiments, specific enzyme properties associated with residue differences compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598, and / or 630 at the residue positions indicated herein include, inter alia, enzymatic activity, substrate tolerance, thermostability, regioselectivity, and stereoselectivity. Improvements in enzyme activity are associated with residue differences at the residue positions indicated in the Examples herein. Improvements in selectivity are associated with residue differences at the residue positions indicated in the Examples herein. Improvements in thermostability are associated with residue differences at the residue positions indicated in the Examples herein. Thus, residue differences at these residue positions can be used individually or in various combinations to produce engineered proline hydroxylase polypeptides with desired improved properties, including, inter alia, enzymatic activity, substrate tolerance, regioselectivity, stereoselectivity, and thermostability. Other residue differences that affect polypeptide expression can be used to increase expression of engineered proline hydroxylases.

[0149] Given the guidance provided herein, it is further contemplated that any of the exemplary engineered polypeptides comprising the even-numbered sequences of SEQ ID NOS:4-658 will find use as starting amino acid sequences for synthesizing other engineered prolyl hydroxylase polypeptides, for example, by subsequent rounds of evolution incorporating new combinations of various amino acid differences from other polypeptides in Tables 4.1, 4.2, 4.3, 4.4, 5.1, 5.2, 5.3, 6.1, 7.1, 7.2, 7.3, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, 11.2, and / or 12.1, as well as other residue positions described herein. Further improvements can be generated by including amino acid differences at residue positions that have been maintained unchanged through earlier rounds of evolution.

[0150] In some embodiments, an engineered polypeptide having proline hydroxylase activity with one or more improved properties compared to SEQ ID NO:4, 116, 162, 322, 412, 492, 562, 598 and / or 630 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the activity relative to SEQ ID NO:4. and one or more residue differences compared to SEQ ID NO:4 at residue positions selected from 21, 28, 58 / 247, 65, 80, 85, 95, 98, 117, 120, 159, 185, 194, 199, 200, 233, 237, 243, 250, 268, 281, 282, 287, 289, 307, 324, 326, 327, 330, 338, 343, 346 and 348. In some embodiments, the engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 include an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 4, as well as 21Q, 28A, 58V / 247V, 65A, 66A, 67A, 68A, 69A, 70A, 71A, 72A, 73A, 74A, 75A, 76A, 77A, 78A, 79A, 80A, 81A, 82A, 83A, 84A, 85A, 86A, 87A, 88A, 89A, 90A, 91A, 92A, 93A, 94A, 95A, 96A, 97A, 98A, 99A, 100A, 101A, 102A, 103A, 104A, 105A, 106A, 107A, 108A, 109A, 110A, 111A, 112A, 113A, 114A, 115A, 116A, 117A, 118A, 119A, 120A, 121A, 122A, 123A, 124A, 125 and 348S.In some embodiments, the engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 include amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 4, as well as R21Q, P28A, E58V / P247V, S65A, K80H, E85L, G95P, G95 and Q348S.

[0151] In some embodiments, the engineered polypeptides having proline hydroxylase activity with one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 have at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173 21, 28, 45, 65, 95, 112, 117, 139, 177, 185, 199, 233, 243, 250, 281, 282, 287, 289, 307, 324, 326, 327, 335, 338, 343, and 346. In some embodiments, an engineered polypeptide having proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 is an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 4. and one or more residue differences (compared to SEQ ID NO:4) selected from 21Q, 28A, 45S, 65A, 95R, 112L, 117S, 139F, 177P, 185D, 199A, 233A, 243V, 250Q, 250T, 281S, 281T, 282E, 282S, 287E, 289D, 307I, 324D, 326G, 326H, 326K, 327Q, 335A, 335M, 338I, 343N, 343P and 346S.In some embodiments, engineered polypeptides having proline hydroxylase activity with one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 include amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 4, as well as R21Q, P2 and one or more residue differences (compared to SEQ ID NO: 4) selected from 8A, Y45S, S65A, G95R, R112L, A117S, M139F, S177P, A185D, T199A, V233A, L243V, V250Q, V250T, R281S, R281T, L282E, L282S, D287E, M289D, V307I, A324D, R326G, R326H, R326K, W327Q, S335A, S335M, M338I, V343N, V343P and A346S.

[0152] In some embodiments, the engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NO:4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprise an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:4 and one or more residue differences at residue positions selected from 48 / 66 / 189 / 194, 48 / 66 / 194 and 66 / 82 / 85 / 135 / 189 / 194 / 267. In some embodiments, the engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NO:4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprise an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:4 and one or more residue differences (relative to SEQ ID NO:4) selected from 48V / 66W / 189N / 194L, 48V / 66W / 194L and 66W / 82P / 85P / 135P / 189N / 194L / 267D. In some embodiments, the engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprise an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 4 and one or more residue differences (relative to SEQ ID NO: 4) selected from A48V / Y66W / A189N / N194L, A48V / Y66W / N194L and Y66W / K82P / E85P / A135P / A189N / N194L / G267D.

[0153] In some embodiments, an engineered polypeptide having proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, % or higher identity, and one or more residue differences at residue positions selected from 20 / 56 / 76 / 168 / 169 / 296, 20 / 56 / 232 / 294, 20 / 119 / 294 / 296, 56 / 76 / 119 / 124 / 147 / 232, 56 / 76 / 294, 76 / 168 / 232 / 294, 76 / 294 / 296, 76 / 296, 147 and 232. In some embodiments, an engineered polypeptide having proline hydroxylase activity with one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 4. and one or more residue differences (compared to SEQ ID NO: 4) selected from 20F / 56P / 76E / 168A / 169L / 296I, 20F / 56P / 232E / 294Y, 20F / 119D / 294Y / 296I, 56P / 76E / 119D / 124F / 147F / 232E, 56P / 76E / 294Y, 76E / 168A / 232E / 294Y, 76E / 294Y / 296I, 76E / 296I, 147F and 232E.In some embodiments, the engineered polypeptides having proline hydroxylase activity with one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 include amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 4, and Y20F / S and Q232E.

[0154] In some embodiments, an engineered polypeptide with proline hydroxylase activity having one or more improved properties compared to SEQ ID NO:4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:116 and one or more residue differences compared to SEQ ID NO:116 at residue positions selected from 123, 189, 195, 233 and 296. In some embodiments, the engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NO: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprise an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 116 and one or more residue differences (relative to SEQ ID NO: 116) selected from 123T, 189A, 189S, 195Y, 233A, 233M and 296V. In some embodiments, the engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NO: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprise an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 116 and one or more residue differences (relative to SEQ ID NO: 116) selected from S123T, N189A, N189S, H195Y, V233A, V233M and L296V.

[0155] In some embodiments, engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 include amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NOs: 116, as well as 20 / 21 / 56, 20 / 21 / 56, 76 / 95 / 232 / 294 / 307 / 335, 20 / 21 / 56 / 76 / 147 / 225 / 232 / 233 / 281 / 294 / 296 / 307 / 335, 20 / 21 / 56 / 95 / 147 / 281 / 294 / 307, 20 / 21 / 56 / 281 / 307, 20 / 21 / 76 / 232 / 243, 20 / 21 / 95 / 2 32 / 307, 20 / 21 / 95 / 281 / 294 / 296, 20 / 21 / 147 / 189 / 233 / 243 / 281 / 307, 20 / 56, 20 / 56 / 76 / 95 / 281 / 307, 20 / 56 / 76 / 147 / 294 / 296 / 307, 20 / 56 / 95 / 147 / 294 , 20 / 56 / 281, 20 / 76, 20 / 76 / 95 / 281 / 294 / 296, 20 / 76 / 95 / 281 / 296 / 307, 20 / 76 / 233 / 294 / 307, 20 / 76 / 243 / 281 / 294, 21 / 76 / 147 / 233 / 294 / 307, 21 / 76 / 14 7 / 243 / 296 / 307 / 335, 21 / 95 / 185 / 189 / 232 / 281 / 296, 21 / 95 / 233 / 243 / 281 / 296, 21 / 95 / 294 / 296 / 307 / 335, 21 / 95 / 307, 21 / 281 / 307, 29 / 76 / 281, 56 / 76 / 95 / 232 / 243 / 281, 56 / 76 / 147 / 281 / 307, 56 / 76 / 243 / 294, 56 / 76 / 281 / 294, 56 / 76 / 296, 56 / 76 / 307, 56 / 95 / 147 / 307 / 335 / 348, 56 / 95 / 232 / 233 / 281 / 294 / 307, 56 / 95 / 243 / 281, 56 / 147 / 281, 56 / 232 / 243 / 281, 56 / 232 / 281, 56 / 232 / 281 / 294 / 296, 56 / 233 / 281 / 294 / 296, 56 / 281 / 307, 76 / 95 / 232 / 243 / 281 / 307,76 / 95 / 243 / 281 / 307 / 335, 76 / 95 / 294 / 307, 76 / 147, 76 / 147 / 233 / 243 / 294, 76 / 147 / 233 / 281 / 294 / 307, 76 / 147 / 243 / 294 / 296 / 307 / 335, 76 / 147 / 281 / 307, 76 / 189 / 296, 76 / 232 / 233 / 243 / 294 / 296 / 307, 76 / 281, 76 / 281 / 294, 76 / 294 / 296, 95 / 120, 95 / 147 / 335, 95 / 232 / 243 / 281 / 294 / 307, 95 / 232 / 281 / 294 / 296, 95 / 281 / 294 / 296, 95 / 335, 147, 147 / 225 / 232 / 243 / 281 / 296 / 307 / 335, 147 / 233 / 243 / 281 / 307, 147 / 233 / 281 / 307 / 335, 147 / 243 / 281, 147 / 307, 232 / 233 / 281 / 294 / 296 / 307, 232 / 281, 232 / 284 / 307, 233 / 243 / 281 / 296 / 307 / 335, 233 / 281 / 296 / 307, 243 / and one or more residue differences at residue positions selected from 281 / 294 / 296, 281, 281 / 294, 281 / 307, 307 and 335, 117, 139, 177, 185, 199, 233, 243, 250, 281, 282, 287, 289, 307, 324, 326, 327, 335, 338, 343 and 346. In some embodiments, an engineered polypeptide having proline hydroxylase activity with one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the activity relative to SEQ ID NO: 116. and 20F / 21Q / 56P, 20F / 21Q / 56P / 76E / 95R / 232E / 294Y / 307I / 335M, 20F / 21Q / 56P / 76E / 147F / 225R / 232E / 233A / 281S / 294Y / 296I / 307L / 335M, 20F / 21Q / 56P / 95R / 147F / 281T / 294Y / 307I, 20F / 21Q / 56P / 281T / 307L,<h2 style=";text-align:left;direction:ltr">20F / 21Q / 76E / 232E / 243V, 20F / 21Q / 95R / 232E / 307I, 20F / 21Q / 95R / 281T / 294Y / 296I, 20F / 21Q / 147F / 189A / 233R / 243V / 281T / 307I, 20F / 56P, 20F / 56P / 76E / 95R / 281S / 307I、20F / 56P / 76E / 147F / 294Y / 296I / 307L、20F / 56P / 95P / 147F / 294Y、20F / 56P / 281S、20F / 76E、20F / 76E / 95R / 281S / 294Y / 296I、20F / 76E / 95R / 281T / 296I / 307I、20F / 76E / 233A / 294Y / 307I、20F / 76E / 243V / 281T / 294Y、21Q / 76E / 147F / 233R / 294Y / 307I、21Q / 76E / 147F / 243V / 296I / 307I / 335M、21Q / 95R / 185L / 189A / 232E / 281T / 296I、21Q / 95R / 233A / 243V / 281T / 296I、21Q / 95R / 294Y / 296I / 307I / 335M、21Q / 95R / 307I、21Q / 281T / 307L、29T / 76E / 281T、56P / 76E / 95R / 232E / 243V / 281T、56P / 76E / 147F / 281T / 307I、56P / 76E / 243V / 294Y、56P / 76E / 281T / 294Y、56P / 76E / 296I、56P / 76E / 307I、56 P / 95P / 147F / 307I / 335M / 348K、56P / 95R / 232E / 233R / 281S / 294Y / 307L、56P / 95R / 243V / 281T、56P / 147F / 281T、56P / 232E / 243V / 281S、56P / 232E / 281S、5 6P / 232E / 281S / 294Y / 296I,56P / 233R / 281S / 294Y / 296I,56P / 281T / 307I,76E / 95P / 232E / 243V / 281S / 307L,76E / 95R / 243V / 281S / 307I / 335M,76E / 95R / 294Y / 307L、76E / 147F、76E / 147F / 233A / 243V / 294Y、76E / 147F / 233R / 281T / 294Y / 307L、76E / 147F / 243V / 294Y / 296I / 307L / 335M、76E / 147F / 281S / 307L、76E / 189A / 296I, 76E / 232E / 233R / 243V / 294Y / 296I / 307I, 76E / 281S, 76E / 281T / 294Y, 76E / 294Y / 296I, 95P / 232E / 281T / 294Y / 296I, 95P / 335M, 95R / 120P, 95R / 147F / 335M, 95R / 232E / 243V / 281T / 294Y / 307I, 95R / 281T / 294Y / 296I, 95R / 335 M, 147F, 147F / 225R / 232E / 243V / 281S / 296I / 307L / 335M, 147F / 233A / 243V / 281S / 307L, 147F / 233R / 281T / 307L / 335M, 147F / 243V / 281S, 147F / 307I, 232E / 233A / 281T / 294Y / 296I / 307I, 232E / 281T, 232E / 284R / 307I, 233A / 243V / 281S / 296I / 307I / 335M, 233A / 281T / 296I / 307I, 243V / 281S / 294Y / 296I, 281T, 281T / 294Y, 281T / 307I, 281T / 307L, 307I and 335M. In some embodiments, the engineered polypeptides having proline hydroxylase activity with one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 include amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 116, as well as Y20F / R21Q / S56P, Y20F / R21Q / S56P / H76E / G95R / Q232E / H294Y / V307I / S335M, Y20F / R21Q / S56P / H76E / Y147F / Q225R / Q232E / V233A / R281S / H294Y / L29 6I / V307L / S335M, Y20F / R21Q / S56P / G95R / Y147F / R281T / H294Y / V307I, Y20F / R21Q / S56P / R281T / V307L, Y20F / R21Q / H76E / Q232E / L243V,<h2 style=";text-align:left;direction:ltr">Y20F / R21Q / G95R / Q232E / V307I、Y20F / R21Q / G95R / R281T / H294Y / L296I、Y2 0F / R21Q / Y147F / N189A / V233R / L243V / R281T / V307I、Y20F / S56P、Y20F / S56 P / H76E / G95R / R281S / V307I、Y20F / S56P / H76E / Y147F / H294Y / L296I / V307L 、Y20F / S56P / G95P / Y147F / H294Y、Y20F / S56P / R281S、Y20F / H76E、Y20F / H76 E / G95R / R281S / H294Y / L296I、Y20F / H76E / G95R / R281T / L296I / V307I、Y20F / H76E / V233A / H294Y / V307I、Y20F / H76E / L243V / R281T / H294Y、R21Q / H76E / Y147F / V233R / H294Y / V307I、R21Q / H76E / Y147F / L243V / L296I / V307I / S335M、R21Q / G95R / A185L / N189A / Q232E / R281T / L296I、R21Q / G95R / V233A / L243 V / R281T / L296I、R21Q / G95R / H294Y / L296I / V307I / S335M、R21Q / G95R / V307 I、R21Q / R281T / V307L、A29T / H76E / R281T、S56P / H76E / G95R / Q232E / L243V / R281T, S56P / H76E / Y147F, R281T / V307I, S56P / H76E / L243V / H294Y, S56P / H76E / R281T / H294Y, S56P / H76E / L296I, S56P / H76E / V307I, S56P / G95P / Y147 F / V307I / S335M / Q348K、S56P / G95R / Q232E / V233R / R281S / H294Y / V307L、S5 6P / G95R / L243V / R281T、S56P / Y147F / R281T、S56P / Q232E / L243V / R281S、S5 6P / Q232E / R281S、S56P / Q232E / R281S / H294Y / L296I、S56P / V233R / R281S / H294Y / L296I、S56P / R281T / V307I、H76E / G95P / Q232E / L243V / R281S / V307L、H76E / G95R / L243V / R281S / V307I / S335M, H76E / G95R / H294Y / V307L, H76E / Y147F, H76E / Y147F / V233A / L243V / H294Y, H76E / Y147F / V233R / R281T / H294Y / V307L, H76E / Y147F / L243V / H294Y / , L296I / V307L / S335M, H76E / Y147F / R281S / V307L, H76E / N189A / L296I, H76E / Q232E / V233R / L243V / H2 94Y / L296I / V307I, H76E / R281S, H76E / R281T / H294Y, H76E / H294Y / L296I, G95P / Q232E / R281T / H294Y / L296I, G95P / S335M, G95R / L120P, G95R / Y147F / S335M, G95R / Q232E / L243V / R281T / H294Y / V307I, G9 5R / R281T / H294Y / L296I, G95R / S335M, Y147F, Y147F / Q225R / Q232E / L243V / R281S / L296I / V307L / S335 M, Y147F / V233A / L243V / R281S / V307L, Y147F / V233R / R281T / V307L / S335M, Y147F / L243V / R281S, Y14 7F / V307I, Q232E / V233A / R281T / H294Y / L296I / V307I, Q232E / R281T, Q232E / G284R / V307I, V233A / L2 Contains one or more residue differences (compared to SEQ ID NO: 116) selected from 43V / R281S / L296I / V307I / S335M, V233A / R281T / L296I / V307I, L243V / R281S / H294Y / L296I, R281T, R281T / H294Y, R281T / V307I, R281T / V307L, V307I and S335M.

[0156] In some embodiments, an engineered polypeptide with proline hydroxylase activity having one or more improved properties compared to SEQ ID NO:4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:116 and one or more residue differences at residue positions selected from 21 / 76 / 147 / 243 / 296 / 307 / 335, 56 / 76 / 147 / 281 / 307 and 95 / 147 / 335. In some embodiments, the engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NO: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprise an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 116 and one or more residue differences (relative to SEQ ID NO: 116) selected from 21Q / 76E / 147F / 243V / 296I / 307I / 335M, 56P / 76E / 147F / 281T / 307I and 95R / 147F / 335M. In some embodiments, the engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NO: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprise an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 116 and one or more residue differences (relative to SEQ ID NO: 116) selected from R21Q / H76E / Y147F / L243V / L296I / V307I / S335M, S56P / H76E / Y147F / R281T / V307I and G95R / Y147F / S335M.

[0157] In some embodiments, engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 include amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NOs: 162, 285, 123, 237, 288, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, and 123 / 199 / 200 / 247 / 250 / 338. In some embodiments, engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 include amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 162, as well as 2L / 85L / 123T / 237E, 28A / 115T / 117V / 120I / 123T / 268T / 270L / 343N / 346S / 348S, 45S / 123T / 326G, 65R / 117V / 120I / 123T / 343N / 346G, 85L / 123T / 281T / 282S, 114G / 115T / 117T / 120P / 123T / 268T / 271A / 313F / 326G / 343N / 346S, 123T / 139F / 233A / 237E / 281M / 282S / 289D / 324Q / 326G and 123T / 199A / 200V / 247L / 250Q / 338I.In some embodiments, engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 include amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 162, as well as G2L / E85L / S123T / Q237E, P28A / V115T / A117V / L120I / S123T / R268T / R270L / V343 N / A346S / Q348S, Y45S / S123T / R326G, S65R / A117V / L120I / S123T / V343N / A346G, E85L / S123T / R281T / L282S, E114G / V115T / A117T / L120P / S123T / R268T / S271A / L313F / R326 G / V343N / A346S, S123T / M139F / V233A / Q237E / R281M / L282S / M289D / A324Q / R326G and S123T / T199A / P200V / P247L / V250Q / M338I (compared to SEQ ID NO: 162).

[0158] In some embodiments, the engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprise an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 322 and one or more residue differences compared to SEQ ID NO: 322 at residue positions selected from 26, 54, 61, 129, 132, 149, 156, 175, 189, 201, 209, 228, 236, 248, 262, 272, 277, 291 and 345. In some embodiments, an engineered polypeptide having proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or more of the activity of SEQ ID NO: 322. , 98%, 99% or higher identity, and one or more residue differences (compared to SEQ ID NO: 322) selected from 26N, 54P, 61H, 129I, 132P, 149G, 156S, 175S, 175V, 189S, 201C, 201G, 201T, 209S, 228T, 236T, 248R, 262V, 272S, 277A, 291G and 345R.In some embodiments, an engineered polypeptide having proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the activity of SEQ ID NO: 322. and amino acid sequences with higher identity thereto, as well as one or more residue differences (compared to SEQ ID NO: 322) selected from G26N, G54P, D61H, A129I, E132P, S149G, V156S, L175S, L175V, N189S, A201C, A201G, A201T, C209S, V228T, Q236T, D248R, S262V, V272S, V277A, P291G and T345R.

[0159] In some embodiments, an engineered polypeptide with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 322 and one or more residue differences at residue positions selected from 25, 43, 54, 58, 61, 79, 129, 132, 143, 156, 163, 175, 179, 201, 209, 236, 248, 278, 291, 345 and 347. In some embodiments, the engineered polypeptides having proline hydroxylase activity with one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 have at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 17 Contains an amino acid sequence with 98%, 99% or more identity and one or more residue differences selected from 25K, 43T, 54P, 54S, 58T, 61H, 79T, 129I, 132N, 143L, 156D, 156S, 163L, 175V, 179L, 201C, 209S, 236T, 248R, 278N, 291G, 345R and 347E (compared to SEQ ID NO: 322).In some embodiments, an engineered polypeptide having proline hydroxylase activity that has one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the activity relative to SEQ ID NO: 322. and one or more residue differences (compared to SEQ ID NO: 322) selected from H25K, A43T, G54P, G54S, E58T, D61H, Q79T, A129I, E132N, D143L, V156D, V156S, Q163L, L175V, E179L, A201C, C209S, Q236T, D248R, S278N, P291G, T345R and A347E.

[0160] In some embodiments, an engineered polypeptide having proline hydroxylase activity that has one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the activity relative to SEQ ID NO: 322. and 117 / 120 / 208 / 270 / 324 / 343 / 346, 85 / 117 / 120 / 135 / 208 / 270 / 324 / 343 / 346, 85 / 117 / 120 / 135 / 208 / 281 / 282 / 289, 85 / 117 / 120 / 270 / 281 / 289, 85 / 117 / 135 / 139 / 208 and 117 / 120 / 208 / 270 / 324 / 343 / 346. In some embodiments, the engineered polypeptide with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 is an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 322: and one or more residue differences (relative to SEQ ID NO: 322) selected from 85L / 117T / 120P / 135S / 208E / 281R / 282L / 289M, 85L / 117T / 135S / 139M / 208E, 85L / 117V / 120I / 135S / 208E / 270L / 324A / 343N / 346G, 85L / 117V / 120P / 270L / 281R / 289M and 117T / 120I / 208E / 270L / 324A / 343N / 346G.In some embodiments, the engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 include an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 322, as well as E85L / A117T / L12 0P / A135S / A208E / M281R / S282L / D289M, E85L / A117T / A135S / F139M / A208E, E85L / A117V / L120I / A135S / A208E / R270L / Q324A / V343N / A346G, E85L / A117V / L120P / R270L / M281R / D289M and A117T / L120I / A208E / R270L / Q324A / V343N / A346G.

[0161] In some embodiments, an engineered polypeptide having proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 1610, 1620, 1 %, 99% or more identity and one or more residue differences at residue positions selected from 47, 48, 56 / 118, 85, 95, 95 / 289, 113, 118, 118 / 247, 154, 162, 162 / 204, 164, 164 / 198 / 271, 168, 169, 187, 195, 243, 271, 275, 281, 314, 330 and 342. In some embodiments, engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 include amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 412, as well as 47M, 48G, 56P / 118W, 89M, 99M, 10 ... and 342R.In some embodiments, the engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 include an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 412, as well as any of the following amino acid sequences: F47M, V48G, S56P / A118W, L85P, G95A / M289V, G95A / M289V, G95B / M289V, G95C / M289V, G95D / M289V, G95E / M289V, G95F ... and N342R.

[0162] In some embodiments, an engineered polypeptide having proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 1610, 1620, 1 and amino acid sequences with 8%, 99% or higher identity and one or more residue differences compared to SEQ ID NO: 412 at residue positions selected from 25 / 129 / 163 / 236 / 262 / 345 / 347, 120 / 156 / 175 / 179 / 201, 129 / 189 / 236 / 262 / 277 / 278, 129 / 236 / 262, 156 / 175 / 179 / 228 and 162. In some embodiments, an engineered polypeptide having proline hydroxylase activity with one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the activity relative to SEQ ID NO: 412. and one or more residue differences (compared to SEQ ID NO: 412) selected from 25K / 129I / 163L / 236T / 262V / 345R / 347E, 120V / 156S / 175V / 179L / 201G, 129I / 189S / 236T / 262V / 277A / 278N, 129I / 236T / 262V, 156S / 175V / 179L / 228A, 162L, and 162V.In some embodiments, an engineered polypeptide having proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 is an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 412. and one or more residue differences (compared to SEQ ID NO: 412) selected from H25K / A129I / Q163L / Q236T / S262V / T345R / A347E, P120V / V156S / L175V / E179L / A201G, A129I / N189S / Q236T / S262V / V277A / S278N, A129I / Q236T / S262V, V156S / L175V / E179L / V228A, H162L, and H162V.

[0163] In some embodiments, an engineered polypeptide with proline hydroxylase activity having one or more improved properties compared to SEQ ID NO: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 492 and one or more residue differences at residue positions selected from 15, 17, 28, 29, 65, 135, 167, 177, 199, 208, 228, 235, 287, 294, 307 and 343. In some embodiments, an engineered polypeptide having proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500, 1510, 1520, 1530, 1540, 1550, 1560, 1570, 1580, 1590, 1600, 1610, 1620, 1 Contains an amino acid sequence with 8%, 99% or more identity and one or more residue differences selected from 15V, 17C, 28I, 29S, 65V, 135G, 135N, 135T, 167G, 177A, 177L, 177P, 199C, 208L, 208M, 208S, 228T, 235E, 287E, 294T, 307L, 343S and 343T (compared to SEQ ID NO: 492).In some embodiments, an engineered polypeptide having proline hydroxylase activity with one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the activity relative to SEQ ID NO: 492. and one or more residue differences (compared to SEQ ID NO: 492) selected from I15V, S17C, P28I, A29S, S65V, S135G, S135N, S135T, Q167G, S177A, S177L, S177P, T199C, E208L, E208M, E208S, V228T, D235E, D287E, H294T, I307L, V343S and V343T.

[0164] In some embodiments, an engineered polypeptide having proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the activity of an engineered polypeptide having proline hydroxylase activity compared to SEQ ID NO: 492. contains an amino acid sequence with a higher identity thereto, and one or more residue differences compared to SEQ ID NO: 492 at residue positions selected from 85 / 187 / 281 / 347, 85 / 187 / 347, 118 / 120 / 162 / 175 / 179 / 330, 118 / 120 / 162 / 175 / 330, 162 / 175 / 179 / 330, 175 / 228 / 330, 195 / 347 and 278 / 314 / 347. In some embodiments, an engineered polypeptide having proline hydroxylase activity with one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 492. and one or more residue differences (compared to SEQ ID NO:492) selected from 85P / 187P / 281L / 347E, 85P / 187P / 347E, 118V / 120V / 162V / 175V / 179L / 330H, 118V / 120V / 162V / 175V / 330H, 162V / 175V / 179L / 330H, 175V / 228A / 330H, 195Y / 347E and 278S / 314A / 347E.In some embodiments, an engineered polypeptide having proline hydroxylase activity with one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 492, and Contains one or more residue differences (compared to SEQ ID NO: 492) selected from L85P / C187P / R281L / A347E, L85P / C187P / A347E, A118V / P120V / H162V / L175V / E179L / L330H, A118V / P120V / H162V / L175V / L330H, H162V / L175V / E179L / L330H, L175V / V228A / L330H, H195Y / A347E and N278S / F314A / A347E.

[0165] In some embodiments, an engineered polypeptide with proline hydroxylase activity having one or more improved properties compared to SEQ ID NO: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 562 and one or more residue differences at residue positions selected from 15, 40, 43, 44, 59, 79, 82, 149, 164, 179, 345 and 347. In some embodiments, an engineered polypeptide with proline hydroxylase activity having one or more improved properties compared to SEQ ID NO: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 562 and one or more residue differences (relative to SEQ ID NO: 562) selected from 15F, 40A, 43S, 44R, 44V, 59L, 79E, 82A, 149N, 164Q, 179T, 345D and 347K. In some embodiments, the engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NO: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprise an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 562 and one or more residue differences (relative to SEQ ID NO: 562) selected from I15F, K40A, A43S, G44R, G44V, R59L, Q79E, K82A, S149N, S164Q, L179T, T345D and A347K.

[0166] In some embodiments, an engineered polypeptide having proline hydroxylase activity with one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 562, and or 562 at residue positions selected from 29 / 85 / 177 / 208 / 228 / 347, 29 / 85 / 208 / 228 / 343 / 347, 29 / 177 / 195 / 228 / 343, 29 / 208 / 228 / 278 / 294 / 347, 56 / 195 / 278, 85 / 187 / 205 / 208 / 278, 113 / 177 / 187 / 195 / 208 / 278 / 294 / 343 / 347 and 177 / 205 / 208 / 228. In some embodiments, engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 include amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 562, as well as 29S / 85P / 177A / 208S / 228T / 347E, 29S / 85P / 208L / 228T / 343T / 347E, 29S / 177P / 195Y / 228T / 343T, 29S / 208S / 228T / 278S / 294T / 347E, 56P / 195Y / 278S, 85P / 187P / 205S / 208L / 278S, 113N / 177P / 187P / 195Y / 208S / 278S / 294Y / 343T / 347E and 177A / 205S / 208L / 228T.In some embodiments, engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 include amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 562, as well as A29S / L85P / S177A / E208S / V228T / A347E, A29S / and S177A / A205S / E208L / V228T / V343T / A347E, A29S / S177P / H195Y / V228T / V343T, A29S / E208S / V228T / N278S / H294T / A347E, S56P / H195Y / N278S, L85P / C187P / A205S / E208L / N278S, S113N / S177P / C187P / H195Y / E208S / N278S / H294Y / V343T / A347E, and S177A / A205S / E208L / V228T.

[0167] In some embodiments, an engineered polypeptide with proline hydroxylase activity having one or more improved properties compared to SEQ ID NO: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 598 and one or more residue differences at residue positions selected from 47, 162, 209, 219, 227 and 342. In some embodiments, an engineered polypeptide with proline hydroxylase activity having one or more improved properties compared to SEQ ID NO: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 598 and one or more residue differences (relative to SEQ ID NO: 598) selected from 47Q, 162S, 209H, 219V, 227R, 342L and 342M. In some embodiments, the engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NO: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprise an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 598 and one or more residue differences (relative to SEQ ID NO: 598) selected from F47Q, V162S, C209H, T219V, S227R, N342L and N342M.

[0168] In some embodiments, engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 include amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 598, as well as 17 / 44 / 179 / 195 / 250 / 313 / 330. and 187 / 286.In some embodiments, engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 include amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 598, as well as 17V / 44R / 199C / 313C, 17V / 44V / 179T / 195Y / 250P / 313C / 345D, 43S / 44V / 195Y / 199C, 44R / 179T / 195Y / 199C, 44R / 179T / 195Y / 199C / 345D, 44V / 149N / 164Q / 1 71M / 187P, 44V / 179T / 195Y / 199C / 345D, 79E / 163D / 164Q / 171M / 187N / 201V / 286P / 28 Contains one or more residue differences (compared to SEQ ID NO: 598) selected from 8T, 82A / 163D / 164Q, 82A / 163D / 164Q / 171M / 187P / 201V / 203Q / 208I / 286P / 288T / 320V, 149N / 164Q / 171M / 288T and 187P / 286P.In some embodiments, engineered polypeptides with proline hydroxylase activity having one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 include amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 598, as well as S17V / G44R / T199C / L313C, S17V / G44V / L179T / H195Y / V250P / L313C / T345D, A43S / G44V / H195Y / T199C, G44R / L 179T / H195Y / T199C, G44R / L179T / H195Y / T199C / T345D, G44V / S149N / S164Q / T171M / C187P, G 44V / L179T / H195Y / T199C / T345D, Q79E / Q163D / S164Q / T171M / C187N / A201V / A286P / V288T, K8 Contains one or more residue differences (compared to SEQ ID NO: 598) selected from 2A / Q163D / S164Q, K82A / Q163D / S164Q / T171M / C187P / A201V / S203Q / L208I / A286P / V288T / K320V, S149N / S164Q / T171M / V288T and C187P / A286P.

[0169] In some embodiments, an engineered polypeptide having proline hydroxylase activity that has one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of the activity of an engineered polypeptide having proline hydroxylase activity relative to SEQ ID NO: 630. contains an amino acid sequence with a higher identity thereto, and one or more residue differences compared to SEQ ID NO: 630 at residue positions selected from 82 / 164 / 171 / 203 / 208, 135 / 163 / 164 / 201 / 203 / 208, 162, 162 / 219 / 236, 162 / 219 / 313 / 338, 162 / 236 / 342, 162 / 313 / 342 and 164 / 171 / 201 / 203 / 282. In some embodiments, an engineered polypeptide having proline hydroxylase activity with one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 630. and one or more residue differences (relative to SEQ ID NO: 630) selected from 82A / 164T / 171M / 203Q / 208I, 135P / 163D / 164Q / 201V / 203Q / 208I, 162S, 162S / 219V / 236L, 162S / 219V / 313C / 338I, 162S / 236L / 342M, 162S / 313C / 342M and 164Q / 171M / 201V / 203Q / 282V.In some embodiments, an engineered polypeptide having proline hydroxylase activity with one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 630, and Contains one or more residue differences (compared to SEQ ID NO: 630) selected from K82A / S164T / T171M / S203Q / L208I, S135P / Q163D / S164Q / A201V / S203Q / L208I, V162S, V162S / T219V / T236L, V162S / T219V / L313C / M338I, V162S / T236L / N342M, V162S / L313C / N342M and S164Q / T171M / A201V / S203Q / L282V.

[0170] As will be appreciated by those skilled in the art, in some embodiments, one or a combination of the selected residue differences described above can be held constant (i.e., maintained) in the engineered proline hydroxylase as a core feature, and additional residue differences at other residue positions can be incorporated into the sequence to generate additional engineered proline hydroxylase polypeptides with improved properties. Thus, for any engineered proline hydroxylase containing one or a subset of the residue differences described above, it will be understood that the invention contemplates other engineered proline hydroxylases that include one or a subset of the residue differences and, in addition, one or more residue differences at other residue positions disclosed herein.

[0171] As noted above, engineered polypeptides having proline hydroxylase activity are also capable of converting the substrate compound L-proline to the product compound trans-3-hydroxyproline. In some embodiments, the engineered proline hydroxylase polypeptide is capable of converting the substrate compound L-proline to the product compound trans-3-hydroxyproline with at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more greater activity than the activity of the reference polypeptide of SEQ ID NO: 4, 116, 162, 322, 412, 492, 562, 598, and / or 630. In some embodiments, the engineered proline hydroxylase polypeptides capable of converting the substrate compound L-proline to the product compound trans-3-hydroxyproline with at least 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold or more greater activity than the activity of the reference polypeptide of SEQ ID NO: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprise an amino acid sequence having one or more features selected from improved regioselectivity, improved activity, improved specific activity, and / or improved thermostability.

[0172] In some embodiments, the engineered proline hydroxylase polypeptide is capable of converting the substrate compound L-proline to the product compound trans-3-hydroxyproline with at least 1.2-fold greater activity than SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630, and comprises an amino acid sequence selected from the even-numbered sequences in the following range: SEQ ID NOs: 6-658.

[0173] In some embodiments, the engineered proline hydroxylase polypeptide is capable of converting the substrate compound L-proline to the product compound trans-3-hydroxyproline with at least two-fold greater activity than SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630, and comprises an amino acid sequence having one or more residue differences provided herein (compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630, if applicable).

[0174] In some embodiments, an engineered proline hydroxylase polypeptide capable of converting the substrate compound L-proline to the product compound trans-3-hydroxyproline with at least twice the activity as compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 comprises an amino acid sequence selected from the even-numbered sequences in the following range: SEQ ID NOs: 6-658.

[0175] In some embodiments, the engineered proline hydroxylase polypeptide is capable of converting at least 50% or more, 60% or more, 70% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, or 95% or more of the compound L-proline to the product compound trans-3-hydroxyproline under HTP assay conditions, SFP assay conditions, or DSP assay conditions at a substrate loading of about 100 g / L, about 50 g / L, or about 20 g / L in 120 hours or less, 72 hours or less, 48 hours or less, or 24 hours or less. In some embodiments, the engineered proline hydroxylase polypeptide is capable of converting at least 50% or more of the compound L-proline to the product compound trans-3-hydroxyproline in 24 h or less at a substrate load of about 20 g / L under DSP assay conditions at about 25° C.

[0176] In some embodiments, the engineered proline hydroxylase has an amino acid sequence that includes one or more residue differences compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 that increase expression of the engineered proline hydroxylase activity in a bacterial host cell, particularly E. coli.

[0177] In some embodiments, the engineered proline hydroxylase polypeptide with improved properties in converting the compound L-proline to the product compound trans-3-hydroxyproline has an amino acid sequence that includes a sequence selected from the even-numbered sequences in the following range: SEQ ID NOs: 6-658.

[0178] In some embodiments, the engineered polypeptides having prolyl hydroxylase activity have at least 80 to 100% of the even-numbered sequences in the following ranges: SEQ ID NOs: 6-658, as provided in Tables 4.1, 4.2, 4.3, 4.4, 5.1, 5.2, 5.3, 6.1, 7.1, 7.2, 7.3, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, 11.2 and / or 12.1. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, and amino acid residue differences compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 that are present in any one of the even-numbered sequences in the following ranges: SEQ ID NOs: 6-658.

[0179] In addition to the residue positions specified above, any of the engineered proline hydroxylase polypeptides disclosed herein can further include other residue differences at other residue positions (i.e., residue positions other than those falling within any of the even-numbered sequences in the following ranges: SEQ ID NOS: 6-658) compared to SEQ ID NOS: 4, 116, 162, 322, 412, 492, 562, 598, and / or 630. Residue differences at these other residue positions can provide additional variation in amino acid sequence without adversely affecting the ability of the polypeptide to carry out the conversion of the compound L-proline to the product compound trans-3-hydroxyproline along with the conversion of proline to cis-4-hydroxyproline. Thus, in some embodiments, in addition to the amino acid residue differences present in any one of the engineered proline hydroxylase polypeptides selected from the even-numbered sequences in the following range: SEQ ID NOs: 6-658, the sequence can further include 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45 or 1-50 residue differences at other amino acid residue positions compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630. In some embodiments, the number of amino acid residue differences compared to the reference sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 residue positions. In some embodiments, the number of amino acid residue differences compared to the reference sequence can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 residue positions. Residue differences at these other positions can be conservative or non-conservative changes. In some embodiments, residue differences can include conservative and non-conservative substitutions compared to the naturally occurring proline hydroxylase polypeptides of SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630.

[0180] In some embodiments, the present invention also provides engineered polypeptides, including fragments of any of the engineered proline hydroxylase polypeptides described herein, that retain the functional activity and / or improved properties of the engineered proline hydroxylase. Accordingly, in some embodiments, the present invention provides polypeptide fragments that are capable of converting the compound L-proline to the product compound trans-3-hydroxyproline under suitable reaction conditions, wherein the fragments comprise at least about 80%, 90%, 95%, 96%, 97%, 98%, or 99% of the full-length amino acid sequence of an engineered proline hydroxylase polypeptide of the invention, such as exemplary engineered proline hydroxylase polypeptides selected from the even-numbered sequences in the following range: SEQ ID NOS: 6-658.

[0181] In some embodiments, an engineered proline hydroxylase polypeptide can have an amino acid sequence that includes a deletion in any one of the engineered proline hydroxylase polypeptide sequences described herein, such as the exemplary engineered polypeptides in the even-numbered sequences in the following range: SEQ ID NOs: 6-658. Thus, in any and all embodiments of the engineered proline hydroxylase polypeptides of the present invention, the amino acid sequence can include a deletion of one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids, eight or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids, up to 20% of the total number of amino acids, or up to 30% of the total number of amino acids of the proline hydroxylase polypeptide, provided that the relevant functional activity and / or improved properties of the engineered proline hydroxylases described herein are maintained. In some embodiments, the deletions can comprise 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, or 1 to 50 amino acid residues. In some embodiments, the number of deletions can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residues. In some embodiments, the deletion may comprise the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24 or 25 amino acid residues.

[0182] In some embodiments, the engineered proline hydroxylase polypeptides herein can have an amino acid sequence that includes an insertion compared to any one of the engineered proline hydroxylase polypeptides described herein, such as the exemplary engineered polypeptides of the even-numbered sequences in the following range: SEQ ID NOS: 6-658. Thus, in any and all embodiments of the proline hydroxylase polypeptides of the present invention, the insertion can comprise one or more amino acids, two or more amino acids, three or more amino acids, four or more amino acids, five or more amino acids, six or more amino acids, eight or more amino acids, ten or more amino acids, fifteen or more amino acids, twenty or more amino acids, thirty or more amino acids, forty or more amino acids, or fifty or more amino acids, provided that the relevant functional activity and / or improved properties of the engineered proline hydroxylases described herein are maintained. The insertion can be to the amino or carboxy terminus or to an internal portion of the proline hydroxylase polypeptide.

[0183] In some embodiments, the engineered proline hydroxylase polypeptides herein can have an amino acid sequence comprising a sequence selected from the even-numbered sequences in the following range: SEQ ID NOS: 6-658, and optionally one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence optionally has 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the substitutions may be conservative or non-conservative.

[0184] In the above-described embodiments, suitable reaction conditions for the engineered polypeptides are provided as described in the Examples.

[0185] In some embodiments, the polypeptides of the invention are fusion polypeptides in which the engineered polypeptide is fused to other polypeptides, such as, by way of example and not limitation, an antibody tag (e.g., a myc epitope), a purification sequence (e.g., a His tag for binding to metals), and a cellular localization signal (e.g., a secretion signal). Thus, the engineered polypeptides described herein can be used with or without fusion to other polypeptides.

[0186] It is understood that the polypeptides described herein are not limited to genetically encoded amino acids. In addition to genetically encoded amino acids, the polypeptides described herein may be composed, in whole or in part, of naturally occurring and / or synthetic non-encoded amino acids. Certain commonly encountered non-encoded amino acids that may comprise the polypeptides described herein are D-stereoisomers of the genetically encoded amino acids; 2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava); N-methylglycine or sarcosine (MeGly or Sar); ornithine (Orn); citrulline (Cit); t-butylalanine (Bua); t-butylglycine (Bug); N-methylisoleucine (MeIle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); naphthylalanine (Nal); 2-chlorophenylalanine (Ocf); 3-chlorophenylalanine (Mcf); 4-chlorophenylalanine (Pcf); 2-fluorophenylalanine (Off); 3-fluorophenylalanine (Mff); 4-fluorophenyl Alanine (Pff); 2-bromophenylalanine (Obf); 3-bromophenylalanine (Mbf); 4-bromophenylalanine (Pbf); 2-methylphenylalanine (Omf); 3-methylphenylalanine (Mmf); 4-methylphenylalanine (Pmf); 2-nitrophenylalanine (Onf); 3-nitrophenylalanine (Mnf); 4-nitrophenylalanine (Pnf); 2-cyanophenylalanine (Ocf); 3-cyanophenylalanine (Mcf); 4-cyanophenylalanine (Pcf); 2-trifluoromethylphenylalanine (Otf); 3-trifluoromethylphenylalanine (Mtf); 4-trifluoromethylphenylalanine (Ptf); 4-aminophenylalanine (Paf); 4-iodophenylalanine (Pif); 4-aminomethylphenylalanine (Pamf); 2,4-dichlorophenylalanine (Opef); 3,4-dichlorophenylalanine (Mpcf);2,4-difluorophenylalanine (Opff); 3,4-difluorophenylalanine (Mpff); pyrid-2-ylalanine (2pAla); pyrid-3-ylalanine (3pAla); pyrid-4-ylalanine (4pAla); naphth-1-ylalanine (1nAla); naphth-2-ylalanine (2nAla); thiazolylalanine (taAla); benzothienylalanine (bAla); thienylalanine (tAla); furylalanine (fAla); homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp); pentafluorophenylalanine (5ff); Styrylalanine (sAla); anthrylalanine (aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenylpentanoic acid (Afp); penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic); β-2-thienylalanine (Thi); methionine sulfoxide (Mso); N(w)-nitroarginine (nArg); homolysine (hLys); phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer); phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutamic acid (glutanic acid) (hGlu); 1-aminocyclopent-(2 or 3)-ene-4-carboxylic acid; pipecolic acid (PA), azetidine-3-carboxylic acid (ACA); 1-aminocyclopentane-3-carboxylic acid; allylglycine (aGly); propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal); homoleucine (hLeu), homovaline (hVal); homoisoleucine (hIle); homoarginine (hArg); N-acetyllysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal); homocysteine (hCys); homoserine (hSer);These include, but are not limited to, hydroxyproline (Hyp) and homoproline (hPro). Additional non-encoded amino acids that may comprise the polypeptides described herein will be apparent to those of skill in the art (see, e.g., Fasman, CRC Practical Handbook of Biochemistry and; (See various amino acids presented in Molecular Biology, CRC Press, Boca Raton, FL, pp. 3-70

[1989] and the references cited therein.) These amino acids can be in either the L- or D-configuration.

[0187] Those skilled in the art will recognize that amino acids or residues bearing side chain protecting groups can also comprise the polypeptides described herein. Non-limiting examples of such protected amino acids, in this case belonging to the aromatic category, include, but are not limited to, Arg(tos), Cys(methylbenzyl), Cys(nitropyridine sulfenyl), Glu(δ-benzyl ester), Gln(xanthyl), Asn(N-δ-xanthyl), His(bom), His(benzyl), His(tos), Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr(O-benzyl), and Tyr(O-benzyl) (protecting groups are listed in parentheses).

[0188] Conformationally constrained non-encoded amino acids that can comprise the polypeptides described herein include, but are not limited to, N-methyl amino acids (L-configuration); 1-aminocyclopent-(2 or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylic acid; homoproline (hPro); and 1-aminocyclopentane-3-carboxylic acid.

[0189] In some embodiments, the engineered polypeptide may be in various forms, such as, for example, an isolated preparation, a substantially purified enzyme, whole cells transformed with a gene(s) encoding the enzyme, and / or cell extracts and / or lysates of such cells, etc. The enzyme may be lyophilized, spray dried, precipitated, or in the form of a crude paste, as further described below.

[0190] In some embodiments, the engineered polypeptide can be provided on a solid support, such as a membrane, resin, solid carrier, or other solid phase material. The solid support can be composed of organic polymers, such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as copolymers and grafts thereof. The solid support can be inorganic, such as glass, silica, controlled pore glass (CPG), reverse-phase silica, or metal, such as gold or platinum. The solid support configuration can be in the form of beads, spheres, particles, granules, gels, membranes, or surfaces. Surfaces can be planar, substantially planar, or non-planar. The solid support can be porous or non-porous and can have swelling or non-swelling characteristics. The solid support can be configured in the form of wells, depressions, or other containers, vessels, features, or locations.

[0191] In some embodiments, engineered polypeptides having prolyl hydroxylase activity of the invention can be immobilized on a solid support so as to retain their improved activity, selectivity, and / or other improved properties relative to the reference polypeptides of SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598, and / or 630. In such embodiments, the immobilized polypeptide can facilitate the biocatalytic conversion of a substrate compound or other suitable substrate to a product and, after the reaction is complete, can be easily retained (e.g., by retaining the beads on which the polypeptide is immobilized) and then reused or recycled in a subsequent reaction. Such immobilized enzyme processes allow for greater efficiency and cost savings. Thus, it is further contemplated that any of the methods using the prolyl hydroxylase polypeptides of the invention can be carried out using the same prolyl hydroxylase polypeptide bound or immobilized to a solid support.

[0192] Enzyme immobilization methods are well known in the art. Engineered polypeptides can be attached by non-covalent or covalent bonds. Various methods for conjugation and immobilization of enzymes to solid supports (e.g., resins, membranes, beads, glass, etc.) are well known in the art (e.g., Yi et al., Proc. Biochem., 42(5): 895-898

[2007] ; Martin et al., Appl. Microbiol. Biotechnol., 76(4): 843-851

[2007] ; Koszelewski et al., J. Mol. Cat. B: Enzymatic, 63: 39-44

[2010] ; Truppo et al., Org. Proc. Res. Dev., published online: dx.doi.org / 10.1021 / op200157c; Hermanson, Bioconjugate Techniques, 2 nded., Academic Press, Cambridge, MA

[2008] ; Mateo et al., Biotechnol. Prog., 18(3):629-34

[2002] ; and "Bioconjugation Protocols: Strategies and Methods," In Methods in Molecular Biology, Niemeyer (ed.), Humana Press, New York, NY

[2004] ; the disclosures of each of which are incorporated herein by reference.) Solid supports useful for immobilizing the engineered prolyl hydroxylases of the invention include, but are not limited to, beads or resins comprising epoxide-functionalized polymethacrylate, aminoepoxide-functionalized polymethacrylate, styrene / DVB copolymer, or octadecyl-functionalized polymethacrylate. Exemplary solid supports useful for immobilizing engineered prolyl hydroxylase polypeptides of the invention include, but are not limited to, chitosan beads, Eupergit C, and SEPABEAD (Mitsubishi), including the following different types of SEPABEAD: EC-EP, EC-HFA / S, EXA252, EXE119, and EXE120.

[0193] In some embodiments, the polypeptides described herein are provided in the form of a kit. The enzymes in the kit can be present individually or as multiple enzymes. The kit can further include reagents for performing the enzymatic reaction, substrates for assessing the activity of the enzyme, and reagents for detecting the product. The kit can also include a reagent dispenser and instructions for using the kit.

[0194] In some embodiments, the kit of the present invention comprises an array comprising a plurality of different prolyl hydroxylase polypeptides at different addressable locations, wherein the different polypeptides are different variants of a reference sequence, each having at least one different improved enzymatic property. In some embodiments, the plurality of polypeptides immobilized on the solid support are configured in an array at various locations that are addressable for robotic reagent delivery or by detection methods and / or instruments. The array can be used to test various substrate compounds for conversion by the polypeptides. Such arrays comprising a plurality of engineered polypeptides and methods for their use are known in the art (see, for example, WO2009 / 008908A2).

[0195] Polynucleotides encoding engineered prolyl hydroxylases, expression vectors, and host cells In another aspect, the present invention provides polynucleotides encoding the engineered proline hydroxylase polypeptides described herein. The polynucleotides can be operably linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. An expression construct containing a heterologous polynucleotide encoding an engineered proline hydroxylase is introduced into a suitable host cell to express the corresponding proline hydroxylase polypeptide.

[0196] As will be apparent to those skilled in the art, the availability of a protein sequence and knowledge of the codons corresponding to various amino acids provides a description of all polynucleotides capable of encoding a polypeptide of interest. The degeneracy of the genetic code, in which the same amino acid is encoded by alternative or synonymous codons, allows for the creation of a vast number of nucleic acids that all encode improved prolyl hydroxylase enzymes. Thus, with knowledge of a specific amino acid sequence, one skilled in the art can create a number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not change the amino acid sequence of the protein. In this regard, the present invention specifically contemplates any and all possible variations of polynucleotides that can be made that encode a polypeptide described herein by selecting combinations based on possible codon choices, and all such variations should be considered to be specifically disclosed for any polypeptide described herein, including the amino acid sequences set out in Tables 4.1, 4.2, 4.3, 4.4, 5.1, 5.2, 5.3, 6.1, 7.1, 7.2, 7.3, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, 11.2 and / or 12.1, and disclosed in the Sequence Listing, which is incorporated herein by reference as the even-numbered sequences in the following range: SEQ ID NOs: 6-658.

[0197] In various embodiments, codons are preferably selected to be compatible with the host cell in which the protein is produced. For example, to express a gene in bacteria, the preferred codons used in bacteria are used; the preferred codons used in yeast are used for expression in yeast; and the preferred codons used in mammals are used for expression in mammalian cells. In some embodiments, to optimize the codon usage of a proline hydroxylase, not all codons need to be replaced, since the native sequence will contain the preferred codons, and since the use of preferred codons for all amino acid residues may not be required. Consequently, a codon-optimized polynucleotide encoding a proline hydroxylase enzyme can contain preferred codons at more than about 40%, 50%, 60%, 70%, 80%, or 90% of the codon positions in the full-length coding region.

[0198] In some embodiments, the polynucleotide comprises a codon-optimized nucleotide sequence encoding a naturally occurring prolyl hydroxylase polypeptide amino acid sequence represented by SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598, and / or 630. In some embodiments, the polynucleotide has a nucleic acid sequence that comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a codon-optimized nucleic acid sequence encoding the even-numbered sequences in the following ranges: SEQ ID NOs: 6-658. In some embodiments, the polynucleotide has a nucleic acid sequence that comprises at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a codon-optimized nucleic acid sequence in the odd-numbered sequences in the following ranges: SEQ ID NOs: 5-657. Codon-optimized sequences of the odd-numbered sequences in the following range: SEQ ID NOS: 5-657 enhance expression of the encoded wild-type proline hydroxylase and provide preparations of the enzyme capable of converting greater than 80% of the compound L-proline to the product compound trans-3-hydroxyproline in vitro under mini-DSP assay conditions, and greater than 45% of the compound L-proline to the product compound trans-3-hydroxyproline under DSP assay conditions. In some embodiments, the codon-optimized polynucleotide sequence can enhance expression of the proline hydroxylase by at least 1.2-fold, 1.5-fold, or 2-fold or more compared to the naturally occurring polynucleotide sequence of ANO from fungal species No. 11243.

[0199] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference sequence selected from the odd-numbered sequences in SEQ ID NOs: 3-657, or its complement, and encodes a polypeptide having proline hydroxylase activity.

[0200] In some embodiments, the polynucleotide encodes an engineered polypeptide having proline hydroxylase activity with one or more improved properties compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630, as described above, wherein the polypeptide is selected from a reference sequence selected from SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630. and one or more residue differences compared to SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598, and / or 630, selected from the even-numbered sequences in the following range: SEQ ID NOs: 6-658. In some embodiments, the reference amino acid sequence is selected from the even-numbered sequences in the following range: SEQ ID NOs: 6-658. In some embodiments, the reference amino acid sequence is SEQ ID NO: 4. In some embodiments, the reference amino acid sequence is SEQ ID NO: 116. In some embodiments, the reference amino acid sequence is SEQ ID NO: 162. In some embodiments, the reference amino acid sequence is SEQ ID NO: 322. In some embodiments, the reference amino acid sequence is SEQ ID NO: 412. In some embodiments, the reference amino acid sequence is SEQ ID NO: 492. In some embodiments, the reference amino acid sequence is SEQ ID NO: 562. In some embodiments, the reference amino acid sequence is SEQ ID NO: 598. In some embodiments, the reference amino acid sequence is SEQ ID NO: 630.

[0201] In some embodiments, the polynucleotide encodes an engineered proline hydroxylase polypeptide capable of converting the substrate compound L-proline to the product compound trans-3-hydroxyproline, the polypeptide having improved enzymatic properties compared to a reference polypeptide of SEQ ID NOs: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630, wherein the polypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95% or more of the enzymatic properties of a reference polypeptide selected from any one of the even-numbered sequences in the range: SEQ ID NOs: 6-658. , 96%, 97%, 98% or 99% identity to a polypeptide sequence of any one of the following ranges: SEQ ID NO: 4, 116, 162, 322, 412, 492, 562, 598 and / or 630 contained in any one of the even-numbered sequences of SEQ ID NO: 6-658, as listed in Tables 4.1, 4.2, 4.3, 4.4, 5.1, 5.2, 5.3, 6.1, 7.1, 7.2, 7.3, 8.1, 8.2, 9.1, 9.2, 10.1, 10.2, 11.1, 11.2 and / or 12.1.

[0202] In some embodiments, the polynucleotide encoding the engineered prolyl hydroxylase comprises a polynucleotide sequence selected from the odd-numbered sequences in the following range: SEQ ID NOs: 5-657.

[0203] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from the odd-numbered sequences in the following range: SEQ ID NOs: 5-657, or its complement, and encodes a polypeptide having proline hydroxylase activity with one or more of the improved properties described herein.

[0204] In some embodiments, polynucleotides capable of hybridizing under highly stringent conditions include amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO:4, as well as 21, 28, 58 / 247, 65, 80, 85, or at residue positions selected from 95, 98, 117, 120, 159, 185, 194, 199, 200, 233, 237, 243, 250, 268, 281, 282, 287, 289, 307, 324, 326, 327, 330, 338, 343, 346 and 348; at residue positions selected from 81, 282, 287, 289, 307, 324, 326, 327, 335, 338, 343 and 346, or at residue positions selected from 48 / 66 / 189 / 194, 48 / 66 / 194 and 66 / 82 / 85 / 135 / 189 / 194 / 267, or 20 / 56 / 76 / 168 / 169 / 296, 20 / 56 / 232 / 294, 20 / 119 / and encoding an engineered polypeptide having proline hydroxylase activity with one or more improved properties, comprising one or more residue differences compared to SEQ ID NO:4 at residue positions selected from 294 / 296, 56 / 76 / 119 / 124 / 147 / 232, 56 / 76 / 294, 76 / 168 / 232 / 294, 76 / 294 / 296, 76 / 296, 147 and 232.

[0205] In some embodiments, polynucleotides capable of hybridizing under highly stringent conditions include amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 116, and at residue positions selected from 123, 189, 195, 233 and 296, or 20 / 21 / 56, 20 / 21 / 56, 20 / 21 / 56, 76 / 95, 232, 294, 307, 335, 20 / 21 / 56 / 76 / 147 / 225 / 232 / 233 / 281 / 294 / 296 / 307 / 335, 20 / 21 / 56 / 95 / 147 / 281 / 294 / 307, 20 / 21 / 56 / 281 / 307, 20 / 21 / 76 / 232 / 243, 20 / 21 / 95 / 232 / 307, 20 / 2 1 / 95 / 281 / 294 / 296, 20 / 21 / 147 / 189 / 233 / 243 / 281 / 307, 20 / 56, 20 / 56 / 76 / 95 / 281 / 307, 20 / 56 / 76 / 147 / 294 / 296 / 307, 20 / 56 / 95 / 147 / 294, 20 / 56 / 28 1, 20 / 76, 20 / 76 / 95 / 281 / 294 / 296, 20 / 76 / 95 / 281 / 296 / 307, 20 / 76 / 233 / 294 / 307, 20 / 76 / 243 / 281 / 294, 21 / 76 / 147 / 233 / 294 / 307, 21 / 76 / 147 / 243 / 2 96 / 307 / 335, 21 / 95 / 185 / 189 / 232 / 281 / 296, 21 / 95 / 233 / 243 / 281 / 296, 21 / 95 / 294 / 296 / 307 / 335, 21 / 95 / 307, 21 / 281 / 307, 29 / 76 / 281, 56 / 76 / 95 / 23 2 / 243 / 281, 56 / 76 / 147 / 281 / 307, 56 / 76 / 243 / 294, 56 / 76 / 281 / 294, 56 / 76 / 296, 56 / 76 / 307, 56 / 95 / 147 / 307 / 335 / 348, 56 / 95 / 232 / 233 / 281 / 294 / 307 , 56 / 95 / 243 / 281, 56 / 147 / 281, 56 / 232 / 243 / 281, 56 / 232 / 281, 56 / 232 / 281 / 294 / 296, 56 / 233 / 281 / 294 / 296, 56 / 281 / 307, 76 / 95 / 232 / 243 / 281 / 307,76 / 95 / 243 / 281 / 307 / 335, 76 / 95 / 294 / 307, 76 / 147, 76 / 147 / 233 / 243 / 294, 76 / 147 / 233 / 281 / 294 / 307, 76 / 147 / 243 / 294 / 296 / 307 / 335, 76 / 147 / 281 / 307, 76 / 189 / 296, 76 / 232 / 233 / 243 / 294 / 296 / 307, 76 / 281, 76 / 281 / 294, 76 / 294 / 296, 95 / 120, 95 / 147 / 335, 95 / 232 / 243 / 281 / 294 / 307, 95 / 232 / 281 / 294 / 296, 95 / 281 / 294 / 296, 95 / 335, 147, 147 / 225 / 232 / 243 / 281 / 296 / 307 / 335, 147 / 233 / 243 / 281 / 307, 147 / 2 Select from 33 / 281 / 307 / 335, 147 / 243 / 281, 147 / 307, 232 / 233 / 281 / 294 / 296 / 307, 232 / 281, 232 / 284 / 307, 233 / 243 / 281 / 296 / 307 / 335, 233 / 281 / 296 / 307, 243 / 281 / 294 / 296, 281, 281 / 294, 281 / 307, 307 and 335 and encoding an engineered polypeptide having proline hydroxylase activity having one or more improved properties, comprising one or more residue differences compared to SEQ ID NO: 116 at residue positions selected from 21 / 76 / 147 / 243 / 296 / 307 / 335, 56 / 76 / 147 / 281 / 307 and 95 / 147 / 335.

[0206] In some embodiments, polynucleotides capable of hybridizing under highly stringent conditions include those having an amino acid sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 162, as well as those having 2 / 85 / 123 / 237, 28 / 115 / 117 / 120 / 123 / 268 / 270 / 343 / 346 / 348, 45 / 123 / 326, 65 / 117 / 120 123 / 139 / 233 / 237 / 281 / 282 / 289 / 324 / 326 and 123 / 199 / 200 / 247 / 250 / 338.

[0207] In some embodiments, polynucleotides capable of hybridizing under highly stringent conditions include those having an amino acid sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 322, and at residue positions selected from 26, 54, 61, 129, 132, 149, 156, 175, 189, 201, 209, 228, 236, 248, 262, 272, 277, 291, and 345, or at residue positions selected from 25, 43, 54, 58, 61, 79, 129, 132, 143, 156, 163, 175 , 179, 201, 209, 236, 248, 278, 291, 345 and 347, or at a residue position selected from 85 / 117 / 120 / 135 / 208 / 270 / 324 / 343 / 346, 85 / 117 / 120 / 135 / 208 / 281 / 282 / 289, 85 / 117 / 120 / 270 / 281 / 289, 85 / 117 / 135 / 139 / 208 and 117 / 120 / 208 / 270 / 324 / 343 / 346.

[0208] In some embodiments, polynucleotides capable of hybridizing under highly stringent conditions include those having an amino acid sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 412, as well as those having 47, 48, 56 / 118, 85, 95, 95 / 289, 113, 118, 118 / 247, 154, 162, 162 / 204, 164, 164 / 198 / 271, 168, 169, 187, 195, 206, 210, 212, 214, 216, 218, 218, 219, 220, 222, 224, 226, 228, 229, 230, 232, 234, 236, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278 and 342, or at a residue position selected from 25 / 129 / 163 / 236 / 262 / 345 / 347, 120 / 156 / 175 / 179 / 201, 129 / 189 / 236 / 262 / 277 / 278, 129 / 236 / 262, 156 / 175 / 179 / 228 and 162.

[0209] In some embodiments, polynucleotides capable of hybridizing under highly stringent conditions include those having an amino acid sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 492, and residues selected from 15, 17, 28, 29, 65, 135, 167, 177, 199, 208, 228, 235, 287, 294, 307, and 343. or at residue positions selected from 85 / 187 / 281 / 347, 85 / 187 / 347, 118 / 120 / 162 / 175 / 179 / 330, 118 / 120 / 162 / 175 / 330, 162 / 175 / 179 / 330, 175 / 228 / 330, 195 / 347 and 278 / 314 / 347,

[0210] In some embodiments, polynucleotides capable of hybridizing under highly stringent conditions include those having an amino acid sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 562, and at residue positions selected from 15, 40, 43, 44, 59, 79, 82, 149, 164, 179, 345 and 347, or 29 / 85 / 177 / 208 / 228 / 347, 29 29 / 177 / 195 / 228 / 343, 29 / 208 / 228 / 278 / 294 / 347, 56 / 195 / 278, 85 / 187 / 205 / 208 / 278, 113 / 177 / 187 / 195 / 208 / 278 / 294 / 343 / 347 and 177 / 205 / 208 / 228.

[0211] In some embodiments, polynucleotides capable of hybridizing under highly stringent conditions include those having an amino acid sequence with at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 598, and those having at least one of the following sequences at residue positions selected from 47, 162, 209, 219, 227 and 342, or 17 / 44 / 179 / 195 / 250 / 313 / 345, 17 / 44 / 199 / 313, 43 / 44 / 195 / 199, 44 / and encoding an engineered polypeptide having proline hydroxylase activity with one or more improved properties, comprising one or more residue differences compared to SEQ ID NO:598 at residue positions selected from 149 / 164 / 171 / 187, 44 / 179 / 195 / 199, 44 / 179 / 195 / 199 / 345, 79 / 163 / 164 / 171 / 187 / 201 / 286 / 288, 82 / 163 / 164, 82 / 163 / 164 / 171 / 187 / 201 / 203 / 208 / 286 / 288 / 320, 149 / 164 / 171 / 288 and 187 / 286.

[0212] In some embodiments, polynucleotides capable of hybridizing under highly stringent conditions include amino acid sequences having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to SEQ ID NO: 630, as well as 82 / 164 / 171 / 203 / 208, 135 / 163 / 164 162 / 236 / 342, 162 / 313 / 342 and 164 / 171 / 201 / 203 / 282.

[0213] In some embodiments, the polynucleotide encodes a polypeptide described herein but has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered prolyl hydroxylase. In some embodiments, the reference polynucleotide sequence is selected from the odd-numbered sequences in the range SEQ ID NOs: 3-657.

[0214] In some embodiments, an isolated polynucleotide encoding any of the engineered prolyl hydroxylase polypeptides provided herein is manipulated in various ways to result in expression of the polypeptide. In some embodiments, the polynucleotide encoding the polypeptide is provided as an expression vector in which one or more regulatory sequences are present to regulate expression of the polynucleotide and / or polypeptide. Depending on the expression vector, it may be desirable or necessary to manipulate the isolated polynucleotide before insertion into a vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art.

[0215] In some embodiments, control sequences include, among other sequences, a promoter, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator. As is known in the art, suitable promoters can be selected based on the host cell used. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present application include promoters obtained from the E. coli lac operon, the Streptomyces coelicolor agarase gene (dagA), the Bacillus subtilis levansucrase gene (sacB), the Bacillus licheniformis alpha-amylase gene (amyL), the Bacillus stearothermophilus maltogenic amylase gene (amyM), the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), the Bacillus licheniformis penicillinase gene (penP), the Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase genes (see, e.g., Villa-Kamaroff et al. al., Proc. Natl Acad. Sci. USA 75: 3727-3731

[1978] ), and the tac promoter (see, e.g., DeBoer et al., Proc. Natl Acad. Sci. USA 80: 21-25

[1983] ). Exemplary promoters for filamentous fungal host cells include promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96 / 00787), and the NA2-tpi promoter (Aspergillus niger neutral alpha-amylase and Aspergillus Exemplary yeast cell promoters include promoters derived from the Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces Examples of promoters that can be used include those derived from the genes for Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (see, for example, Romanos et al., Yeast 8:423-488

[1992] ).

[0216] In some embodiments, the control sequence is a suitable transcription terminator sequence, which is a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in the selected host cell finds use in the present invention. For example, exemplary transcription terminators for filamentous fungal host cells include Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, e.g., Romanos et al., supra).

[0217] In some embodiments, the control sequence is a suitable leader sequence, a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used. Exemplary leaders for filamentous fungal host cells are obtained from the Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase genes. Suitable leaders for yeast host cells include, but are not limited to, the leaders obtained from the Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP) genes.

[0218] The control sequence may be a polyadenylation sequence, which is operably linked to the 3' end of a nucleic acid sequence and, when transcribed, is recognized by the host cell as a signal for adding polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the selected host cell may be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, polyadenylation sequences derived from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art (see, e.g., Guo and Sherman, Mol. Cell. Bio., 15:5983-5990

[1995] ).

[0219] In some embodiments, the control sequence is a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the secretory pathway of a cell. The 5' end of the coding sequence of a nucleic acid sequence can inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence can contain a signal peptide coding region that is foreign to the coding sequence. Any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the engineered prolyl hydroxylase polypeptides provided herein. Effective signal peptide coding regions for bacterial host cells include Bacillus NC1B 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus Examples of signal peptide coding regions include, but are not limited to, signal peptide coding regions obtained from the genes for Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 57:109-137

[1993] ). Useful signal peptide coding regions for filamentous fungal host cells include, but are not limited to, signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to, the signal peptides from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.

[0220] In some embodiments, the control sequence is a propeptide-coding region that encodes an amino acid sequence positioned at the amino terminus of a polypeptide. The resulting polypeptide is sometimes referred to as a "proenzyme," "propolypeptide," or "zymogen." A propolypeptide can be converted to a mature, active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Examples of propeptide-coding regions include, but are not limited to, the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (see, e.g., WO 95 / 33836). When both a signal peptide and a propeptide region are present at the amino terminus of a polypeptide, the propeptide region is located next to the amino terminus of the polypeptide, and the signal peptide region is located next to the amino terminus of the propeptide region.

[0221] In some embodiments, regulatory sequences are also utilized. Such sequences facilitate regulation of polypeptide expression relative to the growth of the host cell. Examples of regulatory systems are those that turn gene expression on or off in response to chemical or physical stimuli, including the presence of regulatory compounds. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to, the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to, the TAKA alpha-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter.

[0222] In another aspect, the present invention also provides recombinant expression vectors comprising a polynucleotide encoding an engineered proline hydroxylase polypeptide and, depending on the type of host into which it is to be introduced, one or more expression control regions, such as a promoter and terminator, an origin of replication, etc. In some embodiments, the various nucleic acid and control sequences described above are combined with each other to produce a recombinant expression vector containing one or more convenient restriction sites that allow for the insertion or substitution of a nucleic acid sequence encoding a variant proline hydroxylase polypeptide at that site. Alternatively, the polynucleotide sequence(s) of the invention are expressed by inserting the polynucleotide sequence or a nucleic acid construct comprising the polynucleotide sequence into an appropriate vector for expression. In creating an expression vector, a coding sequence is placed in a vector such that it is operably linked to appropriate control sequences for expression.

[0223] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can result in expression of the variant proline hydroxylase polynucleotide sequence. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0224] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector can contain any means for ensuring self-replication. In some alternative embodiments, the vector can be a vector that, upon introduction into a host cell, integrates into the genome and is replicated along with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or transposons, which together contain the total DNA to be introduced into the genome of the host cell, can be used.

[0225] In some embodiments, expression vectors preferably contain one or more selectable markers that allow for easy selection of transformed cells. A "selectable marker" is a gene the product of which provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc. Examples of bacterial selectable markers include, but are not limited to, the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and equivalents thereof. In another aspect, the invention provides a host cell comprising a polynucleotide encoding at least one engineered proline hydroxylase polypeptide of the present application, wherein the polynucleotide is operably linked to one or more control sequences for expression of the engineered proline hydroxylase enzyme(s) in the host cell.Host cells for use in expressing the polypeptides encoded by the expression vectors of the present invention are well known in the art and include, but are not limited to, bacterial cells such as E. coli, Vibrio fluvialis, Streptomyces, and Salmonella typhimurium cells; fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris [ATCC Accession No. 201178]); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary host cells are Escherichia coli strains (e.g., W3110(ΔfhuA) and BL21).

[0226] Thus, in another aspect, the invention provides methods for producing an engineered proline hydroxylase polypeptide, the method comprising culturing a host cell capable of expressing a polynucleotide encoding the engineered proline hydroxylase polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the method further comprises isolating and / or purifying the proline hydroxylase polypeptide as described herein.

[0227] Appropriate culture media and growth conditions for the above-mentioned host cells are well known in the art. Polynucleotides for expression of proline hydroxylase polypeptides can be introduced into cells by various methods known in the art. Techniques include, inter alia, electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0228] Engineered proline hydroxylases having the properties disclosed herein can be obtained by subjecting polynucleotides encoding naturally occurring or engineered proline hydroxylase polypeptides to mutagenesis and / or directed evolution methods known in the art and described herein. Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling (see, e.g., Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751

[1994] ; WO95 / 22625; WO97 / 0078; WO97 / 35966; WO98 / 27230; WO00 / 42651; WO01 / 75767 and U.S. Patent No. 6,537,746). Other directed evolution procedures that can be used include, among others, the staggered extension process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 16:258-261

[1998] ), mutagenic PCR (see, e.g., Caldwell et al., PCR Methods Appl., 3:S136-S140

[1994] ), and cassette mutagenesis (see, e.g., Black et al., Proc. Natl. Acad. Sci. USA 93:3525-3529

[1996] ).

[0229] In some embodiments, the engineered proline hydroxylase is obtained by subjecting a polynucleotide encoding a naturally occurring proline hydroxylase to mutagenesis and / or directed evolution methods, as described above. Mutagenesis can be performed according to any of the techniques known in the art, including random and site-specific mutagenesis. Directed evolution can be performed by any of the techniques known in the art for screening for improved promoter variants, including shuffling. Mutagenesis and directed evolution methods are well known in the art (e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6, No. 180,406, No. 6,251,674, No. 6,265,201, No. 6,277,638, No. 6,287,861, No. 6,287,862, No. 6,291,242, No. 6,297 ,053, No. 6,303,344, No. 6,309,883, No. 6,319,713, No. 6,319,714, No. 6,323,030, No. 6,326,204, No. 6,335,16 No. 0, No. 6,335,198, No. 6,344,356, No. 6,352,859, No. 6,355,484, No. 6,358,740, No. 6,358,742, No. 6,365,377 , No. 6,365,408, No. 6,368,861, No. 6,372,497, No. 6,337,186, No. 6,376,246, No. 6,379,964, No. 6,387,702, No. No. 6,391,552, No. 6,391,640, No. 6,395,547, No. 6,406,855, No. 6,406,910, No. 6,413,745, No. 6,413,774, No. 6,4 No. 20,175, No. 6,423,542, No. 6,426,224, No. 6,436,675, No. 6,444,468, No. 6,455,253, No. 6,479,652, No. 6,482,647, 6,483,011, 6,484,105, 6,489,146, 6,500,617, 6, 500,639, 6,506,602, 6,506,603, 6,518,065, 6,519,065, No. 6,521,453, No. 6,528,311, No. 6,537,746, No. 6,573,098, No. 6,576,467 No. 6,579,678, No. 6,586,182, No. 6,602,986, No. 6,605,430, No. 6,613, No. 514, No. 6,653,072, No. 6,686,515, No. 6,703,240, No. 6,716,631, No. 6, 825,001, 6,902,922, 6,917,882, 6,946,296, 6,961,664, No. 6,995,017, No. 7,024,312, No. 7,058,515, No. 7,105,297, No. 7,148,054 No. 7,220,566, No. 7,288,375, No. 7,384,387, No. 7,421,347, No. 7,430, No. 477, No. 7,462,469, No. 7,534,564, No. 7,620,500, No. 7,620,502, No. 7, 629,170, 7,702,464, 7,747,391, 7,747,393, 7,751,986, No. 7,776,598, No. 7,783,428, No. 7,795,030, No. 7,853,410, No. 7,868,138 No. 7,783,428, No. 7,873,477, No. 7,873,499, No. 7,904,249, No. 7,957, 912, 7,981,614, 8,014,961, 8,029,988, 8,048,674, 8,058,001, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, and any relevant non-US counterparts; Ling et al.,Anal. Biochem., 254(2):157-78

[1997] ; Dale et al., Meth. Mol. Biol., 57:369-74

[1996] ; Smith, Ann. Rev. Genet., 19:423-462

[1985] ; Botstein et al., Science, 229:1193-1201

[1985] ; Carter, Biochem. J., 237:1-7

[1986] ; Kramer et al., Cell, 38:879-887

[1984] ; Wells et al., Gene, 34:315-323

[1985] ; Minshull et al., Curr. Op. Chem. Biol., 3: 284-290

[1999] ; Christians et al., Nat. Biotechnol., 17: 259-264

[1999] ; Crameri et al., Nature, 391: 288-291

[1998] ; Crameri, et al., Nat. Biotechnol., 15:436-438

[1997] ; Zhang et al., Proc. Nat. Acad. Sci. U.S.A., 94:4504-4509

[1997] ; Crameri et al., Nat. Biotechnol., 14:315-319

[1996] ; see Stemmer, Nature, 370:389-391

[1994] ; Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751

[1994] ; WO95 / 22625; WO97 / 0078; WO97 / 35966; WO98 / 27230; WO00 / 42651; WO01 / 75767; and WO2009 / 152336).

[0230] In some embodiments, enzyme clones obtained after mutagenesis treatment are screened by subjecting the enzyme to defined temperatures (or other assay conditions, such as testing the activity of the enzyme across a wide range of substrates) and measuring the amount of enzyme activity remaining after the heat treatment or other assay conditions. Clones containing polynucleotides encoding prolyl hydroxylase polypeptides are then sequenced to identify nucleotide sequence changes, if any, that are used to express the enzyme in host cells. Measurement of enzyme activity from expression libraries can be performed using any suitable method known in the art (e.g., standard biochemical techniques, such as HPLC analysis).

[0231] In some embodiments, clones obtained after mutagenesis can be screened for engineered prolyl hydroxylases with one or more desired improved enzymatic properties (e.g., improved regioselectivity). Measurement of enzyme activity from expression libraries can be performed using standard biochemical techniques, such as HPLC analysis and / or derivatization (before or after separation) of products with, for example, dansyl chloride or OPA (see, e.g., Yaegaki et al., J. Chromatogr. 356(1):163-70

[1986] ).

[0232] If the sequence of the engineered polypeptide is known, polynucleotides encoding the enzyme can be prepared by standard solid-phase methods according to known synthesis methods. In some embodiments, fragments of up to about 100 bases can be synthesized individually and then linked (e.g., by enzymatic or chemical ligation methods, or polymerase-mediated methods) to form any desired contiguous sequence. For example, polynucleotides and oligonucleotides encoding portions of prolyl hydroxylase can be prepared by chemical synthesis known in the art (e.g., the classical phosphoramidite method of Beaucage et al., Tet. Lett. 22:1859-69

[1981] or the method described by Matthes et al., EMBO J. 3:801-05

[1984] ), as typically performed in automated synthesis methods. According to the phosphoramidite method, oligonucleotides are synthesized (e.g., in an automated DNA synthesizer), purified, annealed, ligated, and cloned into an appropriate vector. In addition, essentially any nucleic acid can be obtained from any of a variety of commercial sources. In some embodiments, additional variations can be created by synthesizing oligonucleotides containing deletions, insertions, and / or substitutions and combining the oligonucleotides in various permutations to create engineered prolyl hydroxylases with one or more improved properties.

[0233] Thus, in some embodiments, methods for preparing an engineered proline hydroxylase polypeptide include (a) an amino acid sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity to an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, including 21, 28, 58 / 247, 65, 80, 85, 95, 98, 117, 118, 120, 122, 124, 126, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 1 and (b) synthesizing a polynucleotide encoding a polypeptide having one or more residue differences compared to SEQ ID NO:4 at residue positions selected from 20, 159, 185, 194, 199, 200, 233, 237, 243, 250, 268, 281, 282, 287, 289, 307, 324, 326, 327, 330, 338, 343, 346, and 348; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0234] Thus, in some embodiments, methods for preparing an engineered proline hydroxylase polypeptide include (a) an amino acid sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity to an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, including 21, 28, 45, 65, 95, 112, 116, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 18 and (b) synthesizing a polynucleotide encoding a polypeptide having one or more residue differences compared to SEQ ID NO:4 at residue positions selected from 117, 139, 177, 185, 199, 233, 243, 250, 281, 282, 287, 289, 307, 324, 326, 327, 335, 338, 343, and 346; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0235] Thus, in some embodiments, methods for preparing an engineered proline hydroxylase polypeptide comprise: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, and having one or more residue differences compared to SEQ ID NO: 4 at residue positions selected from 48 / 66 / 189 / 194, 48 / 66 / 194, and 66 / 82 / 85 / 135 / 189 / 194 / 267; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0236] Thus, in some embodiments, methods for preparing an engineered proline hydroxylase polypeptide include (a) an amino acid sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity to an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, including 20 / 56 / 76 / 168 / 169 / 296, 20 / 56 / 232 / and (b) synthesizing a polynucleotide encoding a polypeptide having one or more residue differences compared to SEQ ID NO:4 at residue positions selected from: 294, 20 / 119 / 294 / 296, 56 / 76 / 119 / 124 / 147 / 232, 56 / 76 / 294, 76 / 168 / 232 / 294, 76 / 294 / 296, 76 / 296, 147 and 232; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0237] Thus, in some embodiments, methods for preparing an engineered proline hydroxylase polypeptide comprise: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, and having one or more residue differences compared to SEQ ID NO: 116 at residue positions selected from 123, 189, 195, 233, and 296; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0238] Thus, in some embodiments, methods for preparing an engineered proline hydroxylase polypeptide include (a) an engineered proline hydroxylase polypeptide comprising an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, including: 20 / 21 / 56, 20 / 21 / 56 / 76 / 95 / 232 / 294 / 307 / 335, 20 / 21 / 56 / 76 / 147 / 225 / 232 / 233 / 281 / 294 / 296 / 307 / 335, 20 / 21 / 56 / 95 / 147 / 281 / 294 / 307, 20 / 21 / 56 / 281 / 307, 20 / 21 / 76 / 232 / 243, 20 / 21 / 95 / 232 / 307, 20 / 21 / 95 / 281 / 294 / 296, 20 / 21 / 147 / 189 / 233 / 243 / 281 / 307, 20 / 56, 20 / 56 / 76 / 95 / 281 / 307, 20 / 56 / 76 / 147 / 294 / 296 / 307, 20 / 56 / 95 / 147 / 294, 2 0 / 56 / 281, 20 / 76, 20 / 76 / 95 / 281 / 294 / 296, 20 / 76 / 95 / 281 / 296 / 307, 20 / 76 / 233 / 294 / 307, 20 / 76 / 243 / 281 / 294, 21 / 76 / 147 / 233 / 294 / 307, 21 / 76 / 147 / 243 / 296 / 307 / 335, 21 / 95 / 185 / 189 / 232 / 281 / 296, 21 / 95 / 233 / 243 / 281 / 296, 21 / 95 / 294 / 296 / 307 / 335, 21 / 95 / 307, 21 / 281 / 307, 29 / 76 / 281, 56 / 76 / 95 / 232 / 243 / 281, 56 / 76 / 147 / 281 / 307, 56 / 76 / 243 / 294, 56 / 76 / 281 / 294, 56 / 76 / 296, 56 / 76 / 307, 56 / 95 / 147 / 307 / 335 / 348, 56 / 95 / 232 / 233 / 281 / 294 / 30 7, 56 / 95 / 243 / 281, 56 / 147 / 281, 56 / 232 / 243 / 281, 56 / 232 / 281, 56 / 232 / 281 / 294 / 296, 56 / 233 / 281 / 294 / 296, 56 / 281 / 307, 76 / 95 / 232 / 243 / 281 / 307, 7 6 / 95 / 243 / 281 / 307 / 335, 76 / 95 / 294 / 307, 76 / 147, 76 / 147 / 233 / 243 / 294, 76 / 147 / 233 / 281 / 294 / 307, 76 / 147 / 243 / 294 / 296 / 307 / 335, 76 / 147 / 281 / 307,76 / 189 / 296, 76 / 232 / 233 / 243 / 294 / 296 / 307, 76 / 281, 76 / 281 / 294, 76 / 294 / 296, 95 / 120, 95 / 147 / 335, 95 / 232 / 243 / 281 / 294 / 307, 95 / 232 / 281 / 294 / 29 6, 95 / 281 / 294 / 296, 95 / 335, 147, 147 / 225 / 232 / 243 / 281 / 296 / 307 / 335, 147 / 233 / 243 / 281 / 307, 147 / 233 / 281 / 307 / 335, 147 / 243 / 281, 147 / 307, 232 / 23 3 / 281 / 294 / 296 / 307, 232 / 281, 232 / 284 / 307, 233 / 243 / 281 / 296 / 307 / 335, 233 / 281 / 296 / 307, 243 / 281 / 294 / 296, 281, 281 / 294, 281 / 307, 307 and 335; and (b) synthesizing a polynucleotide encoding a polypeptide having one or more residue differences compared to SEQ ID NO: 116 at residue positions selected from: 3 / 281 / 294 / 296 / 307, 232 / 281, 232 / 284 / 307, 233 / 243 / 281 / 296 / 307 / 335, 233 / 281 / 296 / 307, 243 / 281 / 294 / 296, 281, 281 / 294, 281 / 307, 307 and 335; and (b) expressing the prolyl hydroxylase polypeptide encoded by the polynucleotide.

[0239] Thus, in some embodiments, a method for preparing an engineered proline hydroxylase polypeptide comprises: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, and having one or more residue differences compared to SEQ ID NO: 116 at residue positions selected from 21 / 76 / 147 / 243 / 296 / 307 / 335, 56 / 76 / 147 / 281 / 307, and 95 / 147 / 335; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0240] Thus, in some embodiments, methods for preparing an engineered proline hydroxylase polypeptide include (a) an engineered proline hydroxylase polypeptide comprising an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, including 2 / 85 / 123 / 237, 28 / 115 / 117 / 120 / 123 / 268 / 270 / 343 / 346 / 348, 45 / 123 / 326, 65 / 117 / 120 / 123 / 343 / 346, 85 / 123 / 281 / 282, 114 / 115 / 117 / 120 / 123 / 26 and (b) synthesizing a polynucleotide encoding a polypeptide having one or more residue differences compared to SEQ ID NO: 162 at residue positions selected from 8 / 271 / 313 / 326 / 343 / 346, 123 / 139 / 233 / 237 / 281 / 282 / 289 / 324 / 326 and 123 / 199 / 200 / 247 / 250 / 338; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0241] Thus, in some embodiments, methods for preparing an engineered proline hydroxylase polypeptide include (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, and having one or more residue differences compared to SEQ ID NO: 322 at residue positions selected from 26, 54, 61, 129, 132, 149, 156, 175, 189, 201, 209, 228, 236, 248, 262, 272, 277, 291, and 345; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0242] Thus, in some embodiments, methods for preparing an engineered proline hydroxylase polypeptide comprise: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, and having one or more residue differences compared to SEQ ID NO: 322 at residue positions selected from 25, 43, 54, 58, 61, 79, 129, 132, 143, 156, 163, 175, 179, 201, 209, 236, 248, 278, 291, 345, and 347; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0243] Thus, in some embodiments, a method for preparing an engineered proline hydroxylase polypeptide comprises: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, and having one or more residue differences compared to SEQ ID NO: 322 at residue positions selected from 85 / 117 / 120 / 135 / 208 / 270 / 324 / 343 / 346, 85 / 117 / 120 / 135 / 208 / 281 / 282 / 289, 85 / 117 / 120 / 270 / 281 / 289, 85 / 117 / 135 / 139 / 208 and 117 / 120 / 208 / 270 / 324 / 343 / 346; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0244] Thus, in some embodiments, methods for preparing an engineered proline hydroxylase polypeptide comprise: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, and having one or more residue differences compared to SEQ ID NO: 412 at residue positions selected from: 47, 48, 56 / 118, 85, 95, 95 / 289, 113, 118, 118 / 247, 154, 162, 162 / 204, 164, 164 / 198 / 271, 168, 169, 187, 195, 243, 271, 275, 281, 314, 330, and 342; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0245] Thus, in some embodiments, a method for preparing an engineered proline hydroxylase polypeptide comprises: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, and having one or more residue differences compared to SEQ ID NO: 412 at residue positions selected from 25 / 129 / 163 / 236 / 262 / 345 / 347, 120 / 156 / 175 / 179 / 201, 129 / 189 / 236 / 262 / 277 / 278, 129 / 236 / 262, 156 / 175 / 179 / 228, and 162; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0246] Thus, in some embodiments, a method for preparing an engineered proline hydroxylase polypeptide comprises: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, and having one or more residue differences compared to SEQ ID NO: 492 at residue positions selected from 15, 17, 28, 29, 65, 135, 167, 177, 199, 208, 228, 235, 287, 294, 307, and 343; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0247] Thus, in some embodiments, a method for preparing an engineered proline hydroxylase polypeptide comprises: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, and having one or more residue differences compared to SEQ ID NO: 492 at residue positions selected from 85 / 187 / 281 / 347, 85 / 187 / 347, 118 / 120 / 162 / 175 / 179 / 330, 118 / 120 / 162 / 175 / 330, 162 / 175 / 179 / 330, 175 / 228 / 330, 195 / 347, and 278 / 314 / 347; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0248] Thus, in some embodiments, a method for preparing an engineered proline hydroxylase polypeptide comprises: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, and having one or more residue differences compared to SEQ ID NO: 562 at residue positions selected from 15, 40, 43, 44, 59, 79, 82, 149, 164, 179, 345, and 347; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0249] Thus, in some embodiments, methods for preparing an engineered proline hydroxylase polypeptide include (a) an engineered proline hydroxylase polypeptide comprising an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, including: 29 / 85 / 177 / 208 / 228 / 347, 29 / 85 / 208 / 228 / 343 / 347, 29 / 177 / 195 / 228 / 343, 29 / 208 / 228 / 278 / 294 / 347, 56 / 195 / 278, 85 / 187 / 2 and (b) synthesizing a polynucleotide encoding a polypeptide having one or more residue differences compared to SEQ ID NO:562 at residue positions selected from 05 / 208 / 278, 113 / 177 / 187 / 195 / 208 / 278 / 294 / 343 / 347 and 177 / 205 / 208 / 228; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0250] Thus, in some embodiments, a method for preparing an engineered proline hydroxylase polypeptide comprises: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, and having one or more residue differences compared to SEQ ID NO: 598 at residue positions selected from 47, 162, 209, 219, 227, and 342; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0251] Thus, in some embodiments, methods for preparing an engineered proline hydroxylase polypeptide include (a) an engineered proline hydroxylase polypeptide comprising an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, including: 17 / 44 / 179 / 195 / 250 / 313 / 345, 17 / 44 / 199 / 313, 43 / 44 / 195 / 199, 44 / 149 / 164 / 171 / 187, 44 / 179 / 195 / 199, 44 / 179 / 195 / 199 / 345, 79 / 163 / 164 / 171 / 187 / 201 163 / 164, 171 / 187 / 201 / 203 / 208 / 286 / 288 / 320, 149 / 164 / 171 / 288 and 187 / 286; and (b) synthesizing a polynucleotide encoding a polypeptide having one or more residue differences compared to SEQ ID NO:598 at residue positions selected from: 163 / 164, 171 / 187 / 201 / 203 / 208 / 286 / 288 / 320, 149 / 164 / 171 / 288 and 187 / 286; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0252] Thus, in some embodiments, a method for preparing an engineered proline hydroxylase polypeptide comprises: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-658, and having one or more residue differences compared to SEQ ID NO: 630 at residue positions selected from 82 / 164 / 171 / 203 / 208, 135 / 163 / 164 / 201 / 203 / 208, 162, 162 / 219 / 236, 162 / 219 / 313 / 338, 162 / 236 / 342, 162 / 313 / 342, and 164 / 171 / 201 / 203 / 282; and (b) expressing the proline hydroxylase polypeptide encoded by the polynucleotide.

[0253] In some embodiments of the method, the polynucleotide encodes an engineered proline hydroxylase, optionally having one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence optionally has 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, or 1 to 50 amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the substitutions may be conservative or non-conservative.

[0254] In some embodiments, any of the engineered prolyl hydroxylase enzymes expressed in host cells can be recovered from the cells and / or culture medium using any one or more of well-known techniques for protein purification, including lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography, among others. Suitable solutions for lysis of bacteria such as E. coli and highly efficient extraction of proteins from bacteria such as E. coli are commercially available (e.g., CelLytic B™, Sigma-Aldrich, St. Louis, MO).

[0255] Chromatographic techniques for isolation of prolyl hydroxylase polypeptides include, among others, reverse-phase chromatography, high performance liquid chromatography, ion-exchange chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those skilled in the art.

[0256] In some embodiments, affinity techniques can be used to isolate improved prolyl hydroxylase enzymes. For affinity chromatography purification, any antibody that specifically binds to a prolyl hydroxylase polypeptide can be used. For antibody production, various host animals, including but not limited to rabbits, mice, rats, etc., can be immunized by injection with a prolyl hydroxylase polypeptide or a fragment thereof. The prolyl hydroxylase polypeptide or fragment can be attached to a suitable support, such as BSA, using a side chain functional group or a linker attached to the side chain functional group. In some embodiments, affinity purification can use a specific ligand bound by the prolyl hydroxylase, such as poly(L-proline) or a dye affinity column (see, e.g., EP 0 641 862; Stellwagen, "Dye Affinity Chromatography," In Current Protocols in Protein Science, Unit 9.2-9.2.16

[2001] ).

[0257] Methods of Using Engineered Prolyl Hydroxylase Enzymes In some embodiments, the proline hydroxylases described herein find use in processes for converting a suitable substrate to its hydroxylated product. Generally, a process for carrying out a hydroxylation reaction involves contacting or incubating a substrate compound with a proline hydroxylase polypeptide of the invention in the presence of a co-substrate, such as α-ketoglutarate, under reaction conditions suitable for the formation of a hydroxylated product, as shown in Scheme 1 above.

[0258] In the embodiments provided herein and illustrated in the Examples, a wide range of suitable reaction conditions can be used in the process, including, but not limited to, substrate loading, co-substrate loading, reducing agent, divalent transition metal, pH, temperature, buffer, solvent system, polypeptide loading, and reaction time. Further suitable reaction conditions for carrying out processes for the biocatalytic conversion of substrate compounds to product compounds using the engineered prolyl hydroxylase polypeptides described herein can be readily optimized in view of the guidance provided herein by routine experimentation, including, but not limited to, contacting the engineered prolyl hydroxylase polypeptide and substrate compound under experimental reaction conditions of concentration, pH, temperature, and solvent conditions, and detecting the product compound.

[0259] Suitable reaction conditions using engineered proline hydroxylase polypeptides typically include a co-substrate used stoichiometrically in the hydroxylation reaction. Generally, the co-substrate for proline hydroxylase is α-ketoglutarate, also known as α-ketoglutarate and 2-oxoglutarate. Other analogs of α-ketoglutarate that can serve as a co-substrate for proline hydroxylase can be used. An exemplary analog that can serve as a co-substrate is α-oxoadipate. Because the co-substrate is used stoichiometrically, the co-substrate is present in an equimolar or greater amount than the substrate compound (i.e., the molar concentration of the co-substrate is equal to or greater than the molar concentration of the substrate compound). In some embodiments, suitable reaction conditions include a molar concentration of the co-substrate that is at least 1, 1.5, 2, 3, 4, or 5 times or more the molar concentration of the substrate compound. In some embodiments, suitable reaction conditions can include a co-substrate concentration, particularly alpha-ketoglutarate, of about 0.001 M to about 2 M, 0.01 M to about 2 M, 0.1 M to about 2 M, 0.2 M to about 2 M, about 0.5 M to about 2 M, or about 1 M to about 2 M. In some embodiments, reaction conditions include a co-substrate concentration of about 0.001, 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 1, 1.5, or 2 M. In some embodiments, additional co-substrate can be added during the reaction.

[0260] The substrate compound in the reaction mixture can be varied, taking into account, for example, the desired amount of product compound, the effect of substrate concentration on enzyme activity, the stability of the enzyme under the reaction conditions, and the percent conversion of substrate to product. In some embodiments, suitable reaction conditions include a substrate compound load of at least about 0.5 to about 200 g / L, 1 to about 200 g / L, 5 to about 150 g / L, about 10 to about 100 g / L, 20 to about 100 g / L, or about 50 to about 100 g / L. In some embodiments, suitable reaction conditions include a substrate compound load of at least about 0.5 g / L, at least about 1 g / L, at least about 5 g / L, at least about 10 g / L, at least about 15 g / L, at least about 20 g / L, at least about 30 g / L, at least about 50 g / L, at least about 75 g / L, at least about 100 g / L, at least about 150 g / L, or at least about 200 g / L or even greater. The substrate loading values provided herein are based on the molecular weight of L-proline, however, it is also contemplated that equimolar amounts of various hydrates and salts of L-proline can also be used in the process.

[0261] In carrying out the prolyl hydroxylase-mediated processes described herein, the engineered polypeptide can be added to the reaction mixture in the form of a purified enzyme, a partially purified enzyme, whole cells transformed with a gene(s) encoding the enzyme, as a cell extract and / or lysate of such cells, and / or as an enzyme immobilized on a solid support. Whole cells transformed with a gene(s) encoding the engineered prolyl hydroxylase enzyme, or the cell extracts, lysates, and isolated enzymes thereof, can be used in a variety of different forms, including solids (e.g., lyophilized, spray-dried, etc.) or semi-solids (e.g., crude pastes). Cell extracts or cell lysates can be partially purified by precipitation (ammonium sulfate, polyethyleneimine, heat treatment, etc.) followed by a desalting procedure (e.g., ultrafiltration, dialysis, etc.) prior to lyophilization. Any of the enzyme preparations (including whole cell preparations) can be stabilized, for example, by cross-linking using known cross-linking agents such as glutaraldehyde, or by immobilization on a solid phase (e.g., Eupergit C, etc.).

[0262] The gene(s) encoding the engineered proline hydroxylase polypeptides can be transformed into host cells separately or together in the same host cell. For example, in some embodiments, one set of host cells can be transformed with gene(s) encoding one engineered proline hydroxylase polypeptide, and another set can be transformed with gene(s) encoding another engineered proline hydroxylase polypeptide. Both sets of transformed cells can be used together in a reaction mixture in the form of whole cells or in the form of a lysate or extract obtained therefrom. In other embodiments, host cells can be transformed with gene(s) encoding multiple engineered proline hydroxylase polypeptides. In some embodiments, the engineered polypeptides can be expressed in the form of secreted polypeptides, and the culture medium containing the secreted polypeptides can be used for the proline hydroxylase reaction.

[0263] In some embodiments, the improved activity and / or selectivity of the engineered prolyl hydroxylase polypeptides disclosed herein provides processes in which higher percentage conversion can be achieved with lower concentrations of the engineered polypeptide. In some embodiments of the process, suitable reaction conditions include an amount of engineered polypeptide of about 1% (w / w), 2% (w / w), 5% (w / w), 10% (w / w), 20% (w / w), 30% (w / w), 40% (w / w), 50% (w / w), 75% (w / w), 100% (w / w) or more of the substrate compound load.

[0264] In some embodiments, the engineered polypeptide is present at about 0.01 g / L to about 50 g / L; about 0.05 g / L to about 50 g / L; about 0.1 g / L to about 40 g / L; about 1 g / L to about 40 g / L; about 2 g / L to about 40 g / L; about 5 g / L to about 40 g / L; about 5 g / L to about 30 g / L; about 0.1 g / L to about 10 g / L; about 0.5 g / L to about 10 g / L; about 1 g / L to about 10 g / L; about 0.1 g / L to about 5 g / L; about 0.5 g / L to about 5 g / L; or about 0.1 g / L to about 2 g / L. In some embodiments, the proline hydroxylase polypeptide is present at about 0.01 g / L, 0.05 g / L, 0.1 g / L, 0.2 g / L, 0.5 g / L, 1, 2 g / L, 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L or 50 g / L.

[0265] In some embodiments, the reaction conditions also include a divalent transition metal that can act as a cofactor in the oxidation reaction. Generally, the divalent transition metal cofactor is a divalent iron ion (i.e., Fe +2). The divalent iron ion can be provided in various forms, such as ferrous sulfate (FeSO4), ferrous chloride (FeCl2), ferrous carbonate (FeCO3), and salts of organic acids such as citrate, lactate, and fumarate. An exemplary source of ferrous sulfate is Mohr's salt, which is ferrous ammonium sulfate (NH4)2Fe(SO4)2, available in anhydrous and hydrated (i.e., hexahydrate) forms. Divalent iron ion is the transition metal cofactor found in naturally occurring prolyl hydroxylases and functions efficiently in engineered enzymes, although it should be understood that other divalent transition metals capable of acting as cofactors can be used in the process. In some embodiments, the divalent transition metal cofactor is Mn +2 and Cr +2 In some embodiments, the reaction conditions may include a divalent transition metal cofactor, particularly Fe, at a concentration of about 0.1 mM to 10 mM, 0.1 mM to about 5 mM, 0.5 mM to about 5 mM, about 0.5 mM to about 3 mM, or about 1 mM to about 2 mM. +2 In some embodiments, the reaction conditions include a divalent transition metal cofactor concentration of about 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 1.5 mM, 2 mM, 3 mM, 5 mM, 7.5 mM, or 10 mM. In some embodiments, higher concentrations of divalent transition metal cofactor can be used, for example, up to 50 mM or up to 100 mM.

[0266] In some embodiments, the reaction conditions include the addition of ferric ions, Fe +3 Divalent iron ion, Fe +2 The enzyme may further comprise a reducing agent capable of reducing the enzyme to hydroxylation reaction. In some embodiments, the reducing agent comprises ascorbic acid, typically L-ascorbic acid. While ascorbic acid is not required for the hydroxylation reaction, enzyme activity is enhanced in its presence. Without being bound by theory, ascorbate acts as a catalyst to reduce enzyme-Fe, which is the active form that mediates the hydroxylation reaction. +2It is believed that the morphology is maintained or restored. Generally, reaction conditions can include a corresponding ascorbic acid concentration proportional to the substrate load. In some embodiments, the ascorbic acid is present at a molar concentration of at least about 0.1, 0.2, 0.3, 0.5, 0.75, 1, 1.5, or at least 2 times the molar amount of the substrate. In some embodiments, the reducing agent, particularly L-ascorbic acid, is at a concentration of about 0.001 M to about 0.5 M, about 0.01 M to about 0.5 M, about 0.01 M to about 0.4 M, about 0.1 to about 0.4 M, or about 0.1 to about 0.3 M. In some embodiments, the reducing agent, particularly ascorbic acid, is at a concentration of about 0.001M, 0.005M, 0.01M, 0.02M, 0.03M, 0.05M, 0.1M, 0.15M, 0.2M, 0.3M, 0.4M, or 0.5M.

[0267] In some embodiments, the reaction conditions include molecular oxygen (i.e., O). Without being bound by theory, one oxygen atom of the molecular oxygen is incorporated into the substrate compound to form a hydroxylated product compound. O can be naturally present in the reaction solution or can be artificially introduced and / or supplemented into the reaction. In some embodiments, the reaction conditions can include forced aeration (e.g., sparging) with air, O gas, or other O-containing gas. In some embodiments, O can be increased in the reaction by increasing the pressure of the reaction with O or an O-containing gas. This can be done by carrying out the reaction in a vessel that can be pressurized with O gas. In some embodiments, O gas can be sparged through the reaction solution at a rate of at least 1 liter per hour (L / h), at least 2 L / h, at least 3 L / h, at least 4 L / h, at least 5 L / h, or faster. In some embodiments, O2 gas can be sparged through the reaction solution at a rate of between about 1 L / h and 10 L / h, between about 2 L / h and 7 L / h, or between about 3 L / h and 5 L / h.

[0268] During the course of the reaction, the pH of the reaction mixture may change. The pH of the reaction mixture can be maintained at a desired pH or within a desired pH range. This can be done by adding an acid or base before and / or during the course of the reaction. Alternatively, the pH can be controlled by using a buffer. Thus, in some embodiments, the reaction conditions include a buffer. Suitable buffers for maintaining a desired pH range are known in the art and include, by way of example and not limitation, borate, phosphate, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), acetate, triethanolamine, and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), among others. In some embodiments, the buffer is a phosphate. In some embodiments of the process, suitable reaction conditions include a buffer (e.g., phosphate) concentration of about 0.01 to about 0.4 M, 0.05 to about 0.4 M, 0.1 to about 0.3 M, or about 0.1 to about 0.2 M. In some embodiments, the reaction conditions include a buffer (e.g., phosphate) concentration of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.07, 0.1, 0.12, 0.14, 0.16, 0.18, 0.2, 0.3, or 0.4 M. In some embodiments, the reaction conditions include water as a suitable solvent and no buffer is present.

[0269] In process embodiments, reaction conditions can include a suitable pH. The desired pH or desired pH range can be maintained by the use of an acid or base, a suitable buffer, or a combination of buffering and acid or base addition. The pH of the reaction mixture can be controlled before and / or during the course of the reaction. In some embodiments, suitable reaction conditions include a solution pH of about 4 to about 10, a pH of about 5 to about 10, a pH of about 5 to about 9, a pH of about 6 to about 9, or a pH of about 6 to about 8. In some embodiments, reaction conditions include a solution pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.

[0270] In embodiments of the processes herein, suitable temperatures for the reaction conditions can be used, taking into account, for example, increased reaction rates at higher temperatures and enzyme activity during the reaction. Thus, in some embodiments, suitable reaction conditions include temperatures of about 10°C to about 60°C, about 10°C to about 55°C, about 15°C to about 60°C, about 20°C to about 60°C, about 20°C to about 55°C, about 25°C to about 55°C, or about 30°C to about 50°C. In some embodiments, suitable reaction conditions include temperatures of about 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C. In some embodiments, the temperature during the enzymatic reaction can be maintained at a specific temperature throughout the course of the reaction. In some embodiments, the temperature during the enzymatic reaction can be adjusted over a temperature profile during the course of the reaction.

[0271] The process of the present invention is generally carried out in a solvent. Suitable solvents include water, aqueous buffer solutions, organic solvents, polymer solvents, and / or co-solvent systems that generally include an aqueous solvent, an organic solvent, and / or a polymer solvent. The aqueous solvent (water or aqueous co-solvent system) may be pH-buffered or unbuffered. In some embodiments, processes using engineered prolyl hydroxylase polypeptides can be carried out in aqueous co-solvent systems that include organic solvents (e.g., ethanol, isopropanol (IPA), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl tert-butyl ether (MTBE), toluene, etc.), ionic or polar solvents (e.g., 1-ethyl 4-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl 3-methylimidazolium hexafluorophosphate, glycerol, polyethylene glycol, etc.). In some embodiments, the co-solvent may be a polar solvent, such as a polyol, dimethyl sulfoxide (DMSO), or a lower alcohol. The non-aqueous co-solvent component of the aqueous co-solvent system may be miscible with the aqueous component, resulting in a single liquid phase, or may be partially miscible or immiscible with the aqueous component, resulting in two liquid phases. An exemplary aqueous co-solvent system may include water and one or more co-solvents selected from organic solvents, polar solvents, and polyol solvents. Generally, the co-solvent components of the aqueous co-solvent system are selected so as not to adversely inactivate the prolyl hydroxylase enzyme under the reaction conditions. Suitable co-solvent systems can be readily identified by measuring the enzymatic activity of a designated engineered prolyl hydroxylase enzyme in a candidate solvent system using a defined substrate of interest, using an enzyme activity assay, such as those described herein.

[0272] In some process embodiments, suitable reaction conditions include an aqueous co-solvent comprising DMSO at about 1% to about 50% (v / v), about 1 to about 40% (v / v), about 2% to about 40% (v / v), about 5% to about 30% (v / v), about 10% to about 30% (v / v), or about 10% to about 20% (v / v). In some process embodiments, suitable reaction conditions include an aqueous co-solvent comprising DMSO at about 1% (v / v), about 5% (v / v), about 10% (v / v), about 15% (v / v), about 20% (v / v), about 25% (v / v), about 30% (v / v), about 35% (v / v), about 40% (v / v), about 45% (v / v), or about 50% (v / v).

[0273] In some embodiments, the reaction conditions can include surfactants to stabilize or enhance the reaction. Surfactants can include nonionic, cationic, anionic, and / or amphiphilic surfactants. Exemplary surfactants include, by way of example and not limitation, nonylphenoxypolyethoxyethanol (NP40), Triton® X-100, polyoxyethylene-stearylamine, cetyltrimethylammonium bromide, sodium oleylamidosulfate, polyoxyethylene-sorbitan monostearate, hexadecyldimethylamine, and the like. Any surfactant capable of stabilizing or enhancing the reaction can be used. The concentration of the surfactant to be used in the reaction can generally be 0.1 to 50 mg / ml, particularly 1 to 20 mg / ml.

[0274] In some embodiments, the reaction conditions can include an antifoaming agent to help reduce or prevent the formation of bubbles in the reaction solution, such as when the reaction solution is mixed or sparged. Antifoaming agents include non-polar oils (e.g., minerals, silicones, etc.), polar oils (e.g., fatty acids, alkylamines, alkylamides, alkyl sulfates, etc.), and hydrophobic agents (e.g., treated silica, polypropylene, etc.), some of which also function as surfactants. Exemplary antifoaming agents include Y-30® (Dow Corning), poly-glycol copolymers, oxy / ethoxylated alcohols, and polydimethylsiloxane. In some embodiments, the antifoaming agent can be present at about 0.001% (v / v) to about 5% (v / v), about 0.01% (v / v) to about 5% (v / v), about 0.1% (v / v) to about 5% (v / v), or about 0.1% (v / v) to about 2% (v / v). In some embodiments, the antifoaming agent may be present at about 0.001% (v / v), about 0.01% (v / v), about 0.1% (v / v), about 0.5% (v / v), about 1% (v / v), about 2% (v / v), about 3% (v / v), about 4% (v / v), or about 5% (v / v) or more, as desired to facilitate the reaction.

[0275] The amounts of reactants used in the hydroxylase reaction will generally vary depending on the amount of product desired and concomitantly with the amount of proline hydroxylase substrate used. Those skilled in the art will readily understand how to vary these amounts to adjust for the desired level of productivity and scale of production.

[0276] In some embodiments, the order of addition of reactants is not important. The reactants can be added together to a solvent (e.g., a monophasic solvent, a biphasic aqueous co-solvent system, etc.) at the same time, or alternatively, some of the reactants can be added separately and some together at different times. For example, the cofactor, co-substrate, proline hydroxylase, and substrate can be added to the solvent first.

[0277] Solid reactants (e.g., enzymes, salts, etc.) can be provided to the reaction in a variety of different forms, including powders (e.g., lyophilized, spray-dried, etc.), solutions, emulsions, suspensions, etc. Reactants can be readily lyophilized or spray-dried using methods and equipment known to those of skill in the art. For example, a protein solution can be frozen in small aliquots at −80° C. and then added to a pre-chilled lyophilization chamber, followed by application of a vacuum.

[0278] Due to improved mixing efficiency when an aqueous co-solvent system is used, the proline hydroxylase and cofactor can be added and mixed first with the aqueous phase. The organic phase can then be added and mixed, followed by the addition of the proline hydroxylase substrate and co-substrate. Alternatively, the proline hydroxylase substrate can be premixed with the organic phase prior to addition to the aqueous phase.

[0279] The hydroxylation process is generally allowed to proceed until further conversion of the substrate to the hydroxylated product does not change significantly with reaction time (e.g., less than 10% of the substrate is converted, or less than 5% of the substrate is converted). In some embodiments, the reaction is allowed to proceed until complete or nearly complete conversion of the substrate to the product occurs. The conversion of the substrate to the product can be monitored using known methods by detecting the substrate and / or product with or without derivatization. Suitable analytical methods include gas chromatography, HPLC, MS, and the like.

[0280] In some embodiments of the process, suitable reaction conditions include a substrate load of at least about 5 g / L, 10 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 100 g / L or more, and the method results in at least about 50%, 60%, 70%, 80%, 90%, 95% or more conversion of substrate compounds to product compounds in about 48 hours or less, about 36 hours or less, or about 24 hours or less.

[0281] The engineered proline hydroxylase polypeptides of the invention, when used in a process under suitable reaction conditions, result in an excess of trans-3-hydroxylated product over trans-4-hydroxylated product in an isomeric excess of at least 90%, 95%, 96%, 97%, 98%, 99% or more, and in some embodiments, no detectable amounts of the compound trans-4-hydroxylated product are formed.

[0282] In further embodiments of processes for converting substrate compounds to hydroxylated product compounds using engineered proline hydroxylase polypeptides, suitable reaction conditions can include an initial substrate load to a reaction solution, which is then contacted by the polypeptide. The reaction solution is then further supplemented with additional substrate compound as a continuous or batchwise addition over time at a rate of at least about 1 g / L / h, at least about 2 g / L / h, at least about 4 g / L / h, at least about 6 g / L / h, or faster. Thus, according to these suitable reaction conditions, the polypeptide is added to a solution having an initial substrate load of at least about 20 g / L, 30 g / L, or 40 g / L. This polypeptide addition is followed by the continued addition of additional substrate to the solution at a rate of about 2 g / L / h, 4 g / L / h, or 6 g / L / h until a much higher final substrate loading of at least about 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 100 g / L, 150 g / L, 200 g / L, or more is reached. Thus, in some embodiments of the process, suitable reaction conditions include adding the polypeptide to a solution having an initial substrate loading of at least about 20 g / L, 30 g / L, or 40 g / L, followed by the addition of additional substrate to the solution at a rate of about 2 g / L / h, 4 g / L / h, or 6 g / L / h until a final substrate loading of at least about 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 100 g / L, or more is reached. The substrate-replenished reaction conditions allow higher substrate loadings to be achieved while maintaining a high rate of substrate to hydroxylated product conversion of at least about 50%, 60%, 70%, 80%, 90% or more of the substrate. In some embodiments of this process, the added substrate is present in a solution containing α-ketoglutarate at an equimolar or greater concentration of the added substrate.

[0283] In some embodiments of the process, the reaction using the engineered prolyl hydroxylase polypeptide can include the following suitable reaction conditions: (a) a substrate load of about 60 g / L; (b) about 6 g / L of the engineered polypeptide; (c) about 1.2 molar equivalents of α-ketoglutarate in the substrate compound; (d) about 10 mM ascorbic acid; (e) about 4 mM FeSO4; (f) a pH of about 6.8; (g) a temperature of about 20°C; and (h) a reaction time of about 24 hours.

[0284] In some embodiments, additional reaction components or techniques are implemented to supplement the reaction conditions, including stabilizing the enzyme or preventing its inactivation, reducing product inhibition, or taking measures to shift the reaction equilibrium toward hydroxylated product formation.

[0285] In further embodiments, any of the above-described processes for converting a substrate compound to a product compound can further comprise one or more steps selected from extraction, isolation, purification, and crystallization of the product compound. Methods, techniques, and protocols for extracting, isolating, purifying, and / or crystallizing the hydroxylated product from the biocatalytic reaction mixture produced by the processes disclosed above are known to those skilled in the art and / or accessible through routine experimentation. Moreover, illustrative methods are provided in the Examples below.

[0286] Various features and embodiments of the present invention are illustrated in the following representative examples, which are intended to be illustrative and not limiting. [Example]

[0287] experiment The following examples, including the experiments and results achieved, are presented for illustrative purposes only and are not to be construed as limiting the invention.

[0288] In the experimental disclosure that follows, the following abbreviations apply: ppm (parts per million); M (molar); mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol (mole); gm and g (grams); mg (milligrams); ug and μg (micrograms); L and l (liters); ml and mL (milliliters); cm (centimeters); mm (millimeters); um and μm (micrometers); sec. (seconds); min (minutes); h and hr (hours); U (units); MW (molecular weight); rpm (revolutions per minute); °C (degrees Celsius); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); NA (nucleic acid; polynucleotide); AA (amino acid; polypeptide); E. coli W3110 (a commonly used laboratory strain of E. coli available from the Coli Genetic Stock Center [CGSC], New Haven, CT).coli strains); HPLC (high-pressure liquid chromatography); SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis); PES (polyethersulfone); CFSE (carboxyfluorescein succinimidyl ester); IPTG (isopropyl beta-D-1-thiogalactopyranoside); PMBS (polymyxin B sulfate); NADPH (nicotinamide adenine dinucleotide phosphate); GDH (glucose dehydrogenase); polyethyleneimine (PEI); FIOPC (fold improvement over positive control); DO (dissolved oxygen); ESI (electrospray ionization); LB (Luria broth); TB (terrific broth); MeOH (methanol); HTP (high throughput); SFP (shake flask powder); DSP (downstream process powder); Athens Research (Athens Research Technology, Athens, GA); ProSpec (ProSpec Tany Technogene, East Brunswick, NJ); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Ram Scientific (Ram Scientific, Inc., Yonkers, NY); Pall Corp. (Pall, Corp., Pt. Washington, NY); Millipore (Millipore, Corp., Billerica MA); Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); Devices(Molecular Devices, LLC, Sunnyvale, CA);Kuhner(Adolf Kuhner,AG, Basel, Switzerland);Cambridge Isotope Laboratories(Cambridge Isotope Laboratories,Inc., Tewksbury, MA); Applied Biosystems (part of Life Technologies, Corp., Grand Island, NY); Agilent (Agilent Technologies, Inc., Santa Clara, CA); Thermo. Scientific (part of Thermo Fisher Scientific, Waltham, MA); Fisher (Fisher Scientific, Waltham, MA); Corning (Corning, Inc., Palo Alto, CA); Waters (Waters Corp., Milford, MA); GE Healthcare (GE Healthcare Bio-Sciences, Piscataway, NJ); Pierce (Pierce Biotechnology (now part of Thermo Fisher Scientific), Rockford, IL); Phenomenex (Phenomenex, Inc., Torrance, CA); Optimal (Optimal Biotech Group, Belmont, CA); and Bio-Rad (Bio-Rad Laboratories, Hercules, CA).

[0289] Example 1 E. coli expression host containing the recombinant prolyl hydroxylase gene The initial prolyl hydroxylase (PH) enzyme used to produce the variants of the present invention was obtained from the wild-type ANO sequence from fungal species No. 11243 (accession number GAM84982). The wild-type PH protein sequence was codon-optimized for expression in E. coli, and the DNA was cloned into the expression vector pCK110900 (see Figure 3 of U.S. Patent Application Publication No. 2006 / 0195947), operably linked to the lac promoter under the control of the lacI repressor. The expression vector also contains a P15a origin of replication and a chloramphenicol resistance gene. The resulting plasmid was transformed into E. coli W3110 using standard methods known in the art. Transformants were isolated by subjecting the cells to chloramphenicol selection as known in the art (see, for example, U.S. Patent No. 8,383,346 and WO2010 / 144103).

[0290] Example 2 Preparation of wet cell pellet and lysate containing HTP PH E. coli cells containing the recombinant PH-encoding gene obtained from monoclonal colonies were inoculated into 180 μl LB containing 1% glucose and 30 μg / mL chloramphenicol (CAM) in the wells of a 96-well shallow-well microtiter plate. The plate was sealed with an O2-permeable seal, and the cultures were grown overnight at 30°C, 200 rpm, and 85% humidity. Next, 10 μl of each of the cell cultures was transferred to wells of a 96-well deep-well plate containing 390 mL TB and 30 μg / mL CAM. The deep-well plate was sealed with an O2-permeable seal, and the OD 600 The cells were incubated at 30°C, 250 rpm, and 85% humidity until the pH reached 0.6-0.8. The cell cultures were then induced with IPTG to a final concentration of 1 mM and incubated overnight at 20°C or 30°C. The cells were then pelleted using centrifugation at 4000 rpm for 10 min. The supernatant was discarded, and the pellet was frozen at -80°C prior to lysis.

[0291] For lysis, 400 μl of lysis buffer containing 50 mM sodium phosphate buffer, pH 6.5, 1 g / L lysozyme, and 0.5 g / L polymyxin b sulfate (PMBS) was added to the cell paste in each well produced as described in Example 2. The cells were lysed with shaking on a tabletop shaker at room temperature for 2 hours. The plate was then centrifuged for 15 min at 4000 rpm and 4° C. The clear supernatant was then used in a biocatalytic reaction to determine its activity level.

[0292] Example 3 Preparation of freeze-dried lysates from shake flask (SF) cultures Selected HTP cultures grown as described above were plated onto LB agar plates with 1% glucose and 30 μg / ml CAM and grown overnight at 37°C. A single colony from each culture was transferred to 6 ml of LB with 1% glucose and 30 μg / ml CAM. Cultures were grown for 18 h at 30°C and 250 rpm and subcultured approximately 1:50 into 250 ml of TB containing 30 μg / ml CAM to a final OD of 0.05. 600 The culture was grown at 30°C and 250 rpm for approximately 195 minutes to reach an OD between 0.6 and 0.8. 600 The cultures were then grown for 20 h at 20°C or 30°C and 250 rpm. The cultures were centrifuged at 4000 rpm for 20 min. The supernatant was discarded, and the pellet was resuspended in 30 ml of 20 mM triethanolamine, pH 7.5, and lysed at 18,000 psi using a Microfluidizer® processor system (Microfluidics). The lysate was pelleted (10,000 rpm for 60 min), and the supernatant was frozen and lyophilized to generate shake flask (SF) enzyme.

[0293] Example 4 Improvement over SEQ ID NO: 4 in the conversion of proline substrates to trans-3-hydroxyproline Based on the results of screening variants for the conversion of L-proline substrate to trans-3-hydroxyproline, SEQ ID NO:4 was selected as the parent enzyme. SEQ ID NO:4 is identical to SEQ ID NO:2; both sequences are wild-type proline hydroxylase with an N-terminal his-tag, while SEQ ID NO:3 is a codon-optimized polynucleotide encoding the wild-type proline hydroxylase. A library of engineered genes was produced using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations). The polypeptides encoded by each gene were produced in a HTP as described in Example 2 (by overnight protein expression at 20°C). For all variants, cell pellets were lysed by adding 200 μL lysis buffer (containing 50 mM sodium phosphate buffer pH 6.5, 1 g / L lysozyme, and 0.5 g / L PMBS) and shaking on a tabletop shaker at room temperature for 2 hours. The plates were centrifuged at 4000 rpm for 15 minutes at 4°C to remove cell debris.

[0294] In a 300 μL round-bottom plate, 50 μL of E. coli lysate was added to 200 μL of reaction mix (containing 75 μL of 63 g / L α-ketoglutaric acid [in 50 mM sodium phosphate pH 6.5], 50 μL of 20 mM Mohr's salt in 65 mM ascorbic acid [in 50 mM sodium phosphate pH 6.5], and 75 μL of 33 g / L L-proline) in each well. The plate was sealed with an AirPore seal (Qiagen) and the reaction was allowed to proceed overnight (approximately 18 hours) at 30°C, 200 rpm, and 85% relative humidity in a 2" Kuhner tube.

[0295] After overnight incubation, reactions from each well were derivatized and quenched by aliquoting 25 μL of the reaction mix into a 96-well deep-well plate containing 225 μL of derivatization solution (containing 75 μL of saturated sodium bicarbonate, 25 μL of water, and 125 μL of 2.5 mg / mL FmocCl in ACN per well). After 1 hr of shaking at room temperature, the plate was centrifuged at 4000 rpm for 1 minute, and 40 μL of the soluble fraction of the quenched reaction was mixed with 160 μL of 1:1 ACN:0.5 M HCl. The derivatized and diluted samples were analyzed as described in Table 13.1. Selectivity relative to SEQ ID NO:4 (Selectivity FIOP) was calculated as the ratio of trans-3-hydroxyproline:trans-4-hydroxyproline products formed by the variant relative to the ratio produced by SEQ ID NO:4. Activity relative to SEQ ID NO: 4 (active FIOP) was calculated as the ratio of the trans-3-hydroxyproline peak area of the variant compared to the trans-3-hydroxyproline peak area produced by SEQ ID NO: 4. The results are shown in Tables 4.1 and 4.2. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0296] In addition to HTP analysis, a select subset of informative variants from the HTP screen was also prepared at shake-flask scale (via overnight protein expression at 20°C) as described in Example 3. Lyophilized shake-flask lysate powder (SFP) was tested in 1 mL-scale reactions under the following conditions: 10 g / L L-proline, 50 wt% proline hydroxylase variant SFP, 1.5 equivalents a-KG (α-ketoglutarate), 0.15 equivalents ascorbic acid, 4 mM ammonium iron(II) sulfate hexahydrate, 50 mM sodium phosphate pH 6.5, air, and room temperature. Reactions were run overnight and analyzed using the same method described above for HTP reactions. Selectivity relative to SEQ ID NO:4 (Selectivity FIOP) was calculated as the ratio of trans-3-hydroxyproline:trans-4-hydroxyproline products formed by the variant relative to the ratio produced by SEQ ID NO:4. The results are shown in Table 4.3. [Table 4-5] [Table 4-6]

[0297] The prolyl hydroxylase protein produced by SEQ ID NO:4 was not sufficiently stable under standard expression conditions at 30°C. As described above, for all data shown in Tables 4.1, 4.2, and 4.3, HTP and shake flask proteins were produced by expression at 20°C. To select for more stable and active variants, a library of engineered genes derived from SEQ ID NO:4 was produced using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations), and the polypeptides encoded by each gene were produced in HTP by expression at 30°C. Reactions, derivatizations, and analyses were performed as described above. Stability and activity relative to SEQ ID NO:4 (Stability / Activity FIOP) were calculated as the ratio of the trans-3-hydroxyproline peak area of the variant compared to the trans-3-hydroxyproline peak area produced by SEQ ID NO:4, with both enzymes produced at 30°C. The results are shown in Table 4.4. [Table 4-7]

[0298] Example 5 Improvement over SEQ ID NO: 116 in the conversion of proline substrates to trans-3-hydroxyproline Using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations), a library of engineered genes was generated from an engineered polynucleotide (SEQ ID NO: 115) encoding a polypeptide with prolyl hydroxylase activity of SEQ ID NO: 116. The polypeptides encoded by each gene were produced in HTP (by protein expression overnight at 20°C) as described in Example 2. For all variants, cell pellets were lysed by adding 200 μL lysis buffer (containing 50 mM sodium phosphate buffer pH 6.5, 1 g / L lysozyme, and 0.5 g / L PMBS) and shaking on a benchtop shaker at room temperature for 2 hours. Plates were centrifuged at 4000 rpm for 15 minutes at 4°C to remove cell debris.

[0299] In a 300 μL round-bottom plate, 50 μL of E. coli lysate was added to 200 μL of reaction mix (containing 75 μL of 63 g / L α-ketoglutaric acid [in 50 mM sodium phosphate pH 6.5], 50 μL of 20 mM Mohr's salt in 65 mM ascorbic acid [in 50 mM sodium phosphate pH 6.5], and 75 μL of 33 g / L L-proline) in each well. The plate was sealed with an AirPore seal (Qiagen) and the reaction was allowed to proceed overnight (approximately 18 hours) at 30°C, 200 rpm, and 85% relative humidity in a 2" Kuhner tube.

[0300] After overnight incubation, reactions from each well were derivatized and quenched by aliquoting 25 μL of the reaction mix into a 96-well deep-well plate containing 225 μL of derivatization solution (containing 75 μL of saturated sodium bicarbonate, 25 μL of water, and 125 μL of 2.5 mg / mL FmocCl in ACN per well). After 1 hr of shaking at room temperature, the plate was centrifuged at 4000 rpm for 1 minute, and 40 μL of the soluble fraction of the quenched reaction was mixed with 160 μL of 1:1 ACN:0.5M HCl. The derivatized and diluted samples were analyzed as described in Table 13.1. Selectivity relative to SEQ ID NO:116 (Selectivity FIOP) was calculated as the ratio of trans-3-hydroxyproline:trans-4-hydroxyproline products formed by the variant relative to the ratio produced by SEQ ID NO:116. The results are shown in Table 5.1. [Table 5-1]

[0301] The proline hydroxylase protein produced by SEQ ID NO:116 was not sufficiently stable under standard expression conditions at 30°C. To select for more stable and active variants, a library of engineered genes derived from SEQ ID NO:116 was produced using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations), and the polypeptides encoded by each gene were produced in HTP by expression at 30°C. Reactions, derivatizations, and analyses were performed as described above. Stability and activity relative to SEQ ID NO:116 (Stability / Activity FIOP) were calculated as the ratio of the trans-3-hydroxyproline peak area of the variant compared to the trans-3-hydroxyproline peak area produced by SEQ ID NO:116, with both enzymes produced at 30°C. The results are shown in Table 5.2. [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5]

[0302] In addition to HTP analysis, a selected subset of informative variants from the HTP screen was also prepared at a shake flask scale, as described in Example 3. SFP for SEQ ID NO: 116 was produced at 20°C, and variants derived from SEQ ID NO: 116 were produced at 30°C. Lyophilized shake flask lysate powder (SFP) was tested in 1 mL-scale reactions under the following conditions: 20 g / L L-proline, 5 wt% proline hydroxylase variant SFP, 1.5 equivalents a-KG (α-ketoglutarate), 0.15 equivalents ascorbic acid, 4 mM ammonium iron(II) sulfate hexahydrate, 50 mM sodium phosphate pH 6.5, air, and room temperature. Reactions were carried out overnight and analyzed using similar methods as described above for HTP reactions. Stability and activity relative to SEQ ID NO: 116 (Stability / Activity FIOP) was calculated as the ratio of the trans-3-hydroxyproline peak area of the variant compared to the trans-3-hydroxyproline peak area produced by SEQ ID NO: 116, where SEQ ID NO: 116 was produced at 20° C. and the variant derived from SEQ ID NO: 116 was produced at 30° C. The results are shown in Table 5.3. [Table 5-6]

[0303] Example 6 Improvement over SEQ ID NO: 162 in the conversion of proline substrates to trans-3-hydroxyproline Using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations), a library of engineered genes was generated from an engineered polynucleotide (SEQ ID NO: 161) encoding a polypeptide with prolyl hydroxylase activity of SEQ ID NO: 162. The polypeptides encoded by each gene were produced in HTP (by protein expression overnight at 30°C) as described in Example 2. For all variants, cell pellets were lysed by adding 400 μL lysis buffer (containing 50 mM sodium phosphate buffer pH 6.5, 1 g / L lysozyme, and 0.5 g / L PMBS) and shaking on a benchtop shaker at room temperature for 2 hours. Plates were centrifuged at 4000 rpm for 15 minutes at 4°C to remove cell debris.

[0304] In a 300 μL round-bottom plate, 50 μL of E. coli lysate was added to 200 μL of reaction mix (containing 75 μL of 133 g / L α-ketoglutaric acid [in 50 mM sodium phosphate pH 6.5], 50 μL of 20 mM Mohr's salt in 65 mM ascorbic acid [in 50 mM sodium phosphate pH 6.5], and 75 μL of 67 g / L L-proline) in each well. The plate was sealed with an AirPore seal (Qiagen) and the reaction was allowed to proceed overnight (approximately 18 hours) at 30°C, 200 rpm, and 85% relative humidity in a 2" Kühner tube.

[0305] After overnight incubation, reactions from each well were derivatized and quenched by aliquoting 25 μL of the reaction mix into a 96-well deep-well plate containing 225 μL of derivatization solution (containing 75 μL of saturated sodium bicarbonate, 25 μL of water, and 125 μL of 2.5 mg / mL FmocCl in ACN per well). After 1 hr of shaking at room temperature, the plate was centrifuged at 4000 rpm for 1 minute, and 40 μL of the soluble fraction of the quenched reaction was mixed with 160 μL of 1:1 ACN:0.5 M HCl. The derivatized and diluted samples were analyzed as described in Table 13.1. Selectivity relative to SEQ ID NO:162 (Selectivity FIOP) was calculated as the ratio of trans-3-hydroxyproline:trans-4-hydroxyproline products formed by the variant relative to the ratio produced by SEQ ID NO:162.

[0306] In addition to HTP analysis, a select subset of informative variants from the HTP screen was also prepared at shake flask scale, as described in Example 3, by expression at 30°C. Lyophilized shake flask lysate powder (SFP) was tested in 1 mL-scale reactions under the following conditions: 40 g / L L-proline, 5 wt% proline hydroxylase variant SFP, 1.2 equivalents a-KG (α-ketoglutarate), 25 mM ascorbic acid, 4 mM ammonium iron(II) sulfate hexahydrate, 50 mM sodium phosphate pH 6.5, air, and room temperature. Reactions were run overnight and analyzed using the same method described above for HTP reactions. Selectivity relative to SEQ ID NO: 162 (selectivity FIOP) was calculated as the ratio of trans-3-hydroxyproline to trans-4-hydroxyproline products formed by the variant relative to the ratio produced by SEQ ID NO: 162. The results are shown in Table 6.1. [Table 6-1] [Table 6-2]

[0307] Example 7 Improvement over SEQ ID NO: 322 in the conversion of proline substrates to trans-3-hydroxyproline Using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations), a library of engineered genes was generated from an engineered polynucleotide (SEQ ID NO: 321) encoding a polypeptide with prolyl hydroxylase activity of SEQ ID NO: 322. Polypeptides encoded by each gene were produced in HTP (by protein expression overnight at 30°C) as described in Example 2. For all variants, cell pellets were lysed by adding 400 μL lysis buffer (containing 50 mM sodium phosphate buffer pH 6.5, 1 g / L lysozyme, and 0.5 g / L PMBS) and shaking on a benchtop shaker at room temperature for 2 hours. Plates were centrifuged at 4000 rpm for 15 minutes at 4°C to remove cell debris.

[0308] In a 300 μL round-bottom plate, 50 μL of E. coli lysate was added to 200 μL of reaction mix (containing 75 μL of 266 g / L α-ketoglutaric acid [in 50 mM sodium phosphate pH 6.5], 50 μL of 20 mM Mohr's salts in 65 mM ascorbic acid [in 50 mM sodium phosphate pH 6.5], and 75 μL of 133 g / L L-proline) in each well. The plate was sealed with an AirPore seal (Qiagen) and the reaction was allowed to proceed overnight (approximately 18 hours) at 30°C, 200 rpm, and 85% relative humidity in a 2" Kuhner tube.

[0309] After overnight incubation, reactions from each well were derivatized and quenched by aliquoting 25 μL of the reaction mix into a 96-well deep-well plate containing 225 μL of derivatization solution (containing 75 μL of saturated sodium bicarbonate, 25 μL of water, and 125 μL of 2.5 mg / mL FmocCl in ACN per well). After 1 hr of shaking at room temperature, the plate was centrifuged at 4000 rpm for 1 minute, and 40 μL of the soluble fraction of the quenched reaction was mixed with 160 μL of 1:1 ACN:0.5 M HCl. The derivatized and diluted samples were analyzed as described in Table 13.1. Selectivity relative to SEQ ID NO:322 (Selectivity FIOP) was calculated as the ratio of trans-3-hydroxyproline:trans-4-hydroxyproline products formed by the variant relative to the ratio produced by SEQ ID NO:322. Activity relative to SEQ ID NO: 322 (active FIOP) was calculated as the ratio of the trans-3-hydroxyproline peak area of the variant compared to the trans-3-hydroxyproline peak area produced by SEQ ID NO: 322. The results are shown in Tables 7.1 and 7.2, respectively. [Table 7-1] [Table 7-2] [Table 7-3]

[0310] In addition to HTP analysis, a select subset of informative variants from the HTP screen was also prepared at shake flask scale, as described in Example 3, by expression at 30°C. Lyophilized shake flask lysate powder (SFP) was tested in 1 mL-scale reactions under the following conditions: 40 g / L L-proline, 5 wt% proline hydroxylase variant SFP, 1.2 equivalents a-KG (α-ketoglutarate), 10 mM ascorbic acid, 4 mM ammonium iron(II) sulfate hexahydrate, 50 mM sodium phosphate pH 6.5, air, and room temperature. Reactions were run overnight and analyzed using the same method described above for HTP reactions. Selectivity relative to SEQ ID NO:322 (selectivity FIOP) was calculated as the ratio of trans-3-hydroxyproline:trans-4-hydroxyproline products formed by the variant relative to the ratio produced by SEQ ID NO:322. The results are shown in Table 7.3. [Table 7-4]

[0311] Example 8 Improvement over SEQ ID NO: 412 in the conversion of proline substrates to trans-3-hydroxyproline Using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations), a library of engineered genes was generated from an engineered polynucleotide (SEQ ID NO: 411) encoding a polypeptide with proline hydroxylase activity of SEQ ID NO: 412. Polypeptides encoded by each gene were produced in HTP (by protein expression overnight at 30°C) as described in Example 2. For all variants, cell pellets were lysed by adding 400 μL lysis buffer (containing 50 mM sodium phosphate buffer pH 6.5, 1 g / L lysozyme, and 0.5 g / L PMBS) and shaking on a benchtop shaker at room temperature for 2 hours. Plates were centrifuged at 4000 rpm for 15 minutes at 4°C to remove cell debris.

[0312] In a 300 μL round-bottom plate, 50 μL of E. coli lysate was added to 200 μL of reaction mix (containing 75 μL of 266 g / L α-ketoglutaric acid [in 50 mM sodium phosphate pH 6.5], 50 μL of 20 mM Mohr's salts in 65 mM ascorbic acid [in 50 mM sodium phosphate pH 6.5], and 75 μL of 133 g / L L-proline) in each well. The plate was sealed with an AirPore seal (Qiagen) and the reaction was allowed to proceed overnight (approximately 18 hours) at 30°C, 200 rpm, and 85% relative humidity in a 2" Kuhner tube.

[0313] After overnight incubation, reactions from each well were derivatized and quenched by aliquoting 25 μL of the reaction mix into a 96-well deep-well plate containing 225 μL of derivatization solution (containing 75 μL of saturated sodium bicarbonate, 25 μL of water, and 125 μL of 2.5 mg / mL FmocCl in ACN per well). After 1 hr of shaking at room temperature, the plate was centrifuged at 4000 rpm for 1 minute, and 40 μL of the soluble fraction of the quenched reaction was mixed with 160 μL of 1:1 ACN:0.5M HCl. The derivatized and diluted samples were analyzed as described in Table 13.1. Selectivity relative to SEQ ID NO:412 (Selectivity FIOP) was calculated as the ratio of trans-3-hydroxyproline:trans-4-hydroxyproline products formed by the variant relative to the ratio produced by SEQ ID NO:412. The results are shown in Table 8.1. [Table 8-1] [Table 8-2]

[0314] In addition to HTP analysis, a select subset of informative variants from the HTP screen was also prepared at shake flask scale, as described in Example 3, by expression at 30°C. Lyophilized shake flask lysate powder (SFP) was tested in 1 mL-scale reactions under the following conditions: 60 g / L L-proline, 5 wt% proline hydroxylase variant SFP, 1.2 equivalents a-KG (α-ketoglutarate), 10 mM ascorbic acid, 4 mM ammonium iron(II) sulfate hexahydrate, 50 mM sodium phosphate pH 6.8, air, and room temperature. Reactions were run overnight and analyzed using the same method described above for HTP reactions. Selectivity relative to SEQ ID NO:412 (selectivity FIOP) was calculated as the ratio of trans-3-hydroxyproline:trans-4-hydroxyproline products formed by the variant relative to the ratio produced by SEQ ID NO:412. The results are shown in Table 8.2. [Table 8-3]

[0315] Example 9 Improvement over SEQ ID NO: 492 in the conversion of proline substrates to trans-3-hydroxyproline Using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations), a library of engineered genes was generated from an engineered polynucleotide (SEQ ID NO: 491) encoding a polypeptide with prolyl hydroxylase activity of SEQ ID NO: 492. Polypeptides encoded by each gene were produced in HTP (by protein expression overnight at 30°C) as described in Example 2. For all variants, cell pellets were lysed by adding 600 μL lysis buffer (containing 50 mM sodium phosphate buffer pH 6.5, 1 g / L lysozyme, and 0.5 g / L PMBS) and shaking on a benchtop shaker at room temperature for 2 hours. Plates were centrifuged at 4000 rpm for 15 minutes at 4°C to remove cell debris.

[0316] In a 300 μL round-bottom plate, 50 μL of E. coli lysate was added to 200 μL of reaction mix (containing 75 μL of 667 g / L α-ketoglutaric acid [in 50 mM sodium phosphate pH 6.5], 50 μL of 20 mM Mohr's salts in 65 mM ascorbic acid [in 50 mM sodium phosphate pH 6.5], and 75 μL of 333 g / L L-proline) in each well. The plate was sealed with an AirPore seal (Qiagen) and the reaction was allowed to proceed overnight (approximately 18 hours) at 30°C, 200 rpm, and 85% relative humidity in a 2" Kuhner tube.

[0317] After overnight incubation, reactions from each well were derivatized and quenched by aliquoting 25 μL of the reaction mix into a 96-well deep-well plate containing 225 μL of derivatization solution (containing 75 μL of saturated sodium bicarbonate, 25 μL of water, and 125 μL of 2.5 mg / mL FmocCl in ACN per well). After 1 hr of shaking at room temperature, the plate was centrifuged at 4000 rpm for 1 minute, and 40 μL of the soluble fraction of the quenched reaction was mixed with 160 μL of 1:1 ACN:0.5M HCl. The derivatized and diluted samples were analyzed as described in Table 13.1. Selectivity relative to SEQ ID NO:492 (Selectivity FIOP) was calculated as the ratio of trans-3-hydroxyproline:trans-4-hydroxyproline products formed by the variant relative to the ratio produced by SEQ ID NO:492. The results are shown in Table 9.1. [Table 9-1] [Table 9-2]

[0318] In addition to HTP analysis, a select subset of informative variants from the HTP screen was also prepared at shake flask scale, as described in Example 3, by expression at 30°C. Lyophilized shake flask lysate powder (SFP) was tested in 1 mL-scale reactions under the following conditions: 60 g / L L-proline, 5 wt% proline hydroxylase variant SFP, 1.2 equivalents a-KG (α-ketoglutarate), 10 mM ascorbic acid, 4 mM ammonium iron(II) sulfate hexahydrate, 50 mM sodium phosphate pH 6.8, air, and room temperature. Reactions were run overnight and analyzed using the same method described above for HTP reactions. Selectivity relative to SEQ ID NO:492 (selectivity FIOP) was calculated as the ratio of trans-3-hydroxyproline:trans-4-hydroxyproline products formed by the variant relative to the ratio produced by SEQ ID NO:492. The results are shown in Table 9.2. [Table 9-3] [Table 9-4]

[0319] Example 10 Improvement over SEQ ID NO: 562 in the conversion of proline substrates to trans-3-hydroxyproline Using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations), a library of engineered genes was generated from an engineered polynucleotide (SEQ ID NO: 561) encoding a polypeptide with prolyl hydroxylase activity of SEQ ID NO: 562. Polypeptides encoded by each gene were produced in HTP (by protein expression overnight at 30°C) as described in Example 2. For all variants, cell pellets were lysed by adding 400 μL lysis buffer (containing 50 mM sodium phosphate buffer pH 6.5, 1 g / L lysozyme, and 0.5 g / L PMBS) and shaking on a benchtop shaker at room temperature for 2 hours. Plates were centrifuged at 4000 rpm for 15 minutes at 4°C to remove cell debris.

[0320] In a 300 μL round-bottom plate, 50 μL of E. coli lysate was added to 200 μL of reaction mix (containing 75 μL of 400 g / L α-ketoglutaric acid [in 50 mM sodium phosphate pH 6.5], 50 μL of 20 mM Mohr's salts in 65 mM ascorbic acid [in 50 mM sodium phosphate pH 6.5], and 200 μL of 200 g / L L-proline) in each well. The plate was sealed with an AirPore seal (Qiagen) and the reaction was allowed to proceed overnight (approximately 18 hours) at 30°C, 200 rpm, and 85% relative humidity in a 2" Kuhner tube.

[0321] After overnight incubation, reactions from each well were derivatized and quenched by aliquoting 25 μL of the reaction mix into a 96-well deep-well plate containing 225 μL of derivatization solution (containing 75 μL of saturated sodium bicarbonate, 25 μL of water, and 125 μL of 2.5 mg / mL FmocCl in ACN per well). After 1 hr of shaking at room temperature, the plate was centrifuged at 4000 rpm for 1 minute, and 40 μL of the soluble fraction of the quenched reaction was mixed with 160 μL of 1:1 ACN:0.5M HCl. The derivatized and diluted samples were analyzed as described in Table 13.1. Selectivity relative to SEQ ID NO:562 (Selectivity FIOP) was calculated as the ratio of trans-3-hydroxyproline:trans-4-hydroxyproline products formed by the variant relative to the ratio produced by SEQ ID NO:562. The results are shown in Table 10.1. [Table 10-1]

[0322] In addition to HTP analysis, a select subset of informative variants from the HTP screen was also prepared at shake flask scale, as described in Example 3, by expression at 30°C. Lyophilized shake flask lysate powder (SFP) was tested in 1 mL-scale reactions under the following conditions: 60 g / L L-proline, 5 wt% proline hydroxylase variant SFP, 1.2 equivalents a-KG (α-ketoglutarate), 10 mM ascorbic acid, 4 mM ammonium iron(II) sulfate hexahydrate, 50 mM sodium phosphate pH 6.8, air, and room temperature. Reactions were run overnight and analyzed using the same method described above for HTP reactions. Selectivity relative to SEQ ID NO:562 (selectivity FIOP) was calculated as the ratio of trans-3-hydroxyproline to trans-4-hydroxyproline products formed by the variant relative to the ratio produced by SEQ ID NO:562. The results are shown in Table 10.2. [Table 10-2] [Table 10-3]

[0323] Example 11 Improvement over SEQ ID NO: 598 in the conversion of proline substrates to trans-3-hydroxyproline Using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations), a library of engineered genes was generated from an engineered polynucleotide (SEQ ID NO: 597) encoding a polypeptide with prolyl hydroxylase activity of SEQ ID NO: 598. Polypeptides encoded by each gene were produced in HTP (by protein expression overnight at 30°C) as described in Example 2. For all variants, cell pellets were lysed by adding 200 μL lysis buffer (containing 50 mM sodium phosphate buffer pH 6.5, 1 g / L lysozyme, and 0.5 g / L PMBS) and shaking on a benchtop shaker at room temperature for 2 hours. Plates were centrifuged at 4000 rpm for 15 minutes at 4°C to remove cell debris.

[0324] In a 300 μL round-bottom plate, 50 μL of E. coli lysate was added to 200 μL of reaction mix (containing 75 μL of 267 g / L α-ketoglutaric acid [in 50 mM sodium phosphate pH 6.5], 50 μL of 20 mM Mohr's salts in 65 mM ascorbic acid [in 50 mM sodium phosphate pH 6.5], and 200 μL of 133 g / L L-proline) in each well. The plate was sealed with an AirPore seal (Qiagen) and the reaction was allowed to proceed overnight (approximately 18 hours) at 30°C, 200 rpm, and 85% relative humidity in a 2" Kuhner tube.

[0325] After overnight incubation, reactions from each well were derivatized and quenched by aliquoting 25 μL of the reaction mix into a 96-well deep-well plate containing 225 μL of derivatization solution (containing 75 μL of saturated sodium bicarbonate, 25 μL of water, and 125 μL of 2.5 mg / mL FmocCl in ACN per well). After 1 hr of shaking at room temperature, the plate was centrifuged at 4000 rpm for 1 minute, and 40 μL of the soluble fraction of the quenched reaction was mixed with 160 μL of 1:1 ACN:0.5M HCl. The derivatized and diluted samples were analyzed as described in Table 13.1. Selectivity relative to SEQ ID NO:598 (Selectivity FIOP) was calculated as the ratio of trans-3-hydroxyproline:trans-4-hydroxyproline products formed by the variant relative to the ratio produced by SEQ ID NO:598. The results are shown in Table 11.1. [Table 11-1]

[0326] In addition to HTP analysis, a select subset of informative variants from the HTP screen was also prepared at shake flask scale, as described in Example 3, by expression at 30°C. Lyophilized shake flask lysate powder (SFP) was tested in 1 mL-scale reactions under the following conditions: 60 g / L L-proline, 5 wt% proline hydroxylase variant SFP, 1.2 equivalents a-KG (α-ketoglutarate), 10 mM ascorbic acid, 4 mM ammonium iron(II) sulfate hexahydrate, 50 mM sodium phosphate pH 6.8, air, and room temperature. Reactions were run overnight and analyzed using the same method described above for HTP reactions. Selectivity relative to SEQ ID NO:598 (selectivity FIOP) was calculated as the ratio of trans-3-hydroxyproline:trans-4-hydroxyproline products formed by the variant relative to the ratio produced by SEQ ID NO:598. The results are shown in Table 11.2. [Table 11-2] [Table 11-3]

[0327] Example 12 Improvement over SEQ ID NO: 630 in the conversion of proline substrates to trans-3-hydroxyproline Using well-established techniques (e.g., saturation mutagenesis and recombination of previously identified beneficial mutations), a library of engineered genes was generated from an engineered polynucleotide (SEQ ID NO: 629) encoding a polypeptide with prolyl hydroxylase activity of SEQ ID NO: 630. The polypeptides encoded by each gene were produced in HTP (by protein expression overnight at 30°C) as described in Example 2. For all variants, cell pellets were lysed by adding 200 μL lysis buffer (containing 50 mM sodium phosphate buffer pH 6.5, 1 g / L lysozyme, and 0.5 g / L PMBS) and shaking on a benchtop shaker at room temperature for 2 hours. Plates were centrifuged at 4000 rpm for 15 minutes at 4°C to remove cell debris.

[0328] In a 300 μL round-bottom plate, 50 μL of E. coli lysate was added to 200 μL of reaction mix (containing 75 μL of 267 g / L α-ketoglutaric acid [in 50 mM sodium phosphate pH 6.5], 50 μL of 20 mM Mohr's salts in 65 mM ascorbic acid [in 50 mM sodium phosphate pH 6.5], and 200 μL of 133 g / L L-proline) in each well. The plate was sealed with an AirPore seal (Qiagen) and the reaction was allowed to proceed overnight (approximately 18 hours) at 30°C, 200 rpm, and 85% relative humidity in a 2" Kuhner tube.

[0329] After overnight incubation, reactions from each well were derivatized and quenched by aliquoting 25 μL of the reaction mix into a 96-well deep-well plate containing 225 μL of derivatization solution (containing 75 μL of saturated sodium bicarbonate, 25 μL of water, and 125 μL of 2.5 mg / mL FmocCl in ACN per well). After 1 hr of shaking at room temperature, the plate was centrifuged at 4000 rpm for 1 minute, and 40 μL of the soluble fraction of the quenched reaction was mixed with 160 μL of 1:1 ACN:0.5 M HCl. The derivatized and diluted samples were analyzed as described in Table 13.1. Selectivity relative to SEQ ID NO:630 (Selectivity FIOP) was calculated as the ratio of trans-3-hydroxyproline:trans-4-hydroxyproline products formed by the variant relative to the ratio produced by SEQ ID NO:630.

[0330] In addition to HTP analysis, a select subset of informative variants from the HTP screen was also prepared at shake flask scale, as described in Example 3, by expression at 30°C. Lyophilized shake flask lysate powder (SFP) was tested in 1 mL-scale reactions under the following conditions: 60 g / L L-proline, 5 wt% proline hydroxylase variant SFP, 1.2 equivalents a-KG (α-ketoglutarate), 10 mM ascorbic acid, 4 mM ammonium iron(II) sulfate hexahydrate, 50 mM sodium phosphate pH 6.8, air, and room temperature. Reactions were run overnight and analyzed using the same method described above for HTP reactions. Selectivity relative to SEQ ID NO:630 (selectivity FIOP) was calculated as the ratio of trans-3-hydroxyproline to trans-4-hydroxyproline products formed by the variant relative to the ratio produced by SEQ ID NO:630. The results are shown in Table 12.1. [Table 12]

[0331] Example 13 Analytical detection of trans-3-hydroxyproline produced from proline The data described in Examples 4-12 were collected using the analytical methods in Table 13.1. All of the methods provided herein find use in analyzing variants produced using the present invention. However, the methods described herein are not intended to be the only methods applicable to analyzing the variants provided herein and / or variants produced using the methods provided herein.

[0332] The proline substrate and hydroxylproline product were analyzed as described below. Reactions were derivatized and quenched by aliquoting 25 μL of the reaction mix into a 96-well deep-well plate containing 225 μL of derivatization solution (containing 75 μL of saturated sodium bicarbonate, 25 μL of water, and 125 μL of 2.5 mg / mL FmocCl in ACN per well). After 1 hr of shaking at room temperature, the plate was centrifuged at 4000 rpm for 1 minute, and 40 μL of the soluble fraction of the quenched reaction was mixed with 160 μL of 1:1 ACN:0.5 M HCl. The derivatized and diluted samples were analyzed as described in Table 13.1. [Table 13]

[0333] All publications, patents, patent applications, and other documents cited herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0334] While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention(s).

Claims

[Claim 1] The invention described in the specification.

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