Engineered imine reductases and methods for reductive amination of ketone and amine compounds

Abstract

The present application provides engineered polypeptides having imine reductase activity, polynucleotides encoding the engineered polypeptides, host cells capable of expressing the engineered polypeptides, and methods of using these engineered polypeptides and a series of ketone and amine substrate compounds to prepare secondary and tertiary amine product compounds.

Description

[0001] This application claims priority to U.S. Patent Application Serial No. 62 / 841,633, filed May 1, 2019, which is hereby incorporated by reference in its entirety for all purposes. Technical Field

[0002] The present invention provides engineered polypeptides having imine reductase activity that can be used to produce secondary amines, as well as compositions and methods that utilize these engineered polypeptides.

[0003] Reference to a Sequence Listing, Table, or Computer Program

[0004] A formal copy of the sequence listing is submitted via EFS-Web as an ASCII formatted text file simultaneously with the specification, named "CX2-186WO1_ST25.txt", created on Apr. 21, 2020, and having a size of 1.76 megabytes. The sequence listing submitted via EFS-Web is part of this specification and is hereby incorporated by reference in its entirety.

[0005] Background

[0006] Chiral secondary amines are important building blocks in the pharmaceutical industry. However, there are only a few known biocatalytic pathways for producing such chiral amine compounds. Existing chemical methods use chiral boron reagent-based, transition metal-based reduction methods, or protecting group strategies, which require multi-step syntheses for the overall reduction of imines.

[0007] There are several reports in the literature on the biocatalytic synthesis of stable cyclic amines. Imine reductases or "IREDs" were purified and characterized from Streptomyces sp. strains GF3587 and GF3546 and shown to stereoselectively reduce 2-methyl-1-pyrroline (see, Mitsukura et al., Biosci. Biotech. Biochem., 75:1778-1782

[2011] ); Huber et al., Chem. Cat. Chem., 6:2248-2252

[2014] ). Reduction to acyclic amines and amino acids with alkyl groups using native imine reductases has also been shown (Gand et al., J. Mol. Catal. B: Enzym., 110:126-132

[2014] ). More recently, other authors have reported IREDs, also known as Reductive Aminases (RedAms), which catalyze reductive aminations of synthetic interest (Grogan, Curr. Opin. Chem. Biol., 2018, 43, 15-22; France et al., ChemCatChem., 2018, 10(3), 510-514; Aleku et al., Nat. Chem., 2017, 9, 961-969 and ChemCatChem., 2018, 10(3), 515-519; Sharma et al., ACS Catalysis 2018, 8(12), 11534-11541 and Adv. Synth. Catal., 2017, 359(12), 2011-2015; Roiban et al., ChemCatChem., 2017, 9(24), 4475-4479).

[0008] There is a need in the art for biocatalysts and methods for using them to synthesize chiral secondary and tertiary amines under industrially applicable conditions.

[0009] Overview

[0010] The present invention provides novel biocatalysts and related methods for the synthesis of chiral secondary amines by direct reduction of imine substrates. The biocatalysts of the present disclosure are engineered polypeptide variants of the wild-type gene encoding an opine dehydrogenase having the amino acid sequence of SEQ ID NO:2 from Arthrobacter sp. strain 1C. These engineered polypeptides have imine reductase activity and are capable of catalyzing the conversion of a ketone or aldehyde substrate and a primary or secondary amine substrate to form a secondary or tertiary amine product compound.

[0011] In some embodiments, the present disclosure provides engineered polypeptides having imine reductase activity, the engineered polypeptides comprising an amino acid sequence having at least 80% sequence identity to a reference amino acid sequence selected from the group consisting of SEQ ID NO: 4, 16, 50, 306, 648, and 708, and further comprising one or more residue differences as compared to the reference amino acid sequence. In some embodiments of the engineered polypeptides having imine reductase activity, the imine reductase activity is the activity of: Scheme 1; optionally, the reactions disclosed in Table 2; optionally, the reaction of converting a compound of formula (V) and a compound of formula (VI) into a compound of formula (IV) as a product; and optionally, the reaction of converting compound (2) and compound (3) into product compound (1).

[0012] The present invention provides an engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the reference sequence SEQ ID NO: 4, comprising at least one substitution or set of substitutions at one or more positions selected from: 145, 146, 153, 160, 222, 223, 226, and 261, wherein the positions are numbered with reference to SEQ ID NO: 4. In some additional embodiments, the engineered polypeptide comprises at least one substitution selected from E145I, E145V, R146A, N153L, N153R, D160T, Y222F, Y222W, L223A, L223G, L223I, L223S, L223V, I226M, and E261T, wherein the positions are numbered with reference to SEQ ID NO: 4. In some embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from E145I, E145V, R146A, N153L, N153R, D160T, Y222F, Y222W, L223A, L223G, L223I, L223S, L223V, I226M, and E261T, wherein the positions are numbered with reference to SEQ ID NO: 4. In some additional embodiments, the engineered polypeptide comprises an amino acid sequence having at least 80% sequence identity to any even-numbered sequence listed in SEQ ID NOs: 6 to 802.

[0013] The present invention provides an engineered polypeptide, which comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the reference sequence SEQ ID NO: 16, and comprises at least one substitution or set of substitutions at one or more positions selected from the following: 29 / 94 / 184 / 223 / 232 / 288 / 293, 29 / 94 / 184 / 232 / 287 / 288 / 293 / 311, 29 / 94 / 184 / 232 / 287 / 288 / 311, 29 / 94 / 184 / 232 / 287 / 293 / 332, 29 / 94 / 184 / 232 / 288 / 293 / 311 / 324 / 353, 29 / 94 / 184 / 232 / 288 / 311 / 324 / 332, 29 / 94 / 184 / 232 / 293, 29 / 94 / 184 / 232 / 311, 29 / 94 / 184 / 287 / 293, 29 / 94 / 184 / 287 / 293 / 311 / 353, 29 / 94 / 184 / 287 / 311, 29 / 94 / 184 / 288 / 293 / 324, 29 / 94 / 184 / 288 / 293 / 332 / 353, 29 / 94 / 184 / 288 / 298 / 332, 29 / 94 / 184 / 288 / 353, 29 / 94 / 184 / 293 / 311, 29 / 94 / 184 / 324, 29 / 94 / 223 / 232 / 287, 29 / 94 / 223 / 232 / 293 / 311 / 324, 29 / 94 / 223 / 288 / 293, 29 / 94 / 232 / 287 / 288 / 311, 29 / 94 / 232 / 288 / 293, 29 / 94 / 287 / 288 / 293, 29 / 94 / 287 / 288 / 293 / 353, 29 / 94 / 287 / 311, 29 / 94 / 293 / 311 / 324, 29 / 94 / 293 / 332 / 353, 29 / 94 / 311, 29 / 94 / 324 / 353, 29 / 184, 29 / 184 / 223 / 288 / 293 / 324, 29 / 184 / 232 / 288, 29 / 184 / 287, 29 / 184 / 287 / 288 / 293, 29 / 184 / 287 / 288 / 293 / 311, 29 / 184 / 287 / 311 / 332, 29 / 184 / 288 / 293, 29 / 184 / 288 / 293 / 311, 29 / 184 / 293, 29 / 184 / 293 / 311, 29 / 184 / 293 / 324, 29 / 184 / 353, 29 / 223 / 287 / 288 / 293 / 353, 29 / 232 / 287 / 288 / 293 / 332 / 353,29 / 287 / 288 / 293、29 / 287 / 288 / 293 / 324 / 353、29 / 288 / 293、29 / 311、29 / 311 / 332、29 / 353、72 / 94 / 184 / 288 / 311、86、94 / 96 / 223 / 287 / 288 / 293 / 311 / 324 / 332、94 / 99 / 184 / 293 / 311 / 332 / 353、94 / 184 / 223、94 / 184 / 223 / 232 / 287 / 311、94 / 184 / 223 / 288 / 293、94 / 184 / 223 / 293、94 / 184 / 232 / 287 / 288、94 / 184 / 232 / 287 / 288 / 324 / 332、94 / 184 / 232 / 287 / 293、94 / 184 / 232 / 287 / 293 / 332、94 / 184 / 287 / 288 / 293、94 / 184 / 287 / 288 / 311、94 / 184 / 287 / 293 / 311 / 324 / 353、94 / 184 / 287 / 311、94 / 184 / 287 / 353、94 / 184 / 288 / 293、94 / 184 / 288 / 293 / 311、94 / 184 / 288 / 293 / 311 / 332、94 / 184 / 293、94 / 184 / 293 / 311、94 / 184 / 293 / 332 / 353、94 / 223 / 232 / 311 / 353 / 355 / 356 / 357 / 358 / 359、94 / 223 / 287 / 288 / 293、94 / 223 / 288 / 293 / 311、94 / 232 / 287 / 288 / 293 / 353、94 / 232 / 288 / 293 / 353、94 / 232 / 293、94 / 232 / 293 / 324 / 332、94 / 232 / 311、94 / 287 / 288 / 293、94 / 287 / 288 / 311 / 324、94 / 288 / 293、94 / 288 / 293 / 324、94 / 293 / 324、94 / 311 / 324、153、184 / 223、184 / 223 / 287 / 288 / 353、184 / 232 / 287 / 288 / 293 / 311、184 / 232 / 287 / 288 / 324 / 353、184 / 232 / 287 / 293 / 332、184 / 287 / 288、184 / 287 / 288 / 293、184 / 287 / 288 / 293 / 324 / 353、184 / 287 / 293、184 / 287 / 293 / 311、184 / 288 / 293 / 311 / 324、184 / 332 / 353、205、223 / 232 / 287 / 288 / 332 / 353、223 / 287 / 288, 223 / 287 / 288 / 293 / 311, 223 / 287 / 311 / 353, 223 / 288 / 293, 232 / 287 / 288 / 293, 232 / 287 / 288 / 293 / 311, 232 / 287 / 288 / 293 / 321, 232 / 287 / 311, 232 / 288 / 293, 232 / 288 / 293 / 332, 234 / 287 / 288 / 293 / 311, 287 / 288 / 293, 287 / 288 / 311 / 324, 287 / 293, 287 / 311, 287 / 311 / 332, 287 / 324, 288 / 293, 288 / 293 / 311 / 324 / 353, 288 / 293 / 324, 293 / 311, and 311 / 332, where positions are referenced according to SEQ ID NO:16. In some additional embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from: 29R / 94K / 184Q / 288S / 353E, 29R / 94K / 184R / 232A / 288S / 293F / 311V / 324L / 353E, 29R / 94K / 184R / 287T / 293F, 29R / 94K / 184R / 288S / 293Q / 332V / 353E, 29R / 94K / 184R / 324L, 29R / 94K / 223S / 232A / 287K, 29R / 94K / 232A / 287T / 288S / 311V, 29R / 94K / 232A / 288S / 293F, 29R / 94K / 287K / 311V, 29R / 94K / 287T / 288S / 293F, 29R / 94K / 293H / 311V / 324L, 29R / 94K / 324L / 353E, 29R / 94R / 184Q / 223S / 232A / 288S / 293F, 29R / 94R / 184Q / 232A / 287T / 293F / 332V, 29R / 94R / 184Q / 232A / 288S / 311V / 324L / 332V, 29R / 94R / 184Q / 287K / 293F / 311V / 353E, 29R / 94R / 184Q / 287K / 311V, 29R / 94R / 184Q / 288S / 293F / 324L, 29R / 94R / 184R / 232A / 287K / 288S / 293F / 311V, 29R / 94R / 184R / 232A / 287T / 288S / 311V, 29R / 94R / 184R / 232A / 293F, 29R / 94R / 184R / 232A / 311V, 29R / 94R / 184R / 288S / 298A / 332V,29R / 94R / 184R / 293F / 311V, 29R / 94R / 223S / 288S / 293F, 29R / 94R / 223T / 232A / 293H / 311V / 324L, 29R / 94R / 287K / 288S / 293F, 29R / 94R / 287T / 288S / 293F, 29R / 94R / 287T / 288S / 293F / 353E, 29R / 94R / 293F / 332V / 353E, 29R / 94R / 311V, 29R / 184Q / 223S / 288S / 293F / 324L, 29R / 184Q / 287T / 288S / 293F, 29R / 184R, 29R / 184R / 232A / 288S, 29R / 184R / 287K / 288S / 293F / 311V, 29R / 184R / 287K / 311V / 332V, 29R / 184R / 287T, 29R / 184R / 288S / 293F, 29R / 184R / 288S / 293F / 311V, 29R / 184R / 293F, 29R / 184R / 293F / 311V, 29R / 184R / 293F / 324L, 29R / 184R / 353E, 29R / 223T / 287T / 288S / 293F / 353E, 29R / 232A / 287T / 288S / 293Q / 332V / 353E, 29R / 287T / 288S / 293F, 29R / 287T / 288S / 293F / 324L / 353E, 29R / 288S / 293F, 29R / 311V, 29R / 311V / 332V, 29R / 353E, 72V / 94R / 184R / 288S / 311V, 86Q, 94K / 96V / 223T / 287K / 288S / 293F / 311V / 324L / 332V, 94K / 184Q / 232A / 287T / 293F, 94K / 184Q / 232A / 287T / 293F / 332V, 94K / 184Q / 287T / 293F / 311V / 324L / 353E, 94K / 184R / 223S / 288S / 293F, 94K / 184R / 223T / 232A / 287K / 311V, 94K / 184R / 232A / 287K / 288S, 94K / 184R / 287T / 311V, 94K / 184R / 287T / 353E, 94K / 184R / 288S / 293F / 311V, 94K / 184R / 293H / 311V, 94K / 287K / 288S / 293F, 94K / 287T / 288S / 311V / 324L, 94K / 288S / 293F, 94K / 288S / 293F / 324L94K / 311V / 324L, 94R / 99T / 184R / 293F / 311V / 332V / 353E, 94R / 184Q / 287K / 288S / 311V, 94R / 184Q / 288S / 293F / 311V, 94R / 184Q / 293F / 332V / 353E, 94R / 184R / 223S, 94R / 184R / 223S / 293F, 94R / 184R / 232A / 287K / 288S / 324L / 332V, 94R / 184R / 287K / 288S / 293F, 94R / 184R / 287K / 288S / 311V, 94R / 184R / 288S / 293F, 94R / 184R / 288S / 293F / 311V / 332V, 94R / 184R / 293F, 94R / 223S / 232A / 311V / 353E / 355K / 356K / 357C / 358C / 359-, 94R / 223S / 287K / 288S / 293F, 94R / 223S / 288S / 293F / 311V, 94R / 232A / 287K / 288S / 293F / 353E, 94R / 232A / 288S / 293F / 353E, 94R / 232A / 293F, 94R / 232A / 293F / 324L / 332V, 94R / 232A / 311V, 94R / 287K / 288S / 293F, 94R / 287T / 288S / 293F, 94R / 293F / 324L, 94R / 311V / 324L, 153Y, 184Q / 223T / 287K / 288S / 353E, 184Q / 232A / 287K / 288S / 293F / 311V, 184Q / 232A / 287K / 288S / 324L / 353E, 184Q / 287K / 293F, 184R / 223T, 184R / 232A / 287K / 293F / 332V, 184R / 287K / 293F / 311V, 184R / 287T / 288S, 184R / 287T / 288S / 293F, 184R / 287T / 288S / 293F / 324L / 353E, 184R / 287T / 288S / 293Q, 184R / 288S / 293H / 311V / 324L, 184R / 332V / 353E, 205I, 205V, 223S / 232A / 287K / 288S / 332V / 353E, 223S / 287K / 288S / 293F / 311V, 223S / 287K / 311V / 353E, 223S / 287T / 288S, 223S / 288S / 293F, 232A / 287K / 288S / 293F232A / 287K / 288S / 293F / 311V, 232A / 287K / 311V, 232A / 287T / 288S / 293F / 321V, 232A / 288S / 293F, 232A / 288S / 293F / 332V, 234V / 287K / 288S / 293F / 311V, 287K / 288S / 293F, 287K / 293F, 287K / 311V / 332V, 287K / 324L, 287T / 288S / 311V / 324L, 287T / 311V, 288S / 293F, 288S / 293F / 311V / 324L / 353E, 288S / 293F / 324L, 293F / 311V, and 311V / 332V, wherein positions are referenced according to SEQ ID NO:16. In some embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from: S29R / N94K / V184Q / N288S / G353E, S29R / N94K / V184R / S232A / N288S / Y293F / A311V / D324L / G353E, S29R / N94K / V184R / I287T / Y293F, S29R / N94K / V184R / N288S / Y293Q / T332V / G353E, S29R / N94K / V184R / D324L, S29R / N94K / L223S / S232A / I287K, S29R / N94K / S232A / I287T / N288S / A311V, S29R / N94K / S232A / N288S / Y293F, S29R / N94K / I287K / A311V, S29R / N94K / I287T / N288S / Y293F, S29R / N94K / Y293H / A311V / D324L, S29R / N94K / D324L / G353E, S29R / N94R / V184Q / L223S / S232A / N288S / Y293F, S29R / N94R / V184Q / S232A / I287T / Y293F / T332V, S29R / N94R / V184Q / S232A / N288S / A311V / D324L / T332V, S29R / N94R / V184Q / I287K / Y293F / A311V / G353E, S29R / N94R / V184Q / I287K / A311V, S29R / N94R / V184Q / N288S / Y293F / D324L, S29R / N94R / V184R / S232A / I287K / N288S / Y293F / A311V, S29R / N94R / V184R / S232A / I287T / N288S / A311V,S29R / N94R / V184R / S232A / Y293F, S29R / N94R / V184R / S232A / A311V, S29R / N94R / V184R / N288S / V298A / T332V, S29R / N94R / V184R / Y293F / A311V, S29R / N94R / L223S / N288S / Y293F, S29R / N94R / L223T / S232A / Y293H / A311V / D324L, S29R / N94R / I287K / N288S / Y293F, S29R / N94R / I287T / N288S / Y293F, S29R / N94R / I287T / N288S / Y293F / G353E, S29R / N94R / Y293F / T332V / G353E, S29R / N94R / A311V, S29R / V184Q / L223S / N288S / Y293F / D324L, S29R / V184Q / I287T / N288S / Y293F, S29R / V184R, S29R / V184R / S232A / N288S, S29R / V184R / I287K / N288S / Y293F / A311V, S29R / V184R / I287K / A311V / T332V, S29R / V184R / I287T, S29R / V184R / N288S / Y293F, S29R / V184R / N288S / Y293F / A311V, S29R / V184R / Y293F, S29R / V184R / Y293F / A311V, S29R / V184R / Y293F / D324L, S29R / V184R / G353E, S29R / L223T / I287T / N288S / Y293F / G353E, S29R / S232A / I287T / N288S / Y293Q / T332V / G353E, S29R / I287T / N288S / Y293F, S29R / I287T / N288S / Y293F / D324L / G353E, S29R / N288S / Y293F, S29R / A311V, S29R / A311V / T332V, S29R / G353E, A72V / N94R / V184R / N288S / A311V, I86Q, N94K / A96V / L223T / I287K / N288S / Y293F / A311V / D324L / T332V, N94K / V184Q / S232A / I287T / Y293F, N94K / V184Q / S232A / I287T / Y293F / T332VN94K / V184Q / I287T / Y293F / A311V / D324L / G353E, N94K / V184R / L223S / N288S / Y293F, N94K / V184R / L223T / S232A / I287K / A311V, N94K / V184R / S232A / I287K / N288S, N94K / V184R / I287T / A311V, N94K / V184R / I287T / G353E, N94K / V184R / N288S / Y293F / A311V, N94K / V184R / Y293H / A311V, N94K / I287K / N288S / Y293F, N94K / I287T / N288S / A311V / D324L, N94K / N288S / Y293F, N94K / N288S / Y293F / D324L, N94K / A311V / D324L, N94R / I99T / V184R / Y293F / A311V / T332V / G353E, N94R / V184Q / I287K / N288S / A311V, N94R / V184Q / N288S / Y293F / A311V, N94R / V184Q / Y293F / T332V / G353E, N94R / V184R / L223S, N94R / V184R / L223S / Y293F, N94R / V184R / S232A / I287K / N288S / D324L / T332V, N94R / V184R / I287K / N288S / Y293F, N94R / V184R / I287K / N288S / A311V, N94R / V184R / N288S / Y293F, N94R / V184R / N288S / Y293F / A311V / T332V, N94R / V184R / Y293F, N94R / L223S / S232A / A311V / G353E / R355K / S356K / A357C / V358C / E359-, N94R / L223S / I287K / N288S / Y293F, N94R / L223S / N288S / Y293F / A311V, N94R / S232A / I287K / N288S / Y293F / G353E, N94R / S232A / N288S / Y293F / G353E, N94R / S232A / Y293F, N94R / S232A / Y293F / D324L / T332V, N94R / S232A / A311V, N94R / I287K / N288S / Y293F, N94R / I287T / N288S / Y293F, N94R / Y293F / D324LN94R / A311V / D324L, N153Y, V184Q / L223T / I287K / N288S / G353E, V184Q / S232A / I287K / N288S / Y293F / A311V, V184Q / S232A / I287K / N288S / D324L / G353E, V184Q / I287K / Y293F, V184R / L223T, V184R / S232A / I287K / Y293F / T332V, V184R / I287K / Y293F / A311V, V184R / I287T / N288S, V184R / I287T / N288S / Y293F, V184R / I287T / N288S / Y293F / D324L / G353E, V184R / I287T / N288S / Y293Q, V184R / N288S / Y293H / A311V / D324L, V184R / T332V / G353E, P205I, P205V, L223S / S232A / I287K / N288S / T332V / G353E, L223S / I287K / N288S / Y293F / A311V, L223S / I287K / A311V / G353E, L223S / I287T / N288S, L223S / N288S / Y293F, S232A / I287K / N288S / Y293F, S232A / I287K / N288S / Y293F / A311V, S232A / I287K / A311V, S232A / I287T / N288S / Y293F / A321V, S232A / N288S / Y293F, S232A / N288S / Y293F / T332V, A234V / I287K / N288S / Y293F / A311V, I287K / N288S / Y293F, I287K / Y293F, I287K / A311V / T332V, I287K / D324L, I287T / N288S / A311V / D324L, I287T / A311V, N288S / Y293F, N288S / Y293F / A311V / D324L / G353E, N288S / Y293F / D324L, Y293F / A311V and A311V / T332V, wherein the positions are numbered with reference to SEQ ID NO:16.,

[0014] The present invention provides an engineered polypeptide, the engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the reference sequence SEQ ID NO: 50, comprising at least one substitution or a set of substitutions at one or more positions selected from the following: 29 / 57 / 94 / 153 / 184 / 205 / 261 / 265 / 287, 29 / 57 / 94 / 153 / 205 / 265, 29 / 57 / 94 / 184 / 205 / 277 / 353, 29 / 57 / 94 / 205 / 259 / 261 / 265, 29 / 57 / 94 / 205 / 261 / 353, 29 / 57 / 153 / 184 / 205 / 259 / 261 / 265 / 277, 29 / 57 / 153 / 184 / 205 / 353, 29 / 57 / 153 / 205 / 261 / 265, 29 / 57 / 153 / 205 / 261 / 265 / 277, 29 / 57 / 153 / 205 / 265 / 277, 29 / 57 / 153 / 205 / 277 / 353, 29 / 57 / 184 / 205 / 259 / 261 / 265 / 277 / 287 / 357, 29 / 57 / 184 / 205 / 259 / 261 / 265 / 287, 29 / 57 / 205 / 259 / 261 / 265, 29 / 57 / 205 / 259 / 261 / 265 / 287, 29 / 57 / 205 / 261 / 265 / 353, 29 / 94 / 153 / 184 / 205, 29 / 94 / 153 / 205 / 259 / 261 / 265, 29 / 94 / 153 / 205 / 259 / 261 / 287, 29 / 94 / 153 / 205 / 259 / 261 / 287 / 353, 29 / 94 / 184 / 205 / 259 / 261, 29 / 94 / 184 / 205 / 261, 29 / 94 / 205, 29 / 94 / 205 / 259 / 261 / 287, 29 / 94 / 205 / 261, 29 / 94 / 261 / 265 / 287 / 353, 29 / 153 / 184 / 205, 29 / 153 / 205, 29 / 153 / 205 / 259 / 261 / 265 / 277 / 287, 29 / 153 / 261 / 265 / 287 / 353, 29 / 184 / 205, 29 / 184 / 205 / 259 / 261 / 277, 29 / 184 / 205 / 353, 29 / 184 / 261 / 265, 29 / 184 / 265, 29 / 205, 29 / 205 / 259 / 261, 29 / 205 / 259 / 261 / 265 / 287, 29 / 205 / 259 / 261 / 287, 29 / 205 / 261, 29 / 205 / 261 / 265,29 / 205 / 261 / 265 / 353、29 / 205 / 261 / 353、29 / 205 / 277、29 / 205 / 287、29 / 205 / 287 / 353、29 / 259 / 261 / 265、29 / 261 / 353、57 / 94 / 153 / 184 / 205 / 259 / 261、57 / 94 / 153 / 205 / 261 / 265、57 / 94 / 153 / 259 / 261 / 265、57 / 94 / 153 / 261 / 265、57 / 94 / 184 / 205 / 261 / 265 / 287、57 / 94 / 184 / 261 / 265 / 287、57 / 94 / 205 / 259、57 / 94 / 205 / 259 / 261 / 265 / 277 / 287、57 / 94 / 205 / 261 / 265、57 / 94 / 205 / 277、57 / 153 / 205 / 259 / 261 / 265 / 287、57 / 153 / 205 / 259 / 265 / 277、57 / 153 / 205 / 261、57 / 153 / 205 / 261 / 265、57 / 153 / 205 / 265、57 / 153 / 261 / 265、57 / 153 / 261 / 265 / 287 / 353、57 / 205、57 / 205 / 259 / 261、57 / 205 / 259 / 261 / 265 / 277 / 287 / 353、57 / 205 / 259 / 261 / 287 / 353、57 / 205 / 259 / 265、57 / 205 / 261、57 / 205 / 261 / 265 / 277 / 287 / 353、57 / 205 / 261 / 277、57 / 205 / 261 / 277 / 353、57 / 261 / 265 / 353、94 / 126 / 184 / 205 / 259 / 261 / 265、94 / 153 / 184 / 205、94 / 153 / 184 / 259 / 261 / 265、94 / 153 / 205 / 259 / 261 / 265 / 353、94 / 153 / 205 / 287、94 / 153 / 205 / 287 / 353、94 / 153 / 205 / 353、94 / 184 / 205 / 259 / 261、94 / 184 / 205 / 259 / 261 / 265 / 287、94 / 184 / 205 / 259 / 261 / 353、94 / 184 / 205 / 261 / 265 / 287 / 353、94 / 184 / 353、94 / 205、94 / 205 / 259 / 261 / 265、94 / 205 / 259 / 261 / 265 / 277 / 287、94 / 205 / 259 / 261 / 265 / 353、94 / 205 / 259 / 261 / 287 / 353、94 / 205 / 261 / 265 / 277、94 / 205 / 261 / 265 / 353, 94 / 205 / 261 / 353, 94 / 205 / 277, 94 / 259 / 261 / 265, 153 / 184 / 205 / 261 / 277 / 287, 153 / 184 / 261 / 265 / 287, 153 / 205, 153 / 205 / 261, 153 / 205 / 261 / 265 / 287, 153 / 259 / 261 / 265, 153 / 259 / 265 / 287, 153 / 261 / 265, 153 / 261 / 265 / 287 / 353, 153 / 265, 153 / 265 / 277 / 287 / 353, 184 / 205, 184 / 205 / 259 / 261 / 287, 184 / 205 / 261, 184 / 205 / 261 / 265, 184 / 205 / 261 / 287, 184 / 205 / 277, 184 / 205 / 287 / 353, 184 / 261 / 265 / 287, 202, 205, 205 / 259 / 261, 205 / 259 / 261 / 265, 205 / 259 / 261 / 265 / 277 / 287 / 353, 205 / 259 / 261 / 277 / 287, 205 / 259 / 265, 205 / 261, 205 / 261 / 265, 205 / 261 / 265 / 274, 205 / 261 / 265 / 287, 205 / 261 / 265 / 287 / 353, 205 / 261 / 277, 205 / 261 / 353, 205 / 277 / 287, 205 / 287, 223, 225, 256, 259 / 261 / 265, 261 / 265 / 287, 265 / 287, 283 and 294, where the positions are referenced by SEQ ID NO:50 numbering. In some additional embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from: 29S / 57G / 94K / 153F / 184R / 205C / 261L / 265I / 287T, 29S / 57G / 94K / 153L / 205A / 265I, 29S / 57G / 94K / 184R / 205C / 277V / 353E, 29S / 57G / 94K / 205C / 259F / 261S / 265I, 29S / 57G / 94K / 205C / 261L / 353E, 29S / 57G / 153F / 184R / 205C / 259F / 261V / 265I / 277V, 29S / 57G / 153F / 205C / 261L / 265I / 277V, 29S / 57G / 153F / 205C / 261V / 265I, 29S / 57G / 153F / 205C / 277V / 353E, 29S / 57G / 153L / 184R / 205A / 353E,29S / 57G / 153L / 205A / 265I / 277V, 29S / 57G / 184R / 205C / 259F / 261L / 265I / 277V / 287K / 357S, 29S / 57G / 184R / 205C / 259F / 261V / 265I / 287T, 29S / 57G / 205A / 259F / 261L / 265I / 287T, 29S / 57G / 205A / 259F / 261S / 265I, 29S / 57G / 205C / 261S / 265I / 353E, 29S / 94K / 153F / 205A / 259F / 261S / 287T / 353E, 29S / 94K / 153F / 205C / 259F / 261L / 287K, 29S / 94K / 153L / 184R / 205C, 29S / 94K / 153L / 205C / 259F / 261R / 265I, 29S / 94K / 184R / 205C / 259F / 261S, 29S / 94K / 184R / 205C / 261V, 29S / 94K / 205A, 29S / 94K / 205A / 261V, 29S / 94K / 205C / 259F / 261L / 287T, 29S / 94K / 261R / 265I / 287T / 353E, 29S / 153F / 184R / 205C, 29S / 153F / 205A, 29S / 153F / 261R / 265I / 287K / 353E, 29S / 153L / 205A / 259F / 261L / 265I / 277V / 287T, 29S / 184R / 205A, 29S / 184R / 205A / 353E, 29S / 184R / 205C / 259F / 261V / 277V, 29S / 184R / 261L / 265I, 29S / 184R / 265I, 29S / 205A, 29S / 205A / 259F / 261L / 265I / 287K, 29S / 205A / 259F / 261R, 29S / 205A / 261V, 29S / 205C / 259F / 261L, 29S / 205C / 259F / 261L / 265I / 287T, 29S / 205C / 259F / 261L / 287K, 29S / 205C / 259F / 261L / 287T, 29S / 205C / 259F / 261S, 29S / 205C / 261L, 29S / 205C / 261L / 265I / 353E, 29S / 205C / 261R / 353E, 29S / 205C / 261S, 29S / 205C / 261S / 265I, 29S / 205C / 261V / 265I, 29S / 205C / 277V, 29S / 205C / 287T29S / 205C / 287T / 353E, 29S / 259F / 261L / 265I, 29S / 261V / 353E, 57G / 94K / 153F / 184R / 205A / 259F / 261L, 57G / 94K / 153F / 205A / 261L / 265I, 57G / 94K / 153F / 259F / 261V / 265I, 57G / 94K / 153F / 261V / 265I, 57G / 94K / 184R / 205C / 261V / 265I / 287T, 57G / 94K / 184R / 261S / 265I / 287T, 57G / 94K / 205A / 259F, 57G / 94K / 205A / 259F / 261V / 265I / 277V / 287T, 57G / 94K / 205A / 277V, 57G / 94K / 205C / 259F, 57G / 94K / 205C / 261S / 265I, 57G / 153F / 205A / 261L / 265I, 57G / 153F / 205A / 261S, 57G / 153F / 205C / 259F / 261L / 265I / 287T, 57G / 153F / 205C / 259F / 265I / 277V, 57G / 153F / 205C / 265I, 57G / 153F / 261L / 265I / 287K / 353E, 57G / 153F / 261R / 265I, 57G / 205A, 57G / 205A / 259F / 261L / 265I / 277V / 287K / 353E, 57G / 205A / 259F / 261L / 287T / 353E, 57G / 205A / 259F / 261V, 57G / 205A / 261R, 57G / 205C, 57G / 205C / 259F / 265I, 57G / 205C / 261L / 265I / 277V / 287T / 353E, 57G / 205C / 261L / 277V, 57G / 205C / 261S / 277V / 353E, 57G / 261L / 265I / 353E, 94K / 126C / 184R / 205A / 259F / 261L / 265I, 94K / 153F / 184R / 259F / 261S / 265I, 94K / 153F / 205A / 287K / 353E, 94K / 153F / 205A / 353E, 94K / 153F / 205C / 287K, 94K / 153F / 205C / 287T / 353E, 94K / 153L / 184R / 205C, 94K / 153L / 205C / 259F / 261S / 265I / 353E, 94K / 184R / 205A / 259F / 261L / 353E,94K / 184R / 205A / 259F / 261S、94K / 184R / 205A / 261V / 265I / 287T / 353E、94K / 184R / 205C / 259F / 261S / 265I / 287K、94K / 184R / 353E、94K / 205A、94K / 205A / 259F / 261L / 265I、94K / 205A / 259F / 261L / 265I / 277V / 287K、94K / 205A / 259F / 261R / 265I、94K / 205A / 259F / 261V / 287K / 353E、94K / 205A / 277V、94K / 205C、94K / 205C / 259F / 261S / 265I、94K / 205C / 259F / 261S / 265I / 353E、94K / 205C / 259F / 261V / 265I / 353E、94K / 205C / 261L / 353E、94K / 205C / 261S / 265I / 277V、94K / 205C / 261V / 265I / 353E、94K / 259F / 261S / 265I、94K / 259F / 261V / 265I、153F / 184R / 205A / 261L / 277V / 287T、153F / 184R / 261S / 265I / 287T、153F / 205A、153F / 205A / 261R / 265I / 287T、153F / 205C、153F / 205C / 261L、153F / 205C / 261V、153F / 259F / 261V / 265I、153F / 259F / 265I / 287K、153F / 261L / 265I、153F / 261R / 265I、153F / 261S / 265I / 287T / 353E、153F / 265I、153F / 265I / 277V / 287T / 353E、184R / 205A / 261R / 265I、184R / 205A / 287T / 353E、184R / 205C、184R / 205C / 259F / 261L / 287K、184R / 205C / 261L、184R / 205C / 261R / 287K、184R / 205C / 277V、184R / 261L / 265I / 287T、202L、202M、205A、205A / 259F / 261V / 265I、205A / 259F / 265I、205A / 261L、205A / 261R / 265I / 287K、205C、205C / 259F / 261L、205C / 259F / 261R / 265I / 277V / 287T / 353E、205C / 259F / 261S、205C / 259F / 261S / 265I, 205C / 259F / 261V / 277V / 287T, 205C / 259F / 265I, 205C / 261L / 265I / 274A, 205C / 261L / 277V, 205C / 261L / 353E, 205C / 261R, 205C / 261R / 265I / 287K / 353E, 205C / 261S, 205C / 261V / 265I, 205C / 277V / 287K, 205C / 287K, 205C / 287T, 205S, 205T, 223G, 225F, 225Y, 256L, 256V, 259F / 261L / 265I, 259F / 261V / 265I, 261R / 265I / 287K, 261S / 265I / 287T, 265I / 287T, 283L, and 294I, wherein positions are referenced according to the numbering of SEQ ID NO:50. In some embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from: R29S / A57G / N94K / N153F / Q184R / P205C / E261L / Q265I / I287T, R29S / A57G / N94K / N153L / P205A / Q265I, R29S / A57G / N94K / Q184R / P205C / N277V / G353E, R29S / A57G / N94K / P205C / Y259F / E261S / Q265I, R29S / A57G / N94K / P205C / E261L / G353E, R29S / A57G / N153F / Q184R / P205C / Y259F / E261V / Q265I / N277V, R29S / A57G / N153F / P205C / E261L / Q265I / N277V, R29S / A57G / N153F / P205C / E261V / Q265I, R29S / A57G / N153F / P205C / N277V / G353E, R29S / A57G / N153L / Q184R / P205A / G353E, R29S / A57G / N153L / P205A / Q265I / N277V, R29S / A57G / Q184R / P205C / Y259F / E261L / Q265I / N277V / I287K / A357S, R29S / A57G / Q184R / P205C / Y259F / E261V / Q265I / I287T, R29S / A57G / P205A / Y259F / E261L / Q265I / I287T, R29S / A57G / P205A / Y259F / E261S / Q265I,R29S / A57G / P205C / E261S / Q265I / G353E, R29S / N94K / N153F / P205A / Y259F / E261S / I287T / G353E, R29S / N94K / N153F / P205C / Y259F / E261L / I287K, R29S / N94K / N153L / Q184R / P205C, R29S / N94K / N153L / P205C / Y259F / E261R / Q265I, R29S / N94K / Q184R / P205C / Y259F / E261S, R29S / N94K / Q184R / P205C / E261V, R29S / N94K / P205A, R29S / N94K / P205A / E261V, R29S / N94K / P205C / Y259F / E261L / I287T, R29S / N94K / E261R / Q265I / I287T / G353E, R29S / N153F / Q184R / P205C, R29S / N153F / P205A, R29S / N153F / E261R / Q265I / I287K / G353E, R29S / N153L / P205A / Y259F / E261L / Q265I / N277V / I287T, R29S / Q184R / P205A, R29S / Q184R / P205A / G353E, R29S / Q184R / P205C / Y259F / E261V / N277V, R29S / Q184R / E261L / Q265I, R29S / Q184R / Q265I, R29S / P205A, R29S / P205A / Y259F / E261L / Q265I / I287K, R29S / P205A / Y259F / E261R, R29S / P205A / E261V, R29S / P205C / Y259F / E261L, R29S / P205C / Y259F / E261L / Q265I / I287T, R29S / P205C / Y259F / E261L / I287K, R29S / P205C / Y259F / E261L / I287T, R29S / P205C / Y259F / E261S, R29S / P205C / E261L, R29S / P205C / E261L / Q265I / G353E, R29S / P205C / E261R / G353E, R29S / P205C / E261S, R29S / P205C / E261S / Q265I, R29S / P205C / E261V / Q265I, R29S / P205C / N277V, R29S / P205C / I287TR29S / P205C / I287T / G353E, R29S / Y259F / E261L / Q265I, R29S / E261V / G353E, A57G / N94K / N153F / Q184R / P205A / Y259F / E261L, A57G / N94K / N153F / P205A / E261L / Q265I, A57G / N94K / N153F / Y259F / E261V / Q265I, A57G / N94K / N153F / E261V / Q265I, A57G / N94K / Q184R / P205C / E261V / Q265I / I287T, A57G / N94K / Q184R / E261S / Q265I / I287T, A57G / N94K / P205A / Y259F, A57G / N94K / P205A / Y259F / E261V / Q265I / N277V / I287T, A57G / N94K / P205A / N277V, A57G / N94K / P205C / Y259F, A57G / N94K / P205C / E261S / Q265I, A57G / N153F / P205A / E261L / Q265I, A57G / N153F / P205A / E261S, A57G / N153F / P205C / Y259F / E261L / Q265I / I287T, A57G / N153F / P205C / Y259F / Q265I / N277V, A57G / N153F / P205C / Q265I, A57G / N153F / E261L / Q265I / I287K / G353E, A57G / N153F / E261R / Q265I, A57G / P205A, A57G / P205A / Y259F / E261L / Q265I / N277V / I287K / G353E, A57G / P205A / Y259F / E261L / I287T / G353E, A57G / P205A / Y259F / E261V, A57G / P205A / E261R, A57G / P205C, A57G / P205C / Y259F / Q265I, A57G / P205C / E261L / Q265I / N277V / I287T / G353E, A57G / P205C / E261L / N277V, A57G / P205C / E261S / N277V / G353E, A57G / E261L / Q265I / G353E, N94K / G126C / Q184R / P205A / Y259F / E261L / Q265I, N94K / N153F / Q184R / Y259F / E261S / Q265IN94K / N153F / P205A / I287K / G353E, N94K / N153F / P205A / G353E, N94K / N153F / P205C / I287K, N94K / N153F / P205C / I287T / G353E, N94K / N153L / Q184R / P205C, N94K / N153L / P205C / Y259F / E261S / Q265I / G353E, N94K / Q184R / P205A / Y259F / E261L / G353E, N94K / Q184R / P205A / Y259F / E261S, N94K / Q184R / P205A / E261V / Q265I / I287T / G353E, N94K / Q184R / P205C / Y259F / E261S / Q265I / I287K, N94K / Q184R / G353E, N94K / P205A, N94K / P205A / Y259F / E261L / Q265I, N94K / P205A / Y259F / E261L / Q265I / N277V / I287K, N94K / P205A / Y259F / E261R / Q265I, N94K / P205A / Y259F / E261V / I287K / G353E, N94K / P205A / N277V, N94K / P205C, N94K / P205C / Y259F / E261S / Q265I, N94K / P205C / Y259F / E261S / Q265I / G353E, N94K / P205C / Y259F / E261V / Q265I / G353E, N94K / P205C / E261L / G353E, N94K / P205C / E261S / Q265I / N277V, N94K / P205C / E261V / Q265I / G353E, N94K / Y259F / E261S / Q265I, N94K / Y259F / E261V / Q265I, N153F / Q184R / P205A / E261L / N277V / I287T, N153F / Q184R / E261S / Q265I / I287T, N153F / P205A, N153F / P205A / E261R / Q265I / I287T, N153F / P205C, N153F / P205C / E261L, N153F / P205C / E261V, N153F / Y259F / E261V / Q265I, N153F / Y259F / Q265I / I287K, N153F / E261L / Q265I, N153F / E261R / Q265I, N153F / E261S / Q265I / I287T / G353E,N153F / Q265I, N153F / Q265I / N277V / I287T / G353E, Q184R / P205A / E261R / Q265I, Q184R / P205A / I287T / G353E, Q184R / P205C, Q184R / P205C / Y259F / E261L / I287K, Q184R / P205C / E261L, Q184R / P205C / E261R / I287K, Q184R / P205C / N277V, Q184R / E261L / Q265I / I287T, H202L, H202M, P205A, P205A / Y259F / E261V / Q265I, P205A / Y259F / Q265I, P205A / E261L, P205A / E261R / Q265I / I287K, P205C, P205C / Y259F / E261L, P205C / Y259F / E261R / Q265I / N277V / I287T / G353E, P205C / Y259F / E261S, P205C / Y259F / E261S / Q265I, P205C / Y259F / E261V / N277V / I287T, P205C / Y259F / Q265I, P205C / E261L / Q265I / V274A, P205C / E261L / N277V, P205C / E261L / G353E, P205C / E261R, P205C / E261R / Q265I / I287K / G353E, P205C / E261S, P205C / E261V / Q265I, P205C / N277V / I287K, P205C / I287K, P205C / I287T, P205S, P205T, S223G, G225F, G225Y, C256L, C256V, Y259F / E261L / Q265I, Y259F / E261V / Q265I, E261R / Q265I / I287K, E261S / Q265I / I287T, Q265I / I287T, I283L and F294I, wherein the positions are referenced according to the numbering of SEQ ID NO:50.

[0015] The present invention provides an engineered polypeptide, the engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the reference sequence SEQ ID NO: 306, comprising at least one substitution or one set of substitutions at one or more positions selected from: 197 / 198 / 201 / 259, 198 / 201 / 259 / 280, 202 / 205 / 221 / 223 / 225 / 261, 202 / 205 / 221 / 223 / 261 / 294, 202 / 221 / 222 / 225 / 256 / 261 / 294, 202 / 221 / 223 / 225 / 283 / 294, 205 / 221 / 222, 205 / 221 / 223 / 225 / 256 / 261 / 283, 219, 221 / 222 / 223 / 225, 221 / 223, 221 / 223 / 225, 221 / 223 / 225 / 256 / 261, 221 / 223 / 225 / 261, 221 / 223 / 225 / 294, 221 / 225, 221 / 225 / 256, 221 / 225 / 256 / 261, 221 / 225 / 256 / 261 / 283 / 294, 223 / 225 / 256 / 261, 225 / 256 / 261, 280 and 280 / 306, wherein the positions are numbered with reference to SEQ ID NO: 306.In some additional embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from: 197T / 198S / 201L / 259V, 198A / 201L / 259V / 280L, 202L / 205S / 221S / 223G / 225Y / 261Y, 202L / 221H / 223G / 225F / 283M / 294I, 202M / 205S / 221H / 223G / 261Y / 294I, 202M / 205T / 221L / 223G / 225Y / 261R, 202M / 221S / 222I / 225Y / 256L / 261R / 294I, 205S / 221S / 223G / 225F / 256L / 261T / 283M, 205T / 221H / 222I, 219L, 221H / 222I / 223G / 225Y, 221H / 223G / 225L, 221H / 225F, 221L / 223G / 225L / 256T / 261R, 221L / 225F / 256V, 221S / 223G, 221S / 223G / 225L / 256L / 261R, 221S / 223G / 225L / 261R, 221S / 223G / 225L / 294I, 221S / 225F / 256T / 261R, 221S / 225Y / 256T / 261Y / 283M / 294I, 223G / 225L / 256V / 261R, 225F / 256T / 261T, 280D, 280E, 280I, 280V, 280V / 306F, and 280W, where positions are numbered with reference to SEQ ID NO:306.In some embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from: V197T / H198S / M201L / Y259V, H198A / M201L / Y259V / Y280L, H202L / A205S / Y221S / S223G / G225Y / V261Y, H202L / Y221H / S223G / G225F / I283M / F294I, H202M / A205S / Y221H / S223G / V261Y / F294I, H202M / A205T / Y221L / S223G / G225Y / V261R, H202M / Y221S / F222I / G225Y / C256L / V261R / F294I, A205S / Y221S / S223G / G225F / C256L / V261T / I283M, A205T / Y221H / F222I, F219L, Y221H / F222I / S223G / G225Y, Y221H / S223G / G225L, Y221H / G225F, Y221L / S223G / G225L / C256T / V261R, Y221L / G225F / C256V, Y221S / S223G, Y221S / S223G / G225L / C256L / V261R, Y221S / S223G / G225L / V261R, Y221S / S223G / G225L / F294I, Y221S / G225F / C256T / V261R, Y221S / G225Y / C256T / V261Y / I283M / F294I, S223G / G225L / C256V / V261R, G225F / C256T / V261T, Y280D, Y280E, Y280I, Y280V, Y280V / S306F, and Y280W, wherein positions are referenced according to SEQ ID NO: 306 numbering.

[0016] The present invention provides an engineered polypeptide, the engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity with the reference sequence SEQ ID NO: 648, comprising at least one substitution or a set of substitutions at one or more positions selected from: 17 / 198 / 259 / 280, 197, 197 / 198, 197 / 198 / 223 / 259, 197 / 223 / 277 / 280, 197 / 259, 197 / 277, 198, 198 / 223, 198 / 223 / 259 / 277 / 280, 198 / 259, 198 / 259 / 277, 198 / 259 / 277 / 280, 198 / 277 / 280, 223 / 259, 223 / 259 / 280, 258, 259, 259 / 268 / 277 / 280, 259 / 277, 259 / 280, 263, 277 and 280, wherein the positions are numbered with reference to SEQ ID NO: 648. In some additional embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from: 17P / 198S / 259V / 280F, 17P / 198S / 259W / 280W, 197L, 197L / 198S, 197L / 198S / 223G / 259W, 197L / 223G / 277T / 280K, 197L / 259W, 197L / 277F, 198A, 198S, 198S / 223G, 198S / 223G / 259W / 277W / 280G, 198S / 259V, 198S / 259V / 277T, 198S / 259W / 277F / 280G, 198S / 259W / 277T / 280F, 198S / 277F / 280F, 198S / 277G / 280W, 198S / 277P / 280F, 223G / 259V / 280W, 223G / 259W, 258Y, 259N / 280W, 259W, 259W / 268S / 277T / 280F, 259W / 277W, 263D, 263E, 263F, 263G, 263L, 263M, 263N, 263Q, 263R, 263V, 263W, 277W, 280D, 280F and 280W, wherein the positions are numbered with reference to SEQ ID NO: 648.In some embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from: H17P / H198S / Y259V / Y280F, H17P / H198S / Y259W / Y280W, V197L, V197L / H198S, V197L / H198S / S223G / Y259W, V197L / S223G / N277T / Y280K, V197L / Y259W, V197L / N277F, H198A, H198S, H198S / S223G, H198S / S223G / Y259W / N277W / Y280G, H198S / Y259V, H198S / Y259V / N277T, H198S / Y259W / N277F / Y280G, H198S / Y259W / N277T / Y280F, H198S / N277F / Y280F, H198S / N277G / Y280W, H198S / N277P / Y280F, S223G / Y259V / Y280W, S223G / Y259W, W258Y, Y259N / Y280W, Y259W, Y259W / A268S / N277T / Y280F, Y259W / N277W, Y263D, Y263E, Y263F, Y263G, Y263L, Y263M, Y263N, Y263Q, Y263R, Y263V, Y263W, N277W, Y280D, Y280F, and Y280W, where positions are numbered with reference to SEQ ID NO:648.

[0017] The present invention provides an engineered polypeptide comprising an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the reference sequence SEQ ID NO:708, comprising at least one substitution or set of substitutions at one or more positions selected from 141, 154, 197, 197 / 198, and 278, where positions are numbered with reference to SEQ ID NO:708. In some additional embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from 141D, 154G, 154H, 197A, 197P / 198A, 278E, and 278V, where positions are numbered with reference to SEQ ID NO:708. In some embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from T141D, A154G, A154H, V197A, V197P / S198A, P278E, and P278V, where positions are numbered with reference to SEQ ID NO:708.

[0018] The present invention also provides engineered polynucleotides encoding at least one of the engineered polypeptides described in the foregoing paragraphs. In some embodiments, the engineered polynucleotides comprise the odd-numbered sequences listed in SEQ ID NOs: 5 to 801.

[0019] The present invention also provides vectors, which comprise at least one of the engineered polynucleotides described above. In some embodiments, the vectors further comprise at least one control sequence.

[0020] The present invention also provides host cells, which comprise the vectors provided herein. In some embodiments, the host cells produce at least one of the engineered polypeptides provided herein.

[0021] The present invention also provides a method for producing an engineered imine reductase polypeptide, the method comprising culturing the host cells provided herein under conditions such that the engineered polynucleotide is expressed and the engineered polypeptide is produced. In some embodiments, the method further comprises the step of recovering the engineered polypeptide.

[0022] Description of the Invention

[0023] 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. In general, the nomenclature used herein and the experimental procedures in 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 described in many textbooks and reference works well known to those skilled in the art. Standard techniques or modified forms 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 herein by reference.

[0024] Although any suitable methods and materials similar or equivalent to those described herein may be used 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 methods, protocols, and reagents described, as these may vary depending on how those skilled in the art use them. Accordingly, the terms defined below are more fully described by reference to the present invention as a whole.

[0025] It should be understood that the foregoing general description and the following detailed description are merely exemplary and illustrative, and do not limit the present invention. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Numerical ranges include the numbers defining the range. Thus, each numerical range disclosed herein is intended to include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written out herein. It is also intended that each maximum (or minimum) numerical limitation disclosed herein include every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written out herein.

[0026] Unless the context clearly indicates otherwise, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "a polypeptide" includes more than one polypeptide. Similarly, "comprise", "comprises", and "comprising" are interchangeable with "include", "includes", and "including" and are not intended to be limiting.

[0027] It should be understood that when the description of various embodiments uses the term "comprising", those skilled in the art will understand that in some specific cases, optionally, the language "consisting essentially of" or "consisting of" may be used to describe the embodiments. It should also be understood that in cases where the term "optional" or "optionally" is used in the description of various embodiments, the subsequent described event or situation may or may not occur, and the description includes the case where the event or situation occurs and the case where it does not occur. It should be understood that both the foregoing general description and the following detailed description are merely exemplary and illustrative, and do not limit the present disclosure. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0028] Abbreviations:

[0029] The abbreviations for the amino acids used in genetic coding are conventional and are as follows:

[0030] Amino acid Three-letter abbreviation One-letter abbreviation Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

[0031] When using the three-letter abbreviations, unless specifically preceded by "L" or "D", or clearly apparent from the context in which the abbreviation is used, the amino acid may be with respect to the α-carbon (C α) is of the L-configuration or D-configuration. For example, "Ala" represents alanine without specifying the configuration regarding the α-carbon, while "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively.

[0032] When using single-letter abbreviations, capital letters represent amino acids of the L-configuration regarding the α-carbon, and lowercase letters represent amino acids of the D-configuration regarding the α-carbon. For example, "A" represents L-alanine and "a" represents D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or a mixture thereof), the sequence is presented in the amino (N) to carboxyl (C) direction according to conventional convention.

[0033] 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 specifically described, the abbreviated nucleosides can be ribonucleosides or 2'-deoxyribonucleosides. Nucleosides can be specified as ribonucleosides or 2'-deoxyribonucleosides either individually or collectively. When a nucleic acid sequence is presented as a string of single-letter abbreviations, the sequence is presented in the 5' to 3' direction according to conventional convention, and the phosphates are not shown.

[0034] Definitions:

[0035] Referring to the present invention, the technical and scientific terms used in the description herein will have the meanings commonly understood by those of ordinary skill in the art, unless otherwise clearly defined. Thus, the following terms are intended to have the following meanings.

[0036] The "EC" number refers to the enzyme nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). This IUBMB biochemical classification is an enzyme number classification system based on the chemical reactions catalyzed by enzymes.

[0037] "ATCC" refers to the American Type Culture Collection, whose biological deposit collection includes genes and strains.

[0038] "NCBI" refers to the National Center for Biological Information in the United States and the sequence databases provided therein.

[0039] "Protein", "polypeptide", and "peptide" are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modifications (such as glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.). This definition includes D-amino acids and L-amino acids, as well as mixtures of D-amino acids and L-amino acids, and polymers comprising D-amino acids and L-amino acids and mixtures of D-amino acids and L-amino acids.

[0040] "Amino acid" is referred to herein by its commonly known three-letter symbol or by the single-letter symbol recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Similarly, nucleotides can be referred to by their commonly accepted single-letter codes.

[0041] As used herein, "polynucleotide" and "nucleic acid" refer to two or more nucleosides covalently linked together. Polynucleotides can consist entirely of ribonucleotides (i.e., RNA), consist entirely of 2'-deoxyribonucleotides (i.e., DNA), or contain a mixture of ribonucleotides and 2'-deoxyribonucleotides. Although nucleosides will typically be linked together via standard phosphodiester linkages, polynucleotides can include one or more non-standard linkages. Polynucleotides can be single-stranded or double-stranded, or can include both single-stranded regions and double-stranded regions. Additionally, although polynucleotides typically consist of naturally occurring coding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it can 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 the nucleobases encoding an amino acid sequence.

[0042] "Opine dehydrogenase activity" as used herein refers to the enzymatic activity in which the carbonyl group of a 2-keto acid (such as pyruvate) and the amino group of a neutral L-amino acid (such as L-norvaline) are converted to a secondary amine dicarboxylic acid compound (such as N-[1-(R)-(carboxy)ethyl]-(S)-norvaline).

[0043] As used herein, "imine" refers to an organic compound or functional group containing a nitrogen-carbon double bond, wherein the nitrogen is bonded to hydrogen or an organic group.

[0044] "Imine reductase activity" as used herein refers to the enzymatic activity as illustrated in Scheme 1, in which, in the presence of the cofactor NAD(P)H, the carbonyl group of a ketone or aldehyde and the amino group of a primary or secondary amine (wherein the carbonyl group and the amino group can be on separate compounds or on the same compound) are converted to a secondary or tertiary amine product compound.

[0045] As used herein, "imine reductase" or "IRED" refers to an enzyme having imine reductase activity. It should be understood that imine reductase is not limited to engineered polypeptides derived from the wild-type opine dehydrogenase (L-valine dehydrogenase, a type of opine dehydrogenase) of Arthrobacter sp. strain 1C, but may include other enzymes having imine reductase activity, including engineered polypeptides derived from other opine dehydrogenases, such as octopine dehydrogenase (OpDH) from Pecten maximus, ornithine synthase (CEOS) from Lactococcus lactis K1, β-alanopine dehydrogenase (BADH) from Cellana grata, tauropine dehydrogenase (TauDH) from Suberites domuncula, and N-methyl-L-amino acid dehydrogenase (NMDH) from Pseudomonas putida; or engineered enzymes derived from wild-type enzymes having imine reductase activity. Imine reductase as used herein includes naturally occurring (wild-type) imine reductase and non-naturally occurring engineered polypeptides produced by human manipulation.

[0046] "Coding sequence" refers to the nucleic acid portion (e.g., gene) that encodes the amino acid sequence of a protein.

[0047] "Naturally occurring" or "wild-type" refers to the form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence that exists in an organism that can be isolated from a natural source and has not been intentionally modified by human manipulation.

[0048] As used herein, when used in reference to a cell, nucleic acid, or polypeptide, "recombinant", "engineered", and "non-naturally occurring" refer to a material that has been modified in a manner not found in nature or to a material corresponding to the natural or native form of the material. In some embodiments, a 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 using recombinant techniques. Non-limiting examples include, among others, recombinant cells that express genes not found in cells in their natural (non-recombinant) form or that express natural genes that are otherwise expressed at different levels.

[0049] "Percent sequence identity" and "percent homology" are used interchangeably herein to refer to a comparison between polynucleotides or polypeptides and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence to which it is optimally aligned for the two sequences. The percent may be calculated as follows: determine the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, divide the number of matched positions by the total number of positions in the comparison window, and multiply the result by 100 to yield the percent sequence identity. Optionally, the percent may be calculated as follows: determine the number of positions at which the identical nucleic acid base or amino acid residue or a nucleic acid base or amino acid residue is aligned with a gap occurs in both sequences to yield the number of matched positions, divide the number of matched positions by the total number of positions in the comparison window, and multiply the result by 100 to yield the percent sequence identity. Those skilled in the art understand that there are many established algorithms available for aligning two sequences. The optimal alignment of the sequences to be compared can be conducted, for example, by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482

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

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

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

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

[1977] ). Software for performing BLAST analyses is available to the public through the National Center for Biotechnology Information website. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy a positive-valued threshold score T when aligned with words of the same length in the database sequence.T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits serve as seeds to initiate the search for longer HSPs that contain them. The word hits are then extended in both directions along each sequence until the cumulative alignment score can no longer increase. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a pair of mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. The extension of the word hit in each direction is stopped when: the cumulative alignment score has decreased by amount X from its maximum achieved value; the cumulative score reaches 0 or less than 0 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 the following as defaults: word length (W) of 11, expectation value (E) of 10, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program uses the following as defaults: word length (W) of 3, expectation value (E) of 10, and the BLOSUM62 scoring matrix (see, Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

[1989] ). Exemplary determination of sequence alignment and % sequence identity can be made using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison WI), using the default parameters provided.

[0050] "Reference sequence" means a designated sequence that serves as a basis for sequence comparison. A reference sequence can be a subset of a larger sequence, e.g., a segment of a full-length gene or polypeptide sequence. Typically, 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, or the full length of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides can each (1) contain sequences that are similar between the two sequences (i.e., a portion of the complete sequence), and (2) can also contain sequences that are different between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides typically involves identifying and comparing local regions of sequence similarity by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window". In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more variations in the primary sequence. For example, "a reference sequence based on SEQ ID NO:4 having valine at the residue corresponding to X14" or X14V means a reference sequence in which the corresponding residue (tyrosine) at X14 in SEQ ID NO:4 has been changed to valine.

[0051] "Comparison window" means a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues, wherein a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and wherein the portion of the sequence in the comparison window can contain 20% or fewer additions or deletions (i.e., gaps) compared to the reference sequence for optimal alignment of the two sequences (which does not contain additions or deletions). The comparison window can be longer than 20 contiguous residues and optionally includes windows of 30, 40, 50, 100 or longer.

[0052] As used herein, "substantial identity" means a polynucleotide or polypeptide sequence that has at least 80% sequence identity, at least 85% identity, sequence identity between at least 89% and 95%, or more typically at least 99% sequence identity compared to a reference sequence in a comparison window of at least 20 residue positions, typically in a window of at least 30 - 50 residues, where the percentage of sequence identity is calculated by comparing the reference sequence with a sequence that contains a total of 20% or less deletions or additions relative to the reference sequence in the comparison window. In some specific embodiments applied to polypeptides, the term "substantial identity" means that when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, two polypeptide sequences share at least 80% sequence identity, preferably at least 89% sequence identity, at least 95% sequence identity or more (e.g., 99% sequence identity). In some embodiments, the residue positions that are not identical in the sequences being compared differ by conservative amino acid substitutions.

[0053] When used in the context of the numbering of a given amino acid or polynucleotide sequence, "corresponds to", "reference to", and "relative to" refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue numbering or residue positions of a given polymer are assigned with respect to the reference sequence, rather than by the actual numerical positions of the residues within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence such as the amino acid sequence of an engineered imine reductase can be aligned with a reference sequence by introducing gaps to optimize the residue matches between the two sequences. In these cases, the residues in the given amino acid or polynucleotide sequence are numbered with respect to the reference sequence to which it is aligned, despite the presence of gaps.

[0054] "Amino acid difference" or "residue difference" refers to a change in an amino acid residue at a position in a polypeptide sequence relative to the amino acid residue at the corresponding position in a reference sequence. The position of the amino acid difference in this document is typically referred to 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 X25 compared to SEQ ID NO:2" refers to a change in the amino acid residue at the polypeptide position corresponding to position 25 of SEQ ID NO:2. Thus, if the reference polypeptide SEQ ID NO:2 has valine at position 25, "a residue difference at position X25 compared to SEQ ID NO:2" refers to an amino acid substitution of any residue other than valine at the position of the polypeptide corresponding to position 25 of SEQ ID NO:2. In most instances in this document, a specific amino acid residue difference at a position is indicated 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 different from that in the reference polypeptide). In some embodiments, more than one amino acid may be present at the designated residue position (i.e., alternative amino acids may be listed in the form of XnY / Z, where Y and Z represent the alternative amino acid residues). In some cases (e.g., in Tables 5.1, 6.1, 7.1, 8.1, 9.1, and 10.1), the present invention also provides specific amino acid differences represented by the conventional symbol "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 addition, in some cases, the polypeptides of the present invention may include one or more amino acid residue differences relative to the reference sequence, which are indicated by a list of designated positions that are altered relative to the reference sequence. In some additional embodiments, the present invention provides engineered polypeptide sequences that include both conservative amino acid substitutions and non-conservative amino acid substitutions.

[0055] As used herein, "conservative amino acid substitution" refers to the replacement of a residue with a different residue having a similar side chain, and thus generally includes the replacement of an amino acid in a polypeptide with an amino acid in the same or a similar amino acid-defined category. By way of example and not limitation, an amino acid having an aliphatic side chain is replaced with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid having a hydroxyl side chain is replaced with another amino acid having a hydroxyl side chain (e.g., serine and threonine); an amino acid having an aromatic side chain is replaced with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid having a basic side chain is replaced with another amino acid having a basic side chain (e.g., lysine and arginine); an amino acid having an acidic side chain is replaced with another amino acid having an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic amino acid or a hydrophilic amino acid is replaced with another hydrophobic amino acid or hydrophilic amino acid, respectively. Exemplary conservative substitutions are provided in Table 1 below.

[0056]

[0057] "Non-conservative substitution" refers to the replacement of an amino acid in a polypeptide with an amino acid having significantly different side chain characteristics. Non-conservative substitutions can use amino acids between defined groups rather than within defined groups and affect (a) the structure of the peptide backbone in the substitution region (e.g., replacing glycine with proline), (b) charge or hydrophobicity, or (c) the volume of the side chain. By way of example and not limitation, exemplary non-conservative substitutions can be replacing an acidic amino acid with a basic or aliphatic amino acid; replacing an aromatic amino acid with a small amino acid; and replacing a hydrophilic amino acid with a hydrophobic amino acid.

[0058] "Deletion" refers to a polypeptide modification by removing one or more amino acids from a reference polypeptide. Deletions can include removing 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids composing the reference enzyme, or up to 20% of the total number of amino acids, while retaining enzyme activity and / or retaining the improved properties of the engineered imine reductase. Deletions can involve internal and / or terminal portions of the polypeptide. In various embodiments, deletions can include contiguous segments or can be non-contiguous.

[0059] "Insertion" refers to a polypeptide modification by adding one or more amino acids to a reference polypeptide. In some embodiments, the improved engineered imine reductase includes inserting one or more amino acids into a naturally occurring polypeptide having imine reductase activity, and inserting one or more amino acids into other improved imine reductase polypeptides. The insertion can be in the internal portion of the polypeptide or an insertion at the carboxyl or amino terminus. As used herein, an insertion includes a fusion protein as known in the art. The insertion can be a continuous segment of amino acids, or separated by one or more amino acids in the naturally occurring polypeptide.

[0060] As used herein, a "fragment" refers to a polypeptide having an amino terminus and / or carboxyl terminus deletion but the remaining amino acid sequence being the same as the corresponding position in the sequence. The fragment can be at least 14 amino acids long, at least 20 amino acids long, at least 50 amino acids long or longer, and up to the full-length imine reductase polypeptide such as SEQ ID NO:2 or 70%, 80%, 90%, 95%, 98% and 99% of the engineered imine reductase provided in the even-numbered sequences of SEQ ID NO:6 to SEQ ID NO:802.

[0061] "Isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants such as proteins, lipids, and polynucleotides that naturally accompany it. The term includes polypeptides that have been removed or purified from their natural environment or expression system (e.g., host cell or in vitro synthesis). The engineered imine reductase can be present intracellularly, in cell culture medium, or prepared in various forms such as lysates or isolated products. Thus, in some embodiments, the engineered imine reductase can be an isolated polypeptide.

[0062] "Substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis, it is more abundant than any other individual macromolecular species in the composition), and is typically a substantially pure composition when the target species constitutes at least about 50% of the macromolecular species present on a molar or % weight basis. Generally, a substantially pure imine reductase composition will contain 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 all the macromolecular species present in the composition on a molar or % weight basis. In some embodiments, the target substance is purified to substantially homogeneous (i.e., contaminant substances cannot be detected in the composition by conventional detection methods), where the composition consists essentially of a single macromolecular substance. Solvent substances, small molecules (<500 daltons), and elemental ion substances are not considered macromolecular substances. In some embodiments, the isolated engineered imine reductase polypeptide is a substantially pure polypeptide composition.

[0063] "Stereoselectivity" refers to the preferential formation of one stereoisomer over another in a chemical or enzymatic reaction. Stereoselectivity can be partial, where the formation of one stereoisomer is favored over the other, or stereoselectivity can be complete, where only one stereoisomer is formed. When the stereoisomers are enantiomers, the stereoselectivity is referred to as enantioselectivity, which is the fraction (usually reported as a percentage) of one enantiomer in the sum of the two. Optionally, it is commonly reported in the art as the enantiomeric excess (e.e.) (usually a percentage) calculated according to the formula: [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer]. When the stereoisomers are diastereomers, the stereoselectivity is referred to as diastereoselectivity, which is the fraction (usually reported as a percentage) of one diastereomer in a mixture of two diastereomers, and is usually optionally reported as the diastereomeric excess (d.e.). Enantiomeric excess and diastereomeric excess are types of stereoisomeric excess.

[0064] "Highly stereoselective" refers to a chemical or enzymatic reaction that is capable of converting one or more substrates (e.g., substrate compounds (2) and (3)) to the corresponding amine product (e.g., compound (1)) with at least about 85% stereoisomeric excess.

[0065] As used herein, "improved enzyme properties" refers to at least one improved property of an enzyme. In some embodiments, the present invention provides engineered imine reductase polypeptides that exhibit an improvement in any enzyme property as compared to a reference imine reductase polypeptide and / or a wild-type imine reductase polypeptide and / or another engineered imine reductase polypeptide. For the engineered imine reductase polypeptides described herein, the comparison is typically made to the wild-type enzyme from which the imine reductase is derived, although in some embodiments, the reference enzyme can be another improved engineered imine reductase. Thus, the level of "improvement" can be determined and compared among various imine reductase polypeptides, including wild-type, as well as engineered imine reductases. Improved properties include, but are not limited to, properties such as: enzyme activity (which can be expressed as the percentage of substrate conversion), thermal stability, solvent stability, pH activity profile, cofactor requirement, refractoriness to inhibitors (e.g., substrate or product inhibition), stereospecificity, and stereoselectivity (including enantioselectivity).

[0066] "Increased enzyme activity" refers to an improved property of an engineered imine reductase polypeptide, which can be expressed by an increase in specific activity (e.g., product produced / time / weight of protein) or an increase in the percentage of substrate converted to product (e.g., the percentage of conversion of an initial amount of substrate to product using a specified amount of imine reductase over a specified period of time) compared to a reference imine reductase. Exemplary methods for determining enzyme activity are provided in the Examples. Any property related to enzyme activity can be affected, including the classical enzyme properties K m 、V max or k cat , and changes in them can result in increased enzyme activity. The improvement in enzyme activity can range from about 1.2-fold the enzyme activity of the corresponding wild-type enzyme to up to 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold or more of the activity of the naturally occurring or another engineered imine reductase from which the imine reductase polypeptide is derived. Imine reductase activity can be measured by any one of a standard assays, such as by monitoring changes in the properties of the substrate, cofactor or product. In some embodiments, the amount of product produced can be measured by liquid chromatography-mass spectrometry (LC-MS). Comparison of enzyme activities is carried out using a specified enzyme preparation, a specified assay under set conditions and one or more specified substrates, as described in further detail herein. Generally, when comparing lysates, the number of cells and the amount of protein assayed are determined, and the same expression system and the same host cell are used to minimize variations in the amount of enzyme produced by the host cell and present in the lysate.

[0067] "Conversion" refers to the enzymatic conversion of a substrate to the corresponding product. "Percentage conversion" refers to the percentage of substrate that is converted to product under specified conditions over a certain period of time. Thus, the "enzyme activity" or "activity" of an imine reductase polypeptide can be expressed as the "percentage conversion" of substrate to product.

[0068] "Thermostable" refers to an imine reductase polypeptide that retains similar activity (e.g., more than 60% to 80%) after exposure to the same high temperature for a certain period of time (e.g., 0.5 hour - 24 hours) compared to the wild-type enzyme exposed to the high temperature (e.g., 40°C - 80°C).

[0069] "Solvent-stable" refers to an imine reductase polypeptide that retains similar activity (more than, e.g., 60% to 80%) after exposure to the same concentration of the same solvent for a certain period of time (e.g., 0.5 hour - 24 hours) compared to the wild-type enzyme exposed to different concentrations (e.g., 5% - 99%) of solvents (ethanol, isopropanol, dimethyl sulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.).

[0070] "Thermostable and solvent-stable" refers to an imine reductase polypeptide that is both thermostable and solvent-stable.

[0071] The term "stringent hybridization conditions" is used herein to refer to conditions under which a nucleic acid hybrid is stable. As is known to those skilled in the art, the stability of a hybrid is reflected in the melting temperature (T m ) of the hybrid. Generally, the stability of a hybrid is a function of ionic strength, temperature, G / C content, and the presence of chaotropic agents. The T m value of a polynucleotide can be calculated using known methods for predicting melting temperature (see, 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., 1990, Nucl. Acids Res., 18:6409-6412

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

[1991] ); Sambrook et al., ibid); Suggs et al., 1981, in Developmental Biology Using Purified Genes , Brown 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 a complementary sequence of a sequence encoding an engineered imine reductase of the invention.

[0072] "Hybridization stringency" refers to the hybridization conditions in nucleic acid hybridization, such as washing conditions. Typically, the hybridization reaction is carried out under conditions of lower stringency, followed by washing under different but higher stringency. The term "moderate stringency hybridization" refers to conditions that allow the binding of a target DNA to a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity, and greater than about 90% identity to the target polynucleotide. Exemplary moderate stringency conditions are equivalent to hybridization at 42 °C in 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDS, followed by washing at 42 °C in 0.2×SSPE, 0.2% SDS. "High stringency hybridization" generally refers to conditions that differ from the thermal melting temperature T determined for a defined polynucleotide sequence in solution conditions by about 10 °C or less. In some embodiments, high stringency conditions refer to hybridization of those nucleic acid sequences that allow the formation of stable hybrids only at 65 °C in 0.018 M NaCl (i.e., if the hybrid is unstable at 65 °C in 0.018 M NaCl, it is unstable under the high stringency conditions contemplated herein). High stringency conditions can be provided, for example, by hybridizing in conditions equivalent to 42 °C, 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDS, and then washing at 65 °C in 0.1×SSPE and 0.1% SDS. Another high stringency condition is washing in conditions equivalent to hybridization at 65 °C in 5X SSC containing 0.1% (w:v) SDS and washing at 65 °C in 0.1×SSC containing 0.1% SDS. Other high stringency hybridization conditions as well as moderate stringency conditions are described in the references cited above. m conditions that differ by about 10 °C or less. In some embodiments, high stringency conditions refer to hybridization of those nucleic acid sequences that allow the formation of stable hybrids only at 65 °C in 0.018 M NaCl (i.e., if the hybrid is unstable at 65 °C in 0.018 M NaCl, it is unstable under the high stringency conditions contemplated herein). High stringency conditions can be provided, for example, by hybridizing in conditions equivalent to 42 °C, 50% formamide, 5×Denhart's solution, 5×SSPE, 0.2% SDS, and then washing at 65 °C in 0.1×SSPE and 0.1% SDS. Another high stringency condition is washing in conditions equivalent to hybridization at 65 °C in 5X SSC containing 0.1% (w:v) SDS and washing at 65 °C in 0.1×SSC containing 0.1% SDS. Other high stringency hybridization conditions as well as moderate stringency conditions are described in the references cited above.

[0073] A "heterologous" polynucleotide refers to any polynucleotide that has been introduced into a host cell by laboratory techniques and includes polynucleotides that have been removed from a host cell, subjected to laboratory manipulations, and then reintroduced into the host cell.

[0074] "Codon optimization" refers to the alteration of the codons of a polynucleotide encoding a protein to those codons that are preferentially used in a particular organism, such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate, i.e., most amino acids are represented by several codons called "synonyms" or "synonymous" codons, it is well known that codon usage in a particular organism is non-random and biased for particular codon triplets. This codon usage bias may be higher for a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregated protein-coding regions of the genome of an organism. In some embodiments, the polynucleotide encoding an imine reductase can be codon-optimized for optimal production from a host organism selected for expression.

[0075] As used herein, "preferred, optimal, high codon usage preference codons" are interchangeably meant to refer to codons that have a higher frequency of use in a protein-coding region than other codons encoding the same amino acid. Preferred codons can be determined based on codon usage in an individual gene, a group of genes of common function or origin, highly expressed genes, codon frequencies in the aggregated protein-coding regions of an entire organism, codon frequencies in the aggregated protein-coding regions of related organisms, or combinations thereof. Codons whose frequency increases with the level of gene expression are generally the optimal codons for expression. A variety of methods for determining the frequency of codons (e.g., codon usage, relative synonymous codon usage) and codon preference in a particular organism are known, including multivariate analyses such as using cluster analysis or correlation analysis and 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., 22:2437-46

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

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

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

[2000] ; Duret, et al., ibid.; Henaut and Danchin, at Escherichia coli and Salmonella, Neidhardt, et al. (eds.), ASM Press, Washington D.C., pp. 2047-2066

[1996] ). Data sources for obtaining codon usage can depend on any available nucleotide sequence capable of encoding a protein. These data sets include nucleic acid sequences 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, N.Y.

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

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

[1997] ).

[0076] "Control sequence" is defined herein to include all components that are necessary or advantageous for the expression of the polynucleotides and / or polypeptides of the present invention. Each control sequence can be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, the control sequences include a promoter and transcription and translation termination signals. For the purpose of introducing specific restriction sites, the control sequences can be provided with linkers that facilitate the ligation of the control sequences to the coding region of the nucleic acid sequence encoding the polypeptide.

[0077] "Operably linked" is defined herein as a configuration in which the control sequence is placed appropriately (i.e., in a functional relationship) relative to the polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide of interest and / or the polypeptide.

[0078] "Promoter sequence" refers to a nucleic acid sequence recognized by a host cell for the 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 shows transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0079] "Suitable reaction conditions" refers to those conditions in a biocatalytic reaction solution (e.g., ranges of enzyme loading, substrate loading, cofactor loading, temperature, pH, buffer, cosolvent, etc.) under which the imine reductase polypeptide of the present invention is capable of converting a substrate compound into a product compound (e.g., converting compound (2) into compound (1)). Exemplary "suitable reaction conditions" are provided and illustrated by examples in the present invention.

[0080] "Cofactor regeneration system" or "cofactor recycling system" refers to a set of reactants involved in the reaction for reducing the oxidized form of a cofactor (e.g., NADP + to NADPH). The cofactor oxidized in the reductive amination reaction of a ketone substrate catalyzed by an imine reductase is regenerated in a reduced form by the cofactor regeneration system. The cofactor regeneration system includes a stoichiometric reducing agent, which is a source of a reducing hydrogen equivalent and is capable of reducing the oxidized form of the cofactor. The cofactor regeneration system may also contain a catalyst, such as an enzyme catalyst that catalyzes the reduction of the oxidized form of the cofactor by the reducing agent. Cofactor regeneration systems for regenerating NADH or NADPH from NAD + or NADP + are known in the art and can be used in the methods described herein.

[0081] "Phosphodehydrogenase" and "PDH" are used interchangeably herein to refer to an NAD + or NADP + -dependent enzyme that catalyzes the conversion of phosphite and NAD + or NADP + to carbon dioxide and NADH or NADPH, respectively.

[0082] "Loading" (such as in "compound loading" or "enzyme loading" or "cofactor loading") refers to the concentration or amount of a component in a reaction mixture at the start of the reaction.

[0083] In the context of a biocatalyst-mediated process, a "substrate" refers to a compound or molecule on which the biocatalyst acts. For example, in the imine reductase biocatalyst used in the reductive amination method disclosed herein, there are ketone (or aldehyde) substrates of formula (II) such as cyclohexanone, and amine substrates of formula (III) such as butylamine.

[0084] In the context of a biocatalyst-mediated process, a "product" refers to a compound or molecule produced by the action of the biocatalyst. For example, an exemplary product of the imine reductase biocatalyst used in the method disclosed herein is a secondary or tertiary amine compound, such as a compound of formula (I).

[0085] "Alkyl" refers to a saturated hydrocarbon group that is straight-chain or branched-chain and has from 1 to 18 carbon atoms (including the termini), more preferably 1 to 8 carbon atoms (including the termini), and most preferably 1 to 6 carbon atoms (including the termini). An alkyl group having a specified number of carbon atoms is indicated in parentheses (e.g., (C 1 -C 6 ) alkyl refers to an alkyl group having 1 to 6 carbon atoms).

[0086] "Alkenyl" refers to a hydrocarbon group that is straight-chain or branched-chain, has from 2 to 12 carbon atoms (including the termini), and contains at least one double bond, but optionally contains more than one double bond.

[0087] "Alkynyl" refers to a hydrocarbon group that is straight-chain or branched-chain, has from 2 to 12 carbon atoms (including the termini), contains at least one triple bond, but optionally contains more than one triple bond, and additionally optionally contains one or more double-bonded moieties.

[0088] "Alkylene" refers to a straight-chain or branched-chain divalent hydrocarbon group that has from 1 to 18 carbon atoms (including the termini), more preferably 1 to 8 carbon atoms (including the termini), and most preferably 1 to 6 carbon atoms (including the termini), and is optionally substituted with one or more suitable substituents. Exemplary "alkylenes" include, but are not limited to, methylene, ethylene, propylene, butylene, etc.

[0089] "Alkenylene" refers to a straight-chain or branched-chain divalent hydrocarbon group that has 2 to 12 carbon atoms (including the termini) and one or more carbon-carbon double bonds, more preferably 2 to 8 carbon atoms (including the termini), and most preferably 2 to 6 carbon atoms (including the termini), and is optionally substituted with one or more suitable substituents.

[0090] "Heteroalkyl", "heteroalkenyl", and "heteroalkynyl" respectively refer to an alkyl, alkenyl, and alkynyl as defined herein, in which one or more carbon atoms are each independently replaced by the same or different heteroatoms or heteroatom groups. Heteroatoms and / or heteroatom groups that can replace carbon atoms include, but are not limited to, -O-, -S-, -S-O-, -NR γ -, -PH-, -S(O)-, -S(O) 2 -, -S(O)NR γ -, -S(O) 2 NR γ -, etc., including combinations thereof, where each R γ is independently selected from hydrogen, alkyl, cycloalkyl, heterocyclic hydrocarbon group, aryl, and heteroaryl.

[0091] "Aryl" refers to an unsaturated aromatic carbocyclic group having from 6 to 12 carbon atoms (including termini) in a single ring (such as phenyl) or multiple fused rings (such as naphthyl or anthracenyl). Exemplary aryl groups include phenyl, pyridyl, naphthyl, etc.

[0092] "Arylalkyl" refers to an alkyl group substituted by an aryl group (i.e., an "aryl-alkyl-" group), preferably having from 1 to 6 carbon atoms (including termini) in the alkyl moiety and from 6 to 12 carbon atoms (including termini) in the aryl moiety. Such arylalkyl groups are exemplified by benzyl, phenethyl, etc.

[0093] "Aryloxy" refers to the –OR λ group, where R λ is an aryl group which may be optionally substituted.

[0094] "Cycloalkyl" refers to a cyclic alkyl group having from 3 to 12 carbon atoms (including termini) in a single cyclic ring or multiple fused rings, which may be optionally substituted by 1 to 3 alkyl groups. Exemplary cycloalkyl groups include, but are not limited to, monocyclic structures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, 1-methylcyclopropyl, 2-methylcyclopentyl, 2-methylcyclooctyl, etc., and polycyclic structures including bridged ring systems such as adamantyl, etc.

[0095] "Cycloalkylalkyl" refers to an alkyl group substituted by a cycloalkyl group (i.e., a "cycloalkyl-alkyl-" group), preferably having from 1 to 6 carbon atoms (including termini) in the alkyl moiety and from 3 to 12 carbon atoms (including termini) in the cycloalkyl moiety. Such cycloalkylalkyl groups are exemplified by cyclopropylmethyl, cyclohexylethyl, etc.

[0096] "Amino" refers to the group -NH 2 . A substituted amino refers to the groups –NHR ε , NR ε R ε and NR ε R ε R ε , where each R ε is independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, hydrothio, sulfinyl, sulfonyl, etc. Typical amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylsulfonylamino, furanyl-oxy-sulfamino, etc.

[0097] "Aminoalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by one or more amino groups (including substituted amino groups).

[0098] "aminocarbonyl" means -C(O)NH 2 . Substituted aminocarbonyl means –C(O)NR ε R ε , where the amino group NR ε R ε is as defined herein.

[0099] "oxy" means the divalent group -O-, which may have various substituents to form different oxy groups, including ethers and esters.

[0100] "alkoxy" or "alkyloxy" are used interchangeably herein to mean the group –OR δ , where R δ is an alkyl group, including optionally substituted alkyl groups.

[0101] "carboxyl" means -COOH.

[0102] "carbonyl" means -C(O)-, which may have various substituents to form different carbonyl groups, including acids, acid halides, aldehydes, amides, esters and ketones.

[0103] "carboxyalkyl" means an alkyl in which one or more hydrogen atoms are replaced by one or more carboxyl groups.

[0104] "aminocarbonylalkyl" means an alkyl substituted by an aminocarbonyl group as defined herein.

[0105] "halogen" or "halo" means fluorine, chlorine, bromine and iodine.

[0106] "haloalkyl" means an alkyl group in which one or more hydrogen atoms are replaced by a halogen. Thus, the term "haloalkyl" is meant to include monohaloalkyl, dihaloalkyl, trihaloalkyl, etc. up to perhaloalkyl. For example, the expression "(C 1 -C 2 ) haloalkyl" includes 1-fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

[0107] "hydroxy" means -OH.

[0108] "hydroxyalkyl" means an alkyl group in which one or more hydrogen atoms are replaced by one or more hydroxy groups.

[0109] "thiol" or "sulfanyl" means -SH. Substituted thiol or sulfanyl means –S-R ε , where R εis an alkyl, aryl or other suitable substituent.

[0110] "Alkylthio" means –SR δ , where R δ is an alkyl which may be optionally substituted. Typical alkylthio groups include but are not limited to methylthio, ethylthio, n-propylthio, etc.

[0111] "Alkylthioalkyl" means an alkyl substituted by an alkylthio group –SR δ , where R δ is an alkyl which may be optionally substituted.

[0112] "Sulfonyl" means -SO 2 -. A substituted sulfonyl means –SO 2 -R ε , where R ε is an alkyl, aryl or other suitable substituent.

[0113] "Alkylsulfonyl" means –SO 2 -R δ , where R δ is an alkyl which may be optionally substituted. Typical alkylsulfonyl groups include but are not limited to methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, etc.

[0114] "Alkylsulfonylalkyl" means an alkyl substituted by an alkylsulfonyl group –SO 2 -R δ , where R δ is an alkyl which may be optionally substituted.

[0115] "Heteroaryl" means an aromatic heterocyclic group having 1 to 10 carbon atoms (including the termini) and 1 to 4 heteroatoms (including the termini) selected from oxygen, nitrogen and sulfur within the ring. Such heteroaryl groups may have a monocyclic ring (e.g., pyridyl or furyl) or more than one fused ring (e.g., indolizinyl or benzothienyl).

[0116] "Heteroarylalkyl" means an alkyl substituted by a heteroaryl (i.e., a "heteroaryl-alkyl-" group), preferably having 1 to 6 carbon atoms (including the termini) in the alkyl portion and 5 to 12 ring atoms (including the termini) in the heteroaryl portion. Such heteroarylalkyl groups are exemplified by pyridylmethyl, etc.

[0117] "Heterocycle", "heterocyclic", and interchangeably "heterocycloalkyl" refer to saturated or unsaturated groups having a single ring or multiple fused rings, having from 2 to 10 carbon ring atoms (including termini) and from 1 to 4 hetero ring atoms (including termini) selected from nitrogen, sulfur, or oxygen within the ring. Such heterocyclic groups can have a single ring (e.g., piperidinyl or tetrahydrofuranyl) or more than one fused ring (e.g., dihydroindolyl, dihydrobenzofuran, or quinuclidinyl). Examples of heterocycles include, but are not limited to, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indene, 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.

[0118] "Heterocycloalkylalkyl" refers to an alkyl group substituted by a heterocycloalkyl group (i.e., a "heterocycloalkyl-alkyl-" group), preferably having from 1 to 6 carbon atoms (including termini) in the alkyl portion and from 3 to 12 ring atoms (including termini) in the heterocycloalkyl portion.

[0119] "Membered ring" is intended to include any cyclic structure. The number preceding the term "membered" indicates the number of skeletal atoms making up the ring. Thus, for example, cyclohexyl, pyridine, pyran, and thiopyran are 6-membered rings, and cyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.

[0120] As used herein, "Fused bicyclic ring" refers to unsubstituted and substituted carbocyclic and / or heterocyclic ring moieties having from 5 to 8 atoms in each ring, the rings having 2 common atoms.

[0121] As used herein, "optionally substituted" with respect to the above chemical groups means that the position of the chemical group occupied by hydrogen can be substituted by another atom (unless otherwise specified) or chemical group, said another atom such as but not limited to carbon, oxygen, nitrogen or sulfur, said chemical group such as but not limited to hydroxy, oxo, nitro, methoxy, ethoxy, alkoxy, substituted alkoxy, trifluoromethoxy, haloalkoxy, fluorine, chlorine, bromine, iodine, halogen, methyl, ethyl, propyl, butyl, alkyl, alkenyl, alkynyl, substituted alkyl, trifluoromethyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, thio, alkylthio, acyl, carboxyl, alkoxycarbonyl, carboxamido, substituted carboxamido, alkylsulfonyl, alkylsulfinyl, alkylsulfonylamino, sulfonylamino, substituted sulfonylamino, cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximo, 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, heterocyclic group, (heterocyclic)oxy and (heterocyclic)alkyl; wherein the preferred heteroatoms are oxygen, nitrogen and sulfur. Additionally, in the case where there are open valences on these substituted chemical groups, they can be further substituted by alkyl, cycloalkyl, aryl, heteroaryl and / or heterocyclic groups, and in the case where these open valences are on carbon, they can be further substituted by halogen and substituents bonded by oxygen-, nitrogen- or sulfur-bonds, and in the case where there are more than one such open valences, these groups can be linked by directly forming bonds or by forming bonds with new heteroatoms (preferably oxygen, nitrogen or sulfur) to form rings. It is also contemplated that the above substitutions can be made provided that replacing hydrogen with a substituent does not impart unacceptable instability to the molecules of the present invention and is otherwise chemically reasonable. One of ordinary skill in the art will understand that for any chemical group described as optionally substituted, only those chemical groups that are spatially achievable and / or synthetically feasible are intended to be included. As used herein, "optionally substituted" refers to all subsequent modifiers in the term or series of chemical groups. For example, in the term "optionally substituted arylalkyl", the "alkyl" portion and the "aryl" portion of the molecule can be substituted or can be unsubstituted, and for a series of "optionally substituted alkyl, cycloalkyl, aryl and heteroaryl", the alkyl, cycloalkyl, aryl and heteroaryl groups can be substituted or can be unsubstituted independently of one another.

[0122] Conversion of imine to secondary amine

[0123] The present invention provides novel biocatalysts and related methods for the synthesis of chiral secondary amines by direct reductive amination of carbonyl- and amine-containing substrates. The biocatalysts of the present disclosure are engineered polypeptide variants of wild-type genes from Arthrobacter sp. strain 1C, which encode an imine reductase having the amino acid sequence of SEQ ID NO:2. Variants of the wild-type imine reductase (SEQ ID NO:4) contain the following residue differences compared to SEQ ID NO:2: N198H (U.S. Patent Nos. 9,487,760, 9,695,451), and were used as a starting point for protein engineering. These engineered polypeptides are capable of catalyzing the conversion of carbonyl compounds and amines to secondary amines. The general imine reductase activity of the IRED is shown in Scheme 1 below.

[0124] Scheme 1

[0125]

[0126] The engineered polypeptides of the present invention having imine reductase activity can accept a range of substrates. Thus, in the biocatalytic reaction of Scheme 2, the R 1 group of the substrate is selected from a hydrogen atom or an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, arylalkoxy, hydroxyalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocyclic hydrocarbyl, heteroaryl, and heteroarylalkyl; the R 2 groups of the substrate are independently selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, carboxyl, aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, alkylamino, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocyclic hydrocarbyl, heteroaryl, and heteroarylalkyl; and the R 3 groups of the substrate are independently selected from methyl, d3-methyl, and ethyl.

[0127] As further described herein, the engineered polypeptides having imine reductase activity exhibit stereoselectivity; thus, the imine reductase reaction of Scheme 2 can be used to establish one or more chiral centers of the product in a single biocatalytic reaction.

[0128] Scheme 2

[0129]

[0130] In some embodiments, the present invention provides engineered polypeptides that comprise an amino acid sequence having at least 80% sequence identity to the amino acid reference sequence of SEQ ID NO:4 and further comprise one or more amino acid residue differences compared to the reference amino acid sequence, wherein the engineered polypeptides have imine reductase activity. In some embodiments of the engineered polypeptides, the imine reductase activity is the activity of Scheme 2.

[0131] In particular, the engineered imine reductase polypeptides of the present disclosure have been engineered for the efficient conversion of the ketoester substrate 3-[4-(3-chlorophenyl)phenyl]-2-oxopropanoic acid (referred to herein as chlorobiphenylpyruvic acid and "Compound (2)") and the amine substrate L-alanine ethyl ester (referred to herein as "Compound (3)") to the corresponding chiral amine product Compound 3-[4-(3-chlorophenyl)phenyl]-2-[(2-ethoxy-1-methyl-2-oxoethyl)amino]propanoic acid (referred to herein as "Compound (1)") under aqueous conditions, as shown in Scheme 3.

[0132] Compound (1) is a substituted carbamoylmethylaminoacetic acid derivative, and its chemical synthesis is a multi-step process. The low yield of this multi-step chemical process makes the use of an enzyme with imine reductase activity an attractive alternative, as several steps can be removed from the synthetic sequence.

[0133] Scheme 3

[0134]

[0135] Engineered imine reductase polypeptide

[0136] The present invention provides polypeptides having imine reductase activity, polynucleotides encoding the polypeptides, methods for preparing the polypeptides, and methods for using the polypeptides. In the context of describing the polypeptide, it should be understood that it may describe the polynucleotide encoding the polypeptide.

[0137] The improved properties of the engineered polypeptides described above and the suitable reaction conditions for carrying out the desired reactions can be determined by considering the following: the concentration or amount of the polypeptide, substrate, cosubstrate, buffer, solvent, pH, conditions including temperature and reaction time, and / or conditions for immobilizing the polypeptide on a solid support, as further described below and in the examples.

[0138] In some embodiments, exemplary engineered polypeptides having imine reductase activity with improved properties (particularly in converting Compound (II) and Compound (III) to Compound (I)) comprise an amino acid sequence having one or more residue differences at the residue positions shown in Table 5.1 compared to SEQ ID NO:4.

[0139] In some embodiments, exemplary engineered polypeptides with imine reductase activity having improved properties, particularly in converting compounds of formula (II) and compounds of formula (III) to compounds of formula (I), comprise an amino acid sequence having one or more residue differences at the residue positions shown in Table 6.1 compared to SEQ ID NO:16.

[0140] In some embodiments, exemplary engineered polypeptides with imine reductase activity having improved properties, particularly in converting compounds of formula (II) and compounds of formula (III) to compounds of formula (I), comprise an amino acid sequence having one or more residue differences at the residue positions shown in Table 7.1 compared to SEQ ID NO:50.

[0141] In some embodiments, exemplary engineered polypeptides with imine reductase activity having improved properties, particularly in converting compounds of formula (II) and compounds of formula (III) to compounds of formula (I), comprise an amino acid sequence having one or more residue differences at the residue positions shown in Table 8.1 compared to SEQ ID NO:306.

[0142] In some embodiments, exemplary engineered polypeptides with imine reductase activity having improved properties, particularly in converting compounds of formula (II) and compounds of formula (III) to compounds of formula (I), comprise an amino acid sequence having one or more residue differences at the residue positions shown in Table 9.1 compared to SEQ ID NO:648.

[0143] In some embodiments, exemplary engineered polypeptides with imine reductase activity having improved properties, particularly in converting compounds of formula (II) and compounds of formula (III) to compounds of formula (I), comprise an amino acid sequence having one or more residue differences at the residue positions shown in Table 10.1 compared to SEQ ID NO:708.

[0144] The structural and functional information of exemplary non-naturally occurring (or engineered) polypeptides of the invention is based on the conversion of compounds of formula (II) and formula (III) to compounds of formula (I), the results of which are shown in Tables 5.1, 6.1, 7.1, 8.1, 9.1 and 10.1 below and further described in the Examples. The odd-numbered sequence identifiers (i.e., SEQ ID NO) in these tables refer to the nucleotide sequences encoding the amino acid sequences provided by the even-numbered SEQ ID NO in these tables. Exemplary sequences are provided in the electronic sequence listing file accompanying the present invention, which is hereby incorporated by reference herein. Amino acid residue differences are based on comparison with the reference sequences SEQ ID NO: 4, 16, 50, 306, 648 and 708, as indicated.

[0145] The wild-type Arthrobacter sp. strain 1C (CENDH) enzyme was used to generate variant SEQ ID NO: 4 (U.S. Patent Nos. 9,487,760 and 9,695,451), which contains an N198H substitution relative to SEQ ID NO: 2. SEQ ID NO: 4 was used as the starting sequence for generating variants of the invention. The amino acid sequence of the imine reductase (SEQ ID NO: 4) was codon-optimized for expression and synthesis in Escherichia coli (SEQ ID NO: 3).

[0146] The activity of each engineered polypeptide relative to the reference polypeptides SEQ ID NO: 4, 16, 50, 306, 648 or 708 was determined by the conversion of the substrates described in the Examples herein. In some embodiments, shake flask powder (SFP) was used as a secondary screen to evaluate the properties of the engineered imine reductase, the results of which are provided in the Examples. In some embodiments, the SFP form provides a more pure powder preparation of the engineered polypeptide and can contain up to about 30% of the total protein as the engineered polypeptide.

[0147] In some embodiments, specific enzyme properties are correlated with residue differences at the residue positions shown herein compared to SEQ ID NO: 4, 16, 50, 306, 648 or 708. In some embodiments, residue differences that affect polypeptide expression can be used to increase the expression of the engineered imine reductase.

[0148] In view of the guidance provided herein, it is also contemplated that any of the exemplary engineered polypeptides comprising the even-numbered sequences of SEQ ID NOs: 6 - 802 can be used as a starting amino acid sequence for the synthesis of other engineered imine reductase polypeptides, such as by subsequent rounds of evolution that incorporate new combinations of various amino acid differences from other polypeptides in Tables 5.1, 6.1, 7.1, 8.1, 9.1, and 10.1 and other residue positions described herein. Additional improvements can be generated by incorporating amino acid differences at residue positions that were maintained unchanged throughout earlier rounds of evolution.

[0149] In some embodiments, an engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO: 4 and one or more residue differences selected from the following compared to SEQ ID NO: 4: 145, 146, 153, 160, 222, 223, 226, and 261. In some embodiments, an engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO: 4 and one or more residue differences selected from the following compared to SEQ ID NO: 4: 145I, 145V, 146A, 153L, 153R, 160T, 222F, 222W, 223A, 223G, 223I, 223S, 223V, 226M, and 261T. In some embodiments, an engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO: 4 and one or more residue differences selected from the following compared to SEQ ID NO: 4: E145I, E145V, R146A, N153L, N153R, D160T, Y222F, Y222W, L223A, L223G, L223I, L223S, L223V, I226M, and E261T.

[0150] In some embodiments, an engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO: 16 and one or more residue differences selected from the following compared to SEQ ID NO: 16: 29 / 94 / 184 / 223 / 232 / 288 / 293, 29 / 94 / 184 / 232 / 287 / 288 / 293 / 311, 29 / 94 / 184 / 232 / 287 / 288 / 311, 29 / 94 / 184 / 232 / 287 / 293 / 332, 29 / 94 / 184 / 232 / 288 / 293 / 311 / 324 / 353, 29 / 94 / 184 / 232 / 288 / 311 / 324 / 332, 29 / 94 / 184 / 232 / 293, 29 / 94 / 184 / 232 / 311, 29 / 94 / 184 / 287 / 293, 29 / 94 / 184 / 287 / 293 / 311 / 353, 29 / 94 / 184 / 287 / 311, 29 / 94 / 184 / 288 / 293 / 324, 29 / 94 / 184 / 288 / 293 / 332 / 353, 29 / 94 / 184 / 288 / 298 / 332, 29 / 94 / 184 / 288 / 353, 29 / 94 / 184 / 293 / 311, 29 / 94 / 184 / 324, 29 / 94 / 223 / 232 / 287, 29 / 94 / 223 / 232 / 293 / 311 / 324, 29 / 94 / 223 / 288 / 293, 29 / 94 / 232 / 287 / 288 / 311, 29 / 94 / 232 / 288 / 293, 29 / 94 / 287 / 288 / 293, 29 / 94 / 287 / 288 / 293 / 353, 29 / 94 / 287 / 311, 29 / 94 / 293 / 311 / 324, 29 / 94 / 293 / 332 / 353, 29 / 94 / 311, 29 / 94 / 324 / 353, 29 / 184, 29 / 184 / 223 / 288 / 293 / 324, 29 / 184 / 232 / 288, 29 / 184 / 287, 29 / 184 / 287 / 288 / 293, 29 / 184 / 287 / 288 / 293 / 311, 29 / 184 / 287 / 311 / 332, 29 / 184 / 288 / 293, 29 / 184 / 288 / 293 / 311, 29 / 184 / 293, 29 / 184 / 293 / 311, 29 / 184 / 293 / 324, 29 / 184 / 353, 29 / 223 / 287 / 288 / 293 / 353,29 / 232 / 287 / 288 / 293 / 332 / 353、29 / 287 / 288 / 293、29 / 287 / 288 / 293 / 324 / 353、29 / 288 / 293、29 / 311、29 / 311 / 332、29 / 353、72 / 94 / 184 / 288 / 311、86、94 / 96 / 223 / 287 / 288 / 293 / 311 / 324 / 332、94 / 99 / 184 / 293 / 311 / 332 / 353、94 / 184 / 223、94 / 184 / 223 / 232 / 287 / 311、94 / 184 / 223 / 288 / 293、94 / 184 / 223 / 293、94 / 184 / 232 / 287 / 288、94 / 184 / 232 / 287 / 288 / 324 / 332、94 / 184 / 232 / 287 / 293、94 / 184 / 232 / 287 / 293 / 332、94 / 184 / 287 / 288 / 293、94 / 184 / 287 / 288 / 311、94 / 184 / 287 / 293 / 311 / 324 / 353、94 / 184 / 287 / 311、94 / 184 / 287 / 353、94 / 184 / 288 / 293、94 / 184 / 288 / 293 / 311、94 / 184 / 288 / 293 / 311 / 332、94 / 184 / 293、94 / 184 / 293 / 311、94 / 184 / 293 / 332 / 353、94 / 223 / 232 / 311 / 353 / 355 / 356 / 357 / 358 / 359、94 / 223 / 287 / 288 / 293、94 / 223 / 288 / 293 / 311、94 / 232 / 287 / 288 / 293 / 353、94 / 232 / 288 / 293 / 353、94 / 232 / 293、94 / 232 / 293 / 324 / 332、94 / 232 / 311、94 / 287 / 288 / 293、94 / 287 / 288 / 311 / 324、94 / 288 / 293、94 / 288 / 293 / 324、94 / 293 / 324、94 / 311 / 324、153、184 / 223、184 / 223 / 287 / 288 / 353、184 / 232 / 287 / 288 / 293 / 311、184 / 232 / 287 / 288 / 324 / 353、184 / 232 / 287 / 293 / 332、184 / 287 / 288、184 / 287 / 288 / 293、184 / 287 / 288 / 293 / 324 / 353、184 / 287 / 293、184 / 287 / 293 / 311、184 / 288 / 293 / 311 / 324、184 / 332 / 353、205、223 / 232 / 287 / 288 / 332 / 353, 223 / 287 / 288, 223 / 287 / 288 / 293 / 311, 223 / 287 / 311 / 353, 223 / 288 / 293, 232 / 287 / 288 / 293, 232 / 287 / 288 / 293 / 311, 232 / 287 / 288 / 293 / 321, 232 / 287 / 311, 232 / 288 / 293, 232 / 288 / 293 / 332, 234 / 287 / 288 / 293 / 311, 287 / 288 / 293, 287 / 288 / 311 / 324, 287 / 293, 287 / 311, 287 / 311 / 332, 287 / 324, 288 / 293, 288 / 293 / 311 / 324 / 353, 288 / 293 / 324, 293 / 311, and 311 / 332. In some embodiments, the engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO:16 and one or more residue differences selected from the following compared to SEQ ID NO:16: 29R / 94K / 184Q / 288S / 353E, 29R / 94K / 184R / 232A / 288S / 293F / 311V / 324L / 353E, 29R / 94K / 184R / 287T / 293F, 29R / 94K / 184R / 288S / 293Q / 332V / 353E, 29R / 94K / 184R / 324L, 29R / 94K / 223S / 232A / 287K, 29R / 94K / 232A / 287T / 288S / 311V, 29R / 94K / 232A / 288S / 293F, 29R / 94K / 287K / 311V, 29R / 94K / 287T / 288S / 293F, 29R / 94K / 293H / 311V / 324L, 29R / 94K / 324L / 353E, 29R / 94R / 184Q / 223S / 232A / 288S / 293F, 29R / 94R / 184Q / 232A / 287T / 293F / 332V, 29R / 94R / 184Q / 232A / 288S / 311V / 324L / 332V, 29R / 94R / 184Q / 287K / 293F / 311V / 353E, 29R / 94R / 184Q / 287K / 311V, 29R / 94R / 184Q / 288S / 293F / 324L, 29R / 94R / 184R / 232A / 287K / 288S / 293F / 311V,29R / 94R / 184R / 232A / 287T / 288S / 311V, 29R / 94R / 184R / 232A / 293F, 29R / 94R / 184R / 232A / 311V, 29R / 94R / 184R / 288S / 298A / 332V, 29R / 94R / 184R / 293F / 311V, 29R / 94R / 223S / 288S / 293F, 29R / 94R / 223T / 232A / 293H / 311V / 324L, 29R / 94R / 287K / 288S / 293F, 29R / 94R / 287T / 288S / 293F, 29R / 94R / 287T / 288S / 293F / 353E, 29R / 94R / 293F / 332V / 353E, 29R / 94R / 311V, 29R / 184Q / 223S / 288S / 293F / 324L, 29R / 184Q / 287T / 288S / 293F, 29R / 184R, 29R / 184R / 232A / 288S, 29R / 184R / 287K / 288S / 293F / 311V, 29R / 184R / 287K / 311V / 332V, 29R / 184R / 287T, 29R / 184R / 288S / 293F, 29R / 184R / 288S / 293F / 311V, 29R / 184R / 293F, 29R / 184R / 293F / 311V, 29R / 184R / 293F / 324L, 29R / 184R / 353E, 29R / 223T / 287T / 288S / 293F / 353E, 29R / 232A / 287T / 288S / 293Q / 332V / 353E, 29R / 287T / 288S / 293F, 29R / 287T / 288S / 293F / 324L / 353E, 29R / 288S / 293F, 29R / 311V, 29R / 311V / 332V, 29R / 353E, 72V / 94R / 184R / 288S / 311V, 86Q, 94K / 96V / 223T / 287K / 288S / 293F / 311V / 324L / 332V, 94K / 184Q / 232A / 287T / 293F, 94K / 184Q / 232A / 287T / 293F / 332V, 94K / 184Q / 287T / 293F / 311V / 324L / 353E, 94K / 184R / 223S / 288S / 293F, 94K / 184R / 223T / 232A / 287K / 311V, 94K / 184R / 232A / 287K / 288S, 94K / 184R / 287T / 311V, 94K / 184R / 287T / 353E94K / 184R / 288S / 293F / 311V, 94K / 184R / 293H / 311V, 94K / 287K / 288S / 293F, 94K / 287T / 288S / 311V / 324L, 94K / 288S / 293F, 94K / 288S / 293F / 324L, 94K / 311V / 324L, 94R / 99T / 184R / 293F / 311V / 332V / 353E, 94R / 184Q / 287K / 288S / 311V, 94R / 184Q / 288S / 293F / 311V, 94R / 184Q / 293F / 332V / 353E, 94R / 184R / 223S, 94R / 184R / 223S / 293F, 94R / 184R / 232A / 287K / 288S / 324L / 332V, 94R / 184R / 287K / 288S / 293F, 94R / 184R / 287K / 288S / 311V, 94R / 184R / 288S / 293F, 94R / 184R / 288S / 293F / 311V / 332V, 94R / 184R / 293F, 94R / 223S / 232A / 311V / 353E / 355K / 356K / 357C / 358C / 359-, 94R / 223S / 287K / 288S / 293F, 94R / 223S / 288S / 293F / 311V, 94R / 232A / 287K / 288S / 293F / 353E, 94R / 232A / 288S / 293F / 353E, 94R / 232A / 293F, 94R / 232A / 293F / 324L / 332V, 94R / 232A / 311V, 94R / 287K / 288S / 293F, 94R / 287T / 288S / 293F, 94R / 293F / 324L, 94R / 311V / 324L, 153Y, 184Q / 223T / 287K / 288S / 353E, 184Q / 232A / 287K / 288S / 293F / 311V, 184Q / 232A / 287K / 288S / 324L / 353E, 184Q / 287K / 293F, 184R / 223T, 184R / 232A / 287K / 293F / 332V, 184R / 287K / 293F / 311V, 184R / 287T / 288S, 184R / 287T / 288S / 293F, 184R / 287T / 288S / 293F / 324L / 353E, 184R / 287T / 288S / 293Q, 184R / 288S / 293H / 311V / 324L, 184R / 332V / 353E, 205I, 205V223S / 232A / 287K / 288S / 332V / 353E, 223S / 287K / 288S / 293F / 311V, 223S / 287K / 311V / 353E, 223S / 287T / 288S, 223S / 288S / 293F, 232A / 287K / 288S / 293F, 232A / 287K / 288S / 293F / 311V, 232A / 287K / 311V, 232A / 287T / 288S / 293F / 321V, 232A / 288S / 293F, 232A / 288S / 293F / 332V, 234V / 287K / 288S / 293F / 311V, 287K / 288S / 293F, 287K / 293F, 287K / 311V / 332V, 287K / 324L, 287T / 288S / 311V / 324L, 287T / 311V, 288S / 293F, 288S / 293F / 311V / 324L / 353E, 288S / 293F / 324L, 293F / 311V, and 311V / 332V. In some embodiments, an engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO:16 and one or more residue differences selected from the following compared to SEQ ID NO:16: S29R / N94K / V184Q / N288S / G353E, S29R / N94K / V184R / S232A / N288S / Y293F / A311V / D324L / G353E, S29R / N94K / V184R / I287T / Y293F, S29R / N94K / V184R / N288S / Y293Q / T332V / G353E, S29R / N94K / V184R / D324L, S29R / N94K / L223S / S232A / I287K, S29R / N94K / S232A / I287T / N288S / A311V, S29R / N94K / S232A / N288S / Y293F, S29R / N94K / I287K / A311V, S29R / N94K / I287T / N288S / Y293F, S29R / N94K / Y293H / A311V / D324L, S29R / N94K / D324L / G353E, S29R / N94R / V184Q / L223S / S232A / N288S / Y293F, S29R / N94R / V184Q / S232A / I287T / Y293F / T332V,S29R / N94R / V184Q / S232A / N288S / A311V / D324L / T332V, S29R / N94R / V184Q / I287K / Y293F / A311V / G353E, S29R / N94R / V184Q / I287K / A311V, S29R / N94R / V184Q / N288S / Y293F / D324L, S29R / N94R / V184R / S232A / I287K / N288S / Y293F / A311V, S29R / N94R / V184R / S232A / I287T / N288S / A311V, S29R / N94R / V184R / S232A / Y293F, S29R / N94R / V184R / S232A / A311V, S29R / N94R / V184R / N288S / V298A / T332V, S29R / N94R / V184R / Y293F / A311V, S29R / N94R / L223S / N288S / Y293F, S29R / N94R / L223T / S232A / Y293H / A311V / D324L, S29R / N94R / I287K / N288S / Y293F, S29R / N94R / I287T / N288S / Y293F, S29R / N94R / I287T / N288S / Y293F / G353E, S29R / N94R / Y293F / T332V / G353E, S29R / N94R / A311V, S29R / V184Q / L223S / N288S / Y293F / D324L, S29R / V184Q / I287T / N288S / Y293F, S29R / V184R, S29R / V184R / S232A / N288S, S29R / V184R / I287K / N288S / Y293F / A311V, S29R / V184R / I287K / A311V / T332V, S29R / V184R / I287T, S29R / V184R / N288S / Y293F, S29R / V184R / N288S / Y293F / A311V, S29R / V184R / Y293F, S29R / V184R / Y293F / A311V, S29R / V184R / Y293F / D324L, S29R / V184R / G353E, S29R / L223T / I287T / N288S / Y293F / G353E, S29R / S232A / I287T / N288S / Y293Q / T332V / G353E, S29R / I287T / N288S / Y293F, S29R / I287T / N288S / Y293F / D324L / G353ES29R / N288S / Y293F, S29R / A311V, S29R / A311V / T332V, S29R / G353E, A72V / N94R / V184R / N288S / A311V, I86Q, N94K / A96V / L223T / I287K / N288S / Y293F / A311V / D324L / T332V, N94K / V184Q / S232A / I287T / Y293F, N94K / V184Q / S232A / I287T / Y293F / T332V, N94K / V184Q / I287T / Y293F / A311V / D324L / G353E, N94K / V184R / L223S / N288S / Y293F, N94K / V184R / L223T / S232A / I287K / A311V, N94K / V184R / S232A / I287K / N288S, N94K / V184R / I287T / A311V, N94K / V184R / I287T / G353E, N94K / V184R / N288S / Y293F / A311V, N94K / V184R / Y293H / A311V, N94K / I287K / N288S / Y293F, N94K / I287T / N288S / A311V / D324L, N94K / N288S / Y293F, N94K / N288S / Y293F / D324L, N94K / A311V / D324L, N94R / I99T / V184R / Y293F / A311V / T332V / G353E, N94R / V184Q / I287K / N288S / A311V, N94R / V184Q / N288S / Y293F / A311V, N94R / V184Q / Y293F / T332V / G353E, N94R / V184R / L223S, N94R / V184R / L223S / Y293F, N94R / V184R / S232A / I287K / N288S / D324L / T332V, N94R / V184R / I287K / N288S / Y293F, N94R / V184R / I287K / N288S / A311V, N94R / V184R / N288S / Y293F, N94R / V184R / N288S / Y293F / A311V / T332V, N94R / V184R / Y293F, N94R / L223S / S232A / A311V / G353E / R355K / S356K / A357C / V358C / E359-, N94R / L223S / I287K / N288S / Y293FN94R / L223S / N288S / Y293F / A311V, N94R / S232A / I287K / N288S / Y293F / G353E, N94R / S232A / N288S / Y293F / G353E, N94R / S232A / Y293F, N94R / S232A / Y293F / D324L / T332V, N94R / S232A / A311V, N94R / I287K / N288S / Y293F, N94R / I287T / N288S / Y293F, N94R / Y293F / D324L, N94R / A311V / D324L, N153Y, V184Q / L223T / I287K / N288S / G353E, V184Q / S232A / I287K / N288S / Y293F / A311V, V184Q / S232A / I287K / N288S / D324L / G353E, V184Q / I287K / Y293F, V184R / L223T, V184R / S232A / I287K / Y293F / T332V, V184R / I287K / Y293F / A311V, V184R / I287T / N288S, V184R / I287T / N288S / Y293F, V184R / I287T / N288S / Y293F / D324L / G353E, V184R / I287T / N288S / Y293Q, V184R / N288S / Y293H / A311V / D324L, V184R / T332V / G353E, P205I, P205V, L223S / S232A / I287K / N288S / T332V / G353E, L223S / I287K / N288S / Y293F / A311V, L223S / I287K / A311V / G353E, L223S / I287T / N288S, L223S / N288S / Y293F, S232A / I287K / N288S / Y293F, S232A / I287K / N288S / Y293F / A311V, S232A / I287K / A311V, S232A / I287T / N288S / Y293F / A321V, S232A / N288S / Y293F, S232A / N288S / Y293F / T332V, A234V / I287K / N288S / Y293F / A311V, I287K / N288S / Y293F, I287K / Y293F, I287K / A311V / T332V, I287K / D324L, I287T / N288S / A311V / D324L, I287T / A311V, N288S / Y293FN288S / Y293F / A311V / D324L / G353E, N288S / Y293F / D324L, Y293F / A311V and A311V / T332V.

[0151] In some embodiments, an engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO:50 and one or more residue differences selected from the following compared to SEQ ID NO:50: 29 / 57 / 94 / 153 / 184 / 205 / 261 / 265 / 287, 29 / 57 / 94 / 153 / 205 / 265, 29 / 57 / 94 / 184 / 205 / 277 / 353, 29 / 57 / 94 / 205 / 259 / 261 / 265, 29 / 57 / 94 / 205 / 261 / 353, 29 / 57 / 153 / 184 / 205 / 259 / 261 / 265 / 277, 29 / 57 / 153 / 184 / 205 / 353, 29 / 57 / 153 / 205 / 261 / 265, 29 / 57 / 153 / 205 / 261 / 265 / 277, 29 / 57 / 153 / 205 / 265 / 277, 29 / 57 / 153 / 205 / 277 / 353, 29 / 57 / 184 / 205 / 259 / 261 / 265 / 277 / 287 / 357, 29 / 57 / 184 / 205 / 259 / 261 / 265 / 287, 29 / 57 / 205 / 259 / 261 / 265, 29 / 57 / 205 / 259 / 261 / 265 / 287, 29 / 57 / 205 / 261 / 265 / 353, 29 / 94 / 153 / 184 / 205, 29 / 94 / 153 / 205 / 259 / 261 / 265, 29 / 94 / 153 / 205 / 259 / 261 / 287, 29 / 94 / 153 / 205 / 259 / 261 / 287 / 353, 29 / 94 / 184 / 205 / 259 / 261, 29 / 94 / 184 / 205 / 261, 29 / 94 / 205, 29 / 94 / 205 / 259 / 261 / 287, 29 / 94 / 205 / 261, 29 / 94 / 261 / 265 / 287 / 353, 29 / 153 / 184 / 205, 29 / 153 / 205, 29 / 153 / 205 / 259 / 261 / 265 / 277 / 287, 29 / 153 / 261 / 265 / 287 / 353, 29 / 184 / 205, 29 / 184 / 205 / 259 / 261 / 277, 29 / 184 / 205 / 353, 29 / 184 / 261 / 265, 29 / 184 / 265, 29 / 205, 29 / 205 / 259 / 261, 29 / 205 / 259 / 261 / 265 / 287, 29 / 205 / 259 / 261 / 287,29 / 205 / 261、29 / 205 / 261 / 265、29 / 205 / 261 / 265 / 353、29 / 205 / 261 / 353、29 / 205 / 277、29 / 205 / 287、29 / 205 / 287 / 353、29 / 259 / 261 / 265、29 / 261 / 353、57 / 94 / 153 / 184 / 205 / 259 / 261、57 / 94 / 153 / 205 / 261 / 265、57 / 94 / 153 / 259 / 261 / 265、57 / 94 / 153 / 261 / 265、57 / 94 / 184 / 205 / 261 / 265 / 287、57 / 94 / 184 / 261 / 265 / 287、57 / 94 / 205 / 259、57 / 94 / 205 / 259 / 261 / 265 / 277 / 287、57 / 94 / 205 / 261 / 265、57 / 94 / 205 / 277、57 / 153 / 205 / 259 / 261 / 265 / 287、57 / 153 / 205 / 259 / 265 / 277、57 / 153 / 205 / 261、57 / 153 / 205 / 261 / 265、57 / 153 / 205 / 265、57 / 153 / 261 / 265、57 / 153 / 261 / 265 / 287 / 353、57 / 205、57 / 205 / 259 / 261、57 / 205 / 259 / 261 / 265 / 277 / 287 / 353、57 / 205 / 259 / 261 / 287 / 353、57 / 205 / 259 / 265、57 / 205 / 261、57 / 205 / 261 / 265 / 277 / 287 / 353、57 / 205 / 261 / 277、57 / 205 / 261 / 277 / 353、57 / 261 / 265 / 353、94 / 126 / 184 / 205 / 259 / 261 / 265、94 / 153 / 184 / 205、94 / 153 / 184 / 259 / 261 / 265、94 / 153 / 205 / 259 / 261 / 265 / 353、94 / 153 / 205 / 287、94 / 153 / 205 / 287 / 353、94 / 153 / 205 / 353、94 / 184 / 205 / 259 / 261、94 / 184 / 205 / 259 / 261 / 265 / 287、94 / 184 / 205 / 259 / 261 / 353、94 / 184 / 205 / 261 / 265 / 287 / 353、94 / 184 / 353、94 / 205、94 / 205 / 259 / 261 / 265、94 / 205 / 259 / 261 / 265 / 277 / 287、94 / 205 / 259 / 261 / 265 / 353、94 / 205 / 259 / 261 / 287 / 353、94 / 205 / 261 / 265 / 277, 94 / 205 / 261 / 265 / 353, 94 / 205 / 261 / 353, 94 / 205 / 277, 94 / 259 / 261 / 265, 153 / 184 / 205 / 261 / 277 / 287, 153 / 184 / 261 / 265 / 287, 153 / 205, 153 / 205 / 261, 153 / 205 / 261 / 265 / 287, 153 / 259 / 261 / 265, 153 / 259 / 265 / 287, 153 / 261 / 265, 153 / 261 / 265 / 287 / 353, 153 / 265, 153 / 265 / 277 / 287 / 353, 184 / 205, 184 / 205 / 259 / 261 / 287, 184 / 205 / 261, 184 / 205 / 261 / 265, 184 / 205 / 261 / 287, 184 / 205 / 277, 184 / 205 / 287 / 353, 184 / 261 / 265 / 287, 202, 205, 205 / 259 / 261, 205 / 259 / 261 / 265, 205 / 259 / 261 / 265 / 277 / 287 / 353, 205 / 259 / 261 / 277 / 287, 205 / 259 / 265, 205 / 261, 205 / 261 / 265, 205 / 261 / 265 / 274, 205 / 261 / 265 / 287, 205 / 261 / 265 / 287 / 353, 205 / 261 / 277, 205 / 261 / 353, 205 / 277 / 287, 205 / 287, 223, 225, 256, 259 / 261 / 265, 261 / 265 / 287, 265 / 287, 283 and 294. In some embodiments, the engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO:50 and one or more residue differences selected from the following compared to SEQ ID NO:50: 29S / 57G / 94K / 153F / 184R / 205C / 261L / 265I / 287T, 29S / 57G / 94K / 153L / 205A / 265I, 29S / 57G / 94K / 184R / 205C / 277V / 353E, 29S / 57G / 94K / 205C / 259F / 261S / 265I, 29S / 57G / 94K / 205C / 261L / 353E, 29S / 57G / 153F / 184R / 205C / 259F / 261V / 265I / 277V,29S / 57G / 153F / 205C / 261L / 265I / 277V, 29S / 57G / 153F / 205C / 261V / 265I, 29S / 57G / 153F / 205C / 277V / 353E, 29S / 57G / 153L / 184R / 205A / 353E, 29S / 57G / 153L / 205A / 265I / 277V, 29S / 57G / 184R / 205C / 259F / 261L / 265I / 277V / 287K / 357S, 29S / 57G / 184R / 205C / 259F / 261V / 265I / 287T, 29S / 57G / 205A / 259F / 261L / 265I / 287T, 29S / 57G / 205A / 259F / 261S / 265I, 29S / 57G / 205C / 261S / 265I / 353E, 29S / 94K / 153F / 205A / 259F / 261S / 287T / 353E, 29S / 94K / 153F / 205C / 259F / 261L / 287K, 29S / 94K / 153L / 184R / 205C, 29S / 94K / 153L / 205C / 259F / 261R / 265I, 29S / 94K / 184R / 205C / 259F / 261S, 29S / 94K / 184R / 205C / 261V, 29S / 94K / 205A, 29S / 94K / 205A / 261V, 29S / 94K / 205C / 259F / 261L / 287T, 29S / 94K / 261R / 265I / 287T / 353E, 29S / 153F / 184R / 205C, 29S / 153F / 205A, 29S / 153F / 261R / 265I / 287K / 353E, 29S / 153L / 205A / 259F / 261L / 265I / 277V / 287T, 29S / 184R / 205A, 29S / 184R / 205A / 353E, 29S / 184R / 205C / 259F / 261V / 277V, 29S / 184R / 261L / 265I, 29S / 184R / 265I, 29S / 205A, 29S / 205A / 259F / 261L / 265I / 287K, 29S / 205A / 259F / 261R, 29S / 205A / 261V, 29S / 205C / 259F / 261L, 29S / 205C / 259F / 261L / 265I / 287T, 29S / 205C / 259F / 261L / 287K, 29S / 205C / 259F / 261L / 287T, 29S / 205C / 259F / 261S, 29S / 205C / 261L,29S / 205C / 261L / 265I / 353E, 29S / 205C / 261R / 353E, 29S / 205C / 261S, 29S / 205C / 261S / 265I, 29S / 205C / 261V / 265I, 29S / 205C / 277V, 29S / 205C / 287T, 29S / 205C / 287T / 353E, 29S / 259F / 261L / 265I, 29S / 261V / 353E, 57G / 94K / 153F / 184R / 205A / 259F / 261L, 57G / 94K / 153F / 205A / 261L / 265I, 57G / 94K / 153F / 259F / 261V / 265I, 57G / 94K / 153F / 261V / 265I, 57G / 94K / 184R / 205C / 261V / 265I / 287T, 57G / 94K / 184R / 261S / 265I / 287T, 57G / 94K / 205A / 259F, 57G / 94K / 205A / 259F / 261V / 265I / 277V / 287T, 57G / 94K / 205A / 277V, 57G / 94K / 205C / 259F, 57G / 94K / 205C / 261S / 265I, 57G / 153F / 205A / 261L / 265I, 57G / 153F / 205A / 261S, 57G / 153F / 205C / 259F / 261L / 265I / 287T, 57G / 153F / 205C / 259F / 265I / 277V, 57G / 153F / 205C / 265I, 57G / 153F / 261L / 265I / 287K / 353E, 57G / 153F / 261R / 265I, 57G / 205A, 57G / 205A / 259F / 261L / 265I / 277V / 287K / 353E, 57G / 205A / 259F / 261L / 287T / 353E, 57G / 205A / 259F / 261V, 57G / 205A / 261R, 57G / 205C, 57G / 205C / 259F / 265I, 57G / 205C / 261L / 265I / 277V / 287T / 353E, 57G / 205C / 261L / 277V, 57G / 205C / 261S / 277V / 353E, 57G / 261L / 265I / 353E, 94K / 126C / 184R / 205A / 259F / 261L / 265I, 94K / 153F / 184R / 259F / 261S / 265I, 94K / 153F / 205A / 287K / 353E, 94K / 153F / 205A / 353E, 94K / 153F / 205C / 287K,94K / 153F / 205C / 287T / 353E, 94K / 153L / 184R / 205C, 94K / 153L / 205C / 259F / 261S / 265I / 353E, 94K / 184R / 205A / 259F / 261L / 353E, 94K / 184R / 205A / 259F / 261S, 94K / 184R / 205A / 261V / 265I / 287T / 353E, 94K / 184R / 205C / 259F / 261S / 265I / 287K, 94K / 184R / 353E, 94K / 205A, 94K / 205A / 259F / 261L / 265I, 94K / 205A / 259F / 261L / 265I / 277V / 287K, 94K / 205A / 259F / 261R / 265I, 94K / 205A / 259F / 261V / 287K / 353E, 94K / 205A / 277V, 94K / 205C, 94K / 205C / 259F / 261S / 265I, 94K / 205C / 259F / 261S / 265I / 353E, 94K / 205C / 259F / 261V / 265I / 353E, 94K / 205C / 261L / 353E, 94K / 205C / 261S / 265I / 277V, 94K / 205C / 261V / 265I / 353E, 94K / 259F / 261S / 265I, 94K / 259F / 261V / 265I, 153F / 184R / 205A / 261L / 277V / 287T, 153F / 184R / 261S / 265I / 287T, 153F / 205A, 153F / 205A / 261R / 265I / 287T, 153F / 205C, 153F / 205C / 261L, 153F / 205C / 261V, 153F / 259F / 261V / 265I, 153F / 259F / 265I / 287K, 153F / 261L / 265I, 153F / 261R / 265I, 153F / 261S / 265I / 287T / 353E, 153F / 265I, 153F / 265I / 277V / 287T / 353E, 184R / 205A / 261R / 265I, 184R / 205A / 287T / 353E, 184R / 205C, 184R / 205C / 259F / 261L / 287K, 184R / 205C / 261L, 184R / 205C / 261R / 287K, 184R / 205C / 277V, 184R / 261L / 265I / 287T, 202L, 202M, 205A, 205A / 259F / 261V / 265I, 205A / 259F / 265I,205A / 261L, 205A / 261R / 265I / 287K, 205C, 205C / 259F / 261L, 205C / 259F / 261R / 265I / 277V / 287T / 353E, 205C / 259F / 261S, 205C / 259F / 261S / 265I, 205C / 259F / 261V / 277V / 287T, 205C / 259F / 265I, 205C / 261L / 265I / 274A, 205C / 261L / 277V, 205C / 261L / 353E, 205C / 261R, 205C / 261R / 265I / 287K / 353E, 205C / 261S, 205C / 261V / 265I, 205C / 277V / 287K, 205C / 287K, 205C / 287T, 205S, 205T, 223G, 225F, 225Y, 256L, 256V, 259F / 261L / 265I, 259F / 261V / 265I, 261R / 265I / 287K, 261S / 265I / 287T, 265I / 287T, 283L and 294I. In some embodiments, the engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO:50 and one or more residue differences selected from the following compared to SEQ ID NO:50: R29S / A57G / N94K / N153F / Q184R / P205C / E261L / Q265I / I287T, R29S / A57G / N94K / N153L / P205A / Q265I, R29S / A57G / N94K / Q184R / P205C / N277V / G353E, R29S / A57G / N94K / P205C / Y259F / E261S / Q265I, R29S / A57G / N94K / P205C / E261L / G353E, R29S / A57G / N153F / Q184R / P205C / Y259F / E261V / Q265I / N277V, R29S / A57G / N153F / P205C / E261L / Q265I / N277V, R29S / A57G / N153F / P205C / E261V / Q265I, R29S / A57G / N153F / P205C / N277V / G353E, R29S / A57G / N153L / Q184R / P205A / G353E, R29S / A57G / N153L / P205A / Q265I / N277V,R29S / A57G / Q184R / P205C / Y259F / E261L / Q265I / N277V / I287K / A357S, R29S / A57G / Q184R / P205C / Y259F / E261V / Q265I / I287T, R29S / A57G / P205A / Y259F / E261L / Q265I / I287T, R29S / A57G / P205A / Y259F / E261S / Q265I, R29S / A57G / P205C / E261S / Q265I / G353E, R29S / N94K / N153F / P205A / Y259F / E261S / I287T / G353E, R29S / N94K / N153F / P205C / Y259F / E261L / I287K, R29S / N94K / N153L / Q184R / P205C, R29S / N94K / N153L / P205C / Y259F / E261R / Q265I, R29S / N94K / Q184R / P205C / Y259F / E261S, R29S / N94K / Q184R / P205C / E261V, R29S / N94K / P205A, R29S / N94K / P205A / E261V, R29S / N94K / P205C / Y259F / E261L / I287T, R29S / N94K / E261R / Q265I / I287T / G353E, R29S / N153F / Q184R / P205C, R29S / N153F / P205A, R29S / N153F / E261R / Q265I / I287K / G353E, R29S / N153L / P205A / Y259F / E261L / Q265I / N277V / I287T, R29S / Q184R / P205A, R29S / Q184R / P205A / G353E, R29S / Q184R / P205C / Y259F / E261V / N277V, R29S / Q184R / E261L / Q265I, R29S / Q184R / Q265I, R29S / P205A, R29S / P205A / Y259F / E261L / Q265I / I287K, R29S / P205A / Y259F / E261R, R29S / P205A / E261V, R29S / P205C / Y259F / E261L, R29S / P205C / Y259F / E261L / Q265I / I287T, R29S / P205C / Y259F / E261L / I287K, R29S / P205C / Y259F / E261L / I287T, R29S / P205C / Y259F / E261SR29S / P205C / E261L, R29S / P205C / E261L / Q265I / G353E, R29S / P205C / E261R / G353E, R29S / P205C / E261S, R29S / P205C / E261S / Q265I, R29S / P205C / E261V / Q265I, R29S / P205C / N277V, R29S / P205C / I287T, R29S / P205C / I287T / G353E, R29S / Y259F / E261L / Q265I, R29S / E261V / G353E, A57G / N94K / N153F / Q184R / P205A / Y259F / E261L, A57G / N94K / N153F / P205A / E261L / Q265I, A57G / N94K / N153F / Y259F / E261V / Q265I, A57G / N94K / N153F / E261V / Q265I, A57G / N94K / Q184R / P205C / E261V / Q265I / I287T, A57G / N94K / Q184R / E261S / Q265I / I287T, A57G / N94K / P205A / Y259F, A57G / N94K / P205A / Y259F / E261V / Q265I / N277V / I287T, A57G / N94K / P205A / N277V, A57G / N94K / P205C / Y259F, A57G / N94K / P205C / E261S / Q265I, A57G / N153F / P205A / E261L / Q265I, A57G / N153F / P205A / E261S, A57G / N153F / P205C / Y259F / E261L / Q265I / I287T, A57G / N153F / P205C / Y259F / Q265I / N277V, A57G / N153F / P205C / Q265I, A57G / N153F / E261L / Q265I / I287K / G353E, A57G / N153F / E261R / Q265I, A57G / P205A, A57G / P205A / Y259F / E261L / Q265I / N277V / I287K / G353E, A57G / P205A / Y259F / E261L / I287T / G353E, A57G / P205A / Y259F / E261V, A57G / P205A / E261R, A57G / P205C, A57G / P205C / Y259F / Q265I, A57G / P205C / E261L / Q265I / N277V / I287T / G353EA57G / P205C / E261L / N277V, A57G / P205C / E261S / N277V / G353E, A57G / E261L / Q265I / G353E, N94K / G126C / Q184R / P205A / Y259F / E261L / Q265I, N94K / N153F / Q184R / Y259F / E261S / Q265I, N94K / N153F / P205A / I287K / G353E, N94K / N153F / P205A / G353E, N94K / N153F / P205C / I287K, N94K / N153F / P205C / I287T / G353E, N94K / N153L / Q184R / P205C, N94K / N153L / P205C / Y259F / E261S / Q265I / G353E, N94K / Q184R / P205A / Y259F / E261L / G353E, N94K / Q184R / P205A / Y259F / E261S, N94K / Q184R / P205A / E261V / Q265I / I287T / G353E, N94K / Q184R / P205C / Y259F / E261S / Q265I / I287K, N94K / Q184R / G353E, N94K / P205A, N94K / P205A / Y259F / E261L / Q265I, N94K / P205A / Y259F / E261L / Q265I / N277V / I287K, N94K / P205A / Y259F / E261R / Q265I, N94K / P205A / Y259F / E261V / I287K / G353E, N94K / P205A / N277V, N94K / P205C, N94K / P205C / Y259F / E261S / Q265I, N94K / P205C / Y259F / E261S / Q265I / G353E, N94K / P205C / Y259F / E261V / Q265I / G353E, N94K / P205C / E261L / G353E, N94K / P205C / E261S / Q265I / N277V, N94K / P205C / E261V / Q265I / G353E, N94K / Y259F / E261S / Q265I, N94K / Y259F / E261V / Q265I, N153F / Q184R / P205A / E261L / N277V / I287T, N153F / Q184R / E261S / Q265I / I287T, N153F / P205A, N153F / P205A / E261R / Q265I / I287T, N153F / P205C,N153F / P205C / E261L, N153F / P205C / E261V, N153F / Y259F / E261V / Q265I, N153F / Y259F / Q265I / I287K, N153F / E261L / Q265I, N153F / E261R / Q265I, N153F / E261S / Q265I / I287T / G353E, N153F / Q265I, N153F / Q265I / N277V / I287T / G353E, Q184R / P205A / E261R / Q265I, Q184R / P205A / I287T / G353E, Q184R / P205C, Q184R / P205C / Y259F / E261L / I287K, Q184R / P205C / E261L, Q184R / P205C / E261R / I287K, Q184R / P205C / N277V, Q184R / E261L / Q265I / I287T, H202L, H202M, P205A, P205A / Y259F / E261V / Q265I, P205A / Y259F / Q265I, P205A / E261L, P205A / E261R / Q265I / I287K, P205C, P205C / Y259F / E261L, P205C / Y259F / E261R / Q265I / N277V / I287T / G353E, P205C / Y259F / E261S, P205C / Y259F / E261S / Q265I, P205C / Y259F / E261V / N277V / I287T, P205C / Y259F / Q265I, P205C / E261L / Q265I / V274A, P205C / E261L / N277V, P205C / E261L / G353E, P205C / E261R, P205C / E261R / Q265I / I287K / G353E, P205C / E261S, P205C / E261V / Q265I, P205C / N277V / I287K, P205C / I287K, P205C / I287T, P205S, P205T, S223G, G225F, G225Y, C256L, C256V, Y259F / E261L / Q265I, Y259F / E261V / Q265I, E261R / Q265I / I287K, E261S / Q265I / I287T, Q265I / I287T, I283L and F294I.

[0152] In some embodiments, an engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO: 306 and one or more residue differences selected from the following compared to SEQ ID NO: 306: 197 / 198 / 201 / 259, 198 / 201 / 259 / 280, 202 / 205 / 221 / 223 / 225 / 261, 202 / 205 / 221 / 223 / 261 / 294, 202 / 221 / 222 / 225 / 256 / 261 / 294, 202 / 221 / 223 / 225 / 283 / 294, 205 / 221 / 222, 205 / 221 / 223 / 225 / 256 / 261 / 283, 219, 221 / 222 / 223 / 225, 221 / 223, 221 / 223 / 225, 221 / 223 / 225 / 256 / 261, 221 / 223 / 225 / 261, 221 / 223 / 225 / 294, 221 / 225, 221 / 225 / 256, 221 / 225 / 256 / 261, 221 / 225 / 256 / 261 / 283 / 294, 223 / 225 / 256 / 261, 225 / 256 / 261, 280 and 280 / 306.In some embodiments, an engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO: 306 and one or more residue differences selected from the following compared to SEQ ID NO: 306: 197T / 198S / 201L / 259V, 198A / 201L / 259V / 280L, 202L / 205S / 221S / 223G / 225Y / 261Y, 202L / 221H / 223G / 225F / 283M / 294I, 202M / 205S / 221H / 223G / 261Y / 294I, 202M / 205T / 221L / 223G / 225Y / 261R, 202M / 221S / 222I / 225Y / 256L / 261R / 294I, 205S / 221S / 223G / 225F / 256L / 261T / 283M, 205T / 221H / 222I, 219L, 221H / 222I / 223G / 225Y, 221H / 223G / 225L, 221H / 225F, 221L / 223G / 225L / 256T / 261R, 221L / 225F / 256V, 221S / 223G, 221S / 223G / 225L / 256L / 261R, 221S / 223G / 225L / 261R, 221S / 223G / 225L / 294I, 221S / 225F / 256T / 261R, 221S / 225Y / 256T / 261Y / 283M / 294I, 223G / 225L / 256V / 261R, 225F / 256T / 261T, 280D, 280E, 280I, 280V, 280V / 306F and 280W.In some embodiments, an engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO: 306 and one or more residue differences selected from the following compared to SEQ ID NO: 306: V197T / H198S / M201L / Y259V, H198A / M201L / Y259V / Y280L, H202L / A205S / Y221S / S223G / G225Y / V261Y, H202L / Y221H / S223G / G225F / I283M / F294I, H202M / A205S / Y221H / S223G / V261Y / F294I, H202M / A205T / Y221L / S223G / G225Y / V261R, H202M / Y221S / F222I / G225Y / C256L / V261R / F294I, A205S / Y221S / S223G / G225F / C256L / V261T / I283M, A205T / Y221H / F222I, F219L, Y221H / F222I / S223G / G225Y, Y221H / S223G / G225L, Y221H / G225F, Y221L / S223G / G225L / C256T / V261R, Y221L / G225F / C256V, Y221S / S223G, Y221S / S223G / G225L / C256L / V261R, Y221S / S223G / G225L / V261R, Y221S / S223G / G225L / F294I, Y221S / G225F / C256T / V261R, Y221S / G225Y / C256T / V261Y / I283M / F294I, S223G / G225L / C256V / V261R, G225F / C256T / V261T, Y280D, Y280E, Y280I, Y280V, Y280V / S306F and Y280W.

[0153] In some embodiments, an engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO: 648 and one or more residue differences selected from the following compared to SEQ ID NO: 648: 17 / 198 / 259 / 280, 197, 197 / 198, 197 / 198 / 223 / 259, 197 / 223 / 277 / 280, 197 / 259, 197 / 277, 198, 198 / 223, 198 / 223 / 259 / 277 / 280, 198 / 259, 198 / 259 / 277, 198 / 259 / 277 / 280, 198 / 277 / 280, 223 / 259, 223 / 259 / 280, 258, 259, 259 / 268 / 277 / 280, 259 / 277, 259 / 280, 263, 277 and 280. In some embodiments, an engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO: 648 and one or more residue differences selected from the following compared to SEQ ID NO: 648: 17P / 198S / 259V / 280F, 17P / 198S / 259W / 280W, 197L, 197L / 198S, 197L / 198S / 223G / 259W, 197L / 223G / 277T / 280K, 197L / 259W, 197L / 277F, 198A, 198S, 198S / 223G, 198S / 223G / 259W / 277W / 280G, 198S / 259V, 198S / 259V / 277T, 198S / 259W / 277F / 280G, 198S / 259W / 277T / 280F, 198S / 277F / 280F, 198S / 277G / 280W, 198S / 277P / 280F, 223G / 259V / 280W, 223G / 259W, 258Y, 259N / 280W, 259W, 259W / 268S / 277T / 280F, 259W / 277W, 263D, 263E, 263F, 263G, 263L, 263M, 263N, 263Q, 263R, 263V, 263W, 277W, 280D, 280F and 280W.In some embodiments, an engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO: 648 and one or more residue differences selected from the following compared to SEQ ID NO: 648: H17P / H198S / Y259V / Y280F, H17P / H198S / Y259W / Y280W, V197L, V197L / H198S, V197L / H198S / S223G / Y259W, V197L / S223G / N277T / Y280K, V197L / Y259W, V197L / N277F, H198A, H198S, H198S / S223G, H198S / S223G / Y259W / N277W / Y280G, H198S / Y259V, H198S / Y259V / N277T, H198S / Y259W / N277F / Y280G, H198S / Y259W / N277T / Y280F, H198S / N277F / Y280F, H198S / N277G / Y280W, H198S / N277P / Y280F, S223G / Y259V / Y280W, S223G / Y259W, W258Y, Y259N / Y280W, Y259W, Y259W / A268S / N277T / Y280F, Y259W / N277W, Y263D, Y263E, Y263F, Y263G, Y263L, Y263M, Y263N, Y263Q, Y263R, Y263V, Y263W, N277W, Y280D, Y280F and Y280W.

[0154] In some embodiments, an engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO:708 and one or more residue differences selected from the following compared to SEQ ID NO:708: 141, 154, 197, 197 / 198, and 278. In some embodiments, an engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO:708 and one or more residue differences selected from the following compared to SEQ ID NO:708: 141D, 154G, 154H, 197A, 197P / 198A, 278E, and 278V. In some embodiments, an engineered polypeptide having imine reductase activity 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 sequence identity to the reference sequence SEQ ID NO:708 and one or more residue differences selected from the following compared to SEQ ID NO:708: T141D, A154G, A154H, V197A, V197P / S198A, P278E, and P278V.

[0155] As will be understood by those skilled in the art, in some embodiments, one or a combination of the above - selected residue differences can be kept constant (i.e., maintained) as a core feature in an engineered imine reductase, and additional residue differences at other residue positions can be incorporated into the sequence to produce additional engineered imine reductase polypeptides with improved properties. Thus, it should be understood that for any engineered imine reductase containing one or a subset of the above residue differences, the present invention contemplates other engineered imine reductases that contain one or a subset of the said residue differences and additionally contain one or more residue differences at other residue positions disclosed herein.

[0156] As mentioned above, engineered polypeptides having imine reductase activity are also capable of converting substrates (such as compound (2) and compound (3)) into products (such as compound (1)). In some embodiments, the engineered imine reductase polypeptide is capable of converting the substrate compound into the product compound with an activity of 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 higher relative to the activity of the reference polypeptides SEQ ID NO: 4, 16, 50, 306, 648, and / or 708.

[0157] In some embodiments, an engineered imine reductase polypeptide capable of converting the substrate compound into the product compound with an activity of at least 2-fold relative to the activity of SEQ ID NO: 4, 16, 50, 306, 648, and / or 708 comprises an amino acid sequence selected from the even-numbered sequences within SEQ ID NO: 6 to 802.

[0158] In some embodiments, the engineered imine reductase has an amino acid sequence comprising one or more residue differences compared to SEQ ID NO: 4, 16, 50, 306, 648, and / or 708, which increases the expression of the engineered imine reductase activity in a bacterial host cell (particularly in Escherichia coli).

[0159] In some embodiments, the engineered imine reductase polypeptide having improved properties has an amino acid sequence comprising a sequence selected from the even-numbered sequences within the range of SEQ ID NO: 6 to 802.

[0160] In some embodiments, the engineered polypeptide having imine reductase activity comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to one of the even-numbered sequences within the range of SEQ ID NO: 6 to 802, and amino acid residue differences present in any of the even-numbered sequences within the range of SEQ ID NO: 6 - 802 compared to SEQ ID NO: 4, 16, 50, 306, 648, and / or 708, as provided in the examples.

[0161] In addition to the residue positions specified above, any engineered imine reductase polypeptide disclosed herein can also contain additional residue differences at other residue positions (i.e., residue positions other than those included herein) relative to SEQ ID NO: 4, 16, 50, 306, 648, and / or 708. Residue differences at these other residue positions can provide additional variation in the amino acid sequence without adversely affecting the ability of the polypeptide to effect substrate-to-product conversion. Thus, in some embodiments, in addition to the amino acid residue differences present in any of the engineered imine reductase polypeptides of the even-numbered sequences selected from within the range of SEQ ID NO: 6 - 802, the sequence can also contain 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 NO: 4, 16, 50, 306, 648, and / or 708. 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. The residue differences at these other positions can be conservative changes or non-conservative changes. In some embodiments, compared to the naturally occurring imine reductase polypeptides of SEQ ID NO: 4, 16, 50, 306, 648, and / or 708, the residue differences can include conservative substitutions and non-conservative substitutions.

[0162] In some embodiments, the present invention also provides engineered polypeptides comprising fragments of the engineered imine reductase polypeptide that retain the functional activity and / or improved properties of any of the engineered imine reductases described herein. Thus, in some embodiments, the present invention provides polypeptide fragments capable of converting a substrate to a product under suitable reaction conditions, wherein the fragment comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the full-length amino acid sequence of the engineered imine reductase polypeptide of the present invention, such as the exemplary engineered imine reductase polypeptides selected from the even-numbered sequences within the range of SEQ ID NOs: 6-802. In some embodiments, the engineered imine reductase polypeptide may have an amino acid sequence that includes deletions in any of the engineered imine reductase polypeptide sequences described herein, such as the exemplary engineered polypeptides of the even-numbered sequences within the range of SEQ ID NOs: 6-802.

[0163] Thus, for each and every embodiment of the engineered imine reductase polypeptide of the present invention, the amino acid sequence may include deletions of 1 or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids of the imine reductase polypeptide, up to 20% of the total number of amino acids, or up to 30% of the total number of amino acids, wherein the relevant functional activity and / or improved properties of the engineered imine reductase described herein are retained. In some embodiments, the deletion may include 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 residues. In some embodiments, the number of deletions may 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 include deletions 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.

[0164] In some embodiments, the engineered imine reductase polypeptides herein can have an amino acid sequence that includes an inserted amino acid sequence as compared to any of the engineered imine reductase polypeptides described herein, such as the exemplary engineered polypeptides of the even-numbered sequences within the range of SEQ ID NOs: 6 - 802. Thus, for each and every embodiment of the imine reductase polypeptides of the present invention, the insertion can include 1 or more amino acids, 2 or more amino acids, 3 or more amino acids, 4 or more amino acids, 5 or more amino acids, 6 or more amino acids, 8 or more amino acids, 10 or more amino acids, 15 or more amino acids, 20 or more amino acids, 30 or more amino acids, 40 or more amino acids, or 50 or more amino acids, wherein the relevant functional activity and / or improved properties of the engineered imine reductase described herein are maintained. The insertion can be at the amino terminus or carboxyl terminus of the imine reductase polypeptide, or in an internal portion.

[0165] In some embodiments, the engineered imine reductase polypeptides herein can have an amino acid sequence that includes a sequence selected from the even-numbered sequences within the range of SEQ ID NOs: 6 - 802 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 substitution can be a conservative substitution or a non-conservative substitution.

[0166] In the above embodiments, suitable reaction conditions for engineering polypeptides are provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1, and 10.1, and as described in the examples herein.

[0167] In some embodiments, the polypeptides of the invention are fusion polypeptides, wherein the engineered polypeptide is fused to other polypeptides, such other polypeptides including, for example but not limited to, antibody tags (e.g., myc epitope), purification sequences (e.g., His tag for binding metals), and cell localization signals (e.g., secretion signal). Thus, the engineered polypeptides described herein can be used with or without fusion to other polypeptides.

[0168] It should be 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 wholly or partially comprise naturally occurring and / or synthetic non-coded amino acids. Some common non-coded amino acids that the polypeptides described herein may comprise include, but are not limited to: D-stereoisomers of 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); tert-butylalanine (Bua); tert-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-fluorophenylalanine (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); pyridin-2-ylalanine (2pAla); pyridin-3-ylalanine (3pAla); pyridin-4-ylalanine (4pAla); naphthalen-1-ylalanine (1nAla); naphthalen-2-ylalanine (2nAla); thiazolylalanine (taAla); benzothiophenylalanine (bAla); thiophenylalanine (tAla); furylalanine (fAla); homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp); pentafluorophenylalanine (5ff); styrylkalanine (sAla); anthracenylalanine (aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenylvaleric 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); homol-aspartic acid (hAsp); homol-glutamic 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); homol-alanine (hAla); norvaline (nVal); homol-leucine (hLeu), homol-valine (hVal); homol-isoleucine (hIle); homol-arginine (hArg); N-acetyllysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal); homol-cysteine (hCys); homol-serine (hSer); hydroxyproline (Hyp) and homol-proline (hPro). Additional non-coded amino acids that can be included in the polypeptides described herein will be apparent to those skilled in the art (see, e.g., Fasman, CRC Practical Handbook of Biochemistry and Molecular Biology , CRC Press, Boca Raton, FL, pp. 3-70

[1989] and the various amino acids provided in the references cited therein, all of which are hereby incorporated by reference). These amino acids can be in the L-configuration or the D-configuration.

[0169] Those skilled in the art will recognize that amino acids or residues with side-chain protecting groups can also constitute the polypeptides described herein. Non-limiting examples of such protected amino acids, which in this case belong to the aromatic class, include, but are not limited to (the protecting groups are listed in parentheses): Arg(tos), Cys(methylbenzyl), Cys(nitropyridinesulfenyl), Glu(δ-benzyl ester), Gln(xanthenyl), Asn(N-δ-xanthenyl), His(bom), His(benzyl), His(tos), Lys(fmoc), Lys(tos), Ser(O-benzyl), Thr(O-benzyl) and Tyr(O-benzyl).

[0170] Conformationally restricted non-coded amino acids that can constitute 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, homol-proline (hPro) and 1-aminocyclopentane-3-carboxylic acid.

[0171] In some embodiments, the engineered polypeptide can be in various forms, e.g., such as an isolated article, as a substantially pure enzyme, a whole cell transformed with the gene encoding the enzyme, and / or as a cell extract and / or lysate of such a cell. The enzyme can be freeze-dried, spray-dried, precipitated, or in the form of a crude paste, as further discussed below.

[0172] 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 include organic polymers such as polystyrene, polyethylene, polypropylene, polyvinyl fluoride, polyethyleneoxy, and polyacrylamide, as well as their copolymers and grafts. The solid support can also be inorganic, such as glass, silica, controlled pore glass (CPG), reversed-phase silica, or a metal such as gold or platinum. The configuration of the solid support can be in the form of beads, spheres, microparticles, granules, gels, membranes, or surfaces. The surface can be flat, substantially flat, or non-flat. The solid support can be porous or non-porous and can have swelling or non-swelling properties. The solid support can be configured in the form of pores, depressions, or other containers, vessels, features, or locations.

[0173] In some embodiments, the engineered polypeptides having imine reductase activity of the present invention can be immobilized on a solid support such that they retain their improved activity and / or other improved properties relative to the reference polypeptides SEQ ID NO: 4, 16, 50, 306, 648, and / or 708. In such embodiments, the immobilized polypeptides can facilitate the biocatalytic conversion of a substrate compound or other suitable substrate to a product and can be readily retained after the reaction is complete (e.g., by retaining the beads to which the polypeptides are immobilized) and then reused or recycled in subsequent reactions. Such immobilized enzyme methods allow for further increased efficiency and reduced costs. Accordingly, it is also contemplated that any method using the imine reductase polypeptides of the present invention can be carried out using the same imine reductase polypeptides bound or immobilized on a solid support.

[0174] Methods of enzyme immobilization are well known in the art. Engineered polypeptides can be bound non-covalently or covalently. A variety of methods for conjugating and immobilizing enzymes to solid supports (such as resins, membranes, beads, glass, etc.) are well known in the art (see, for example, 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., online publication: dx.doi.org / 10.1021 / op200157c; Hermanson, Bioconjugate Techniques , 2nd Edition, 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 reference are incorporated herein by reference). Solid supports that can be used to immobilize the engineered imine reductases of the present invention include, but are not limited to, beads or resins comprising polymethacrylates having epoxide functional groups, polymethacrylates having aminoepoxide functional groups, styrene / DVB copolymers having octadecyl functional groups, or polymethacrylates. Exemplary solid supports that can be used to immobilize the engineered imine reductase polypeptides of the present 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.

[0175] 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 also include reagents for performing enzymatic reactions, substrates for assessing the activity of the enzymes, and reagents for detecting the products. The kit can also include reagent dispensers and instructions for using the kit.

[0176] In some embodiments, the kits of the invention include an array comprising a plurality of different imine reductase polypeptides at different addressable positions, 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 a solid support are arranged at a plurality of positions of the array for automated delivery of reagents or addressable by detection methods and / or instruments. The array can be used to test the conversion of the polypeptides to various substrate compounds. Such arrays comprising a plurality of engineered polypeptides and methods of using the same are known in the art (see, e.g., WO2009 / 008908A2).

[0177] Polynucleotides, expression vectors, and host cells encoding engineered imine reductases

[0178] In another aspect, the invention provides polynucleotides encoding the engineered imine reductase 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 comprising a heterologous polynucleotide encoding an engineered imine reductase is introduced into a suitable host cell to express the corresponding imine reductase polypeptide.

[0179] As will be apparent to the skilled person, the availability of the protein sequence and knowledge of the codons corresponding to the various amino acids provide a description of all polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, wherein the same amino acid is encoded by alternative codons or synonymous codons, allows for the preparation of a vast number of nucleic acids, all of which encode an improved imine reductase. Thus, given a particular amino acid sequence, one of ordinary skill in the art can prepare any number of different nucleic acids by simply changing 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 invention specifically contemplates every possible variation of polynucleotides that can be prepared by selecting combinations based on possible codon choices to encode the polypeptides described herein, and for any polypeptide described herein, all such variations are considered to be specifically disclosed, including the amino acid sequences presented in Tables 5.1, 6.1, 7.1, 8.1, 9.1, and 10.1 and the even-numbered sequences within the range of SEQ ID NO: 6 - 802 in the sequence listing incorporated herein by reference.

[0180] In various embodiments, codons are preferably selected to accommodate the host cell in which the protein is to be produced. For example, the preferred codons used in bacteria are used for gene expression in bacteria; 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, it is not necessary to substitute all codons to optimize the codon usage of imine reductase, since the native sequence will contain preferred codons and since it may not be necessary to use preferred codons for all amino acid residues. Thus, the codon-optimized polynucleotide encoding imine reductase may contain preferred codons at about 40%, 50%, 60%, 70%, 80% or greater than 90% of the codon positions in the full-length coding region.

[0181] In some embodiments, the polynucleotide comprises a codon-optimized nucleotide sequence encoding the amino acid sequence of a naturally-occurring imine reductase polypeptide as represented by SEQ ID NO: 4, 16, 50, 306, 648 and / or 708. In some embodiments, the polynucleotide has a nucleic acid sequence comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to a codon-optimized nucleic acid sequence encoding an even-numbered sequence within the range of SEQ ID NO: 6 - 802. In some embodiments, the polynucleotide has a nucleic acid sequence comprising at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity to a codon-optimized nucleic acid sequence encoding an odd-numbered sequence within the range of SEQ ID NO: 5 - 801. In some embodiments, the codon-optimized sequence of the odd-numbered sequences within the range of SEQ ID NO: 5 - 801 enhances the expression of the encoded wild-type imine reductase, providing an article of manufacture of an enzyme capable of converting a substrate to a product.

[0182] In some embodiments, the polynucleotide is capable of hybridizing under high stringency conditions to a reference sequence selected from the odd-numbered sequences of SEQ ID NO: 5 - 801 or its complementary sequence and encodes a polypeptide having imine reductase activity.

[0183] In some embodiments, as described above, the polynucleotide encodes an engineered polypeptide having imine reductase activity with improved properties compared to SEQ ID NO: 4, 16, 50, 306, 648, and / or 708, wherein the polypeptide 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 a reference sequence selected from SEQ ID NO: 4, 16, 50, 306, 648, and / or 708 and having one or more residue differences compared to SEQ ID NO: 4, 16, 50, 306, 648, and / or 708, wherein the sequence is selected from the even-numbered sequences within the range of SEQ ID NO: 6 - 802. In some embodiments, the reference amino acid sequence is selected from the even-numbered sequences within the range of SEQ ID NO: 6 - 802. In some embodiments, the reference amino acid sequence is SEQ ID NO: 4, while in some other embodiments, the reference sequence is SEQ ID NO: 16, while in some other embodiments, the reference sequence is SEQ ID NO: 50, while in some other embodiments, the reference sequence is SEQ ID NO: 306, while in some other embodiments, the reference sequence is SEQ ID NO: 648, and in still some other embodiments, the reference sequence is SEQ ID NO: 708.

[0184] In some embodiments, the polynucleotide encodes an imine reductase polypeptide capable of converting a substrate into a product with improved properties compared to SEQ ID NO: 4, 16, 50, 306, 648, and / or 708, wherein the polypeptide 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 sequence identity to the reference sequences SEQ ID NO: 4, 16, 50, 306, 648, and / or 708.

[0185] In some embodiments, the polynucleotide encoding the engineered imine reductase comprises a polynucleotide sequence selected from the odd-numbered sequences within the range of SEQ ID NO: 5 - 801.

[0186] In some embodiments, the polynucleotide is capable of hybridizing under high stringency conditions to a reference polynucleotide sequence selected from the odd-numbered sequences within the range of SEQ ID NOs: 5 - 801 or its complementary sequence, and encodes a polypeptide having imine reductase activity with one or more improved properties as described herein. In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an imine reductase polypeptide 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 higher identity to SEQ ID NO: 4, and the imine reductase polypeptide has an amino acid sequence comprising one or more residue differences at residue positions selected from the following compared to SEQ ID NO: 4: 145, 146, 153, 160, 222, 223, 226, and 261.

[0187] In some embodiments, the polynucleotide is capable of hybridizing under high stringency conditions to a reference polynucleotide sequence selected from the odd-numbered sequences within the range of SEQ ID NOs: 5 - 801 or its complementary sequence, and encodes a polypeptide having imine reductase activity with one or more improved properties as described herein. In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an imine reductase polypeptide 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 higher identity to SEQ ID NO: 16, and the imine reductase polypeptide has an amino acid sequence comprising one or more residue differences at residue positions selected from the following compared to SEQ ID NO: 16: 29 / 94 / 184 / 223 / 232 / 288 / 293, 29 / 94 / 184 / 232 / 287 / 288 / 293 / 311, 29 / 94 / 184 / 232 / 287 / 288 / 311, 29 / 94 / 184 / 232 / 287 / 293 / 332, 29 / 94 / 184 / 232 / 288 / 293 / 311 / 324 / 353, 29 / 94 / 184 / 232 / 288 / 311 / 324 / 332, 29 / 94 / 184 / 232 / 293, 29 / 94 / 184 / 232 / 311, 29 / 94 / 184 / 287 / 293, 29 / 94 / 184 / 287 / 293 / 311 / 353, 29 / 94 / 184 / 287 / 311, 29 / 94 / 184 / 288 / 293 / 324, 29 / 94 / 184 / 288 / 293 / 332 / 353, 29 / 94 / 184 / 288 / 298 / 332, 29 / 94 / 184 / 288 / 353, 29 / 94 / 184 / 293 / 311, 29 / 94 / 184 / 324, 29 / 94 / 223 / 232 / 287, 29 / 94 / 223 / 232 / 293 / 311 / 324, 29 / 94 / 223 / 288 / 293, 29 / 94 / 232 / 287 / 288 / 311, 29 / 94 / 232 / 288 / 293, 29 / 94 / 287 / 288 / 293, 29 / 94 / 287 / 288 / 293 / 353, 29 / 94 / 287 / 311, 29 / 94 / 293 / 311 / 324, 29 / 94 / 293 / 332 / 353, 29 / 94 / 311, 29 / 94 / 324 / 353, 29 / 184, 29 / 184 / 223 / 288 / 293 / 324, 29 / 184 / 232 / 288, 29 / 184 / 287, 29 / 184 / 287 / 288 / 293, 29 / 184 / 287 / 288 / 293 / 311,29 / 184 / 287 / 311 / 332、29 / 184 / 288 / 293、29 / 184 / 288 / 293 / 311、29 / 184 / 293、29 / 184 / 293 / 311、29 / 184 / 293 / 324、29 / 184 / 353、29 / 223 / 287 / 288 / 293 / 353、29 / 232 / 287 / 288 / 293 / 332 / 353、29 / 287 / 288 / 293、29 / 287 / 288 / 293 / 324 / 353、29 / 288 / 293、29 / 311、29 / 311 / 332、29 / 353、72 / 94 / 184 / 288 / 311、86、94 / 96 / 223 / 287 / 288 / 293 / 311 / 324 / 332、94 / 99 / 184 / 293 / 311 / 332 / 353、94 / 184 / 223、94 / 184 / 223 / 232 / 287 / 311、94 / 184 / 223 / 288 / 293、94 / 184 / 223 / 293、94 / 184 / 232 / 287 / 288、94 / 184 / 232 / 287 / 288 / 324 / 332、94 / 184 / 232 / 287 / 293、94 / 184 / 232 / 287 / 293 / 332、94 / 184 / 287 / 288 / 293、94 / 184 / 287 / 288 / 311、94 / 184 / 287 / 293 / 311 / 324 / 353、94 / 184 / 287 / 311、94 / 184 / 287 / 353、94 / 184 / 288 / 293、94 / 184 / 288 / 293 / 311、94 / 184 / 288 / 293 / 311 / 332、94 / 184 / 293、94 / 184 / 293 / 311、94 / 184 / 293 / 332 / 353、94 / 223 / 232 / 311 / 353 / 355 / 356 / 357 / 358 / 359、94 / 223 / 287 / 288 / 293、94 / 223 / 288 / 293 / 311、94 / 232 / 287 / 288 / 293 / 353、94 / 232 / 288 / 293 / 353、94 / 232 / 293、94 / 232 / 293 / 324 / 332、94 / 232 / 311、94 / 287 / 288 / 293、94 / 287 / 288 / 311 / 324、94 / 288 / 293、94 / 288 / 293 / 324、94 / 293 / 324、94 / 311 / 324、153、184 / 223、184 / 223 / 287 / 288 / 353、184 / 232 / 287 / 288 / 293 / 311、184 / 232 / 287 / 288 / 324 / 353、184 / 232 / 287 / 293 / 332, 184 / 287 / 288, 184 / 287 / 288 / 293, 184 / 287 / 288 / 293 / 324 / 353, 184 / 287 / 293, 184 / 287 / 293 / 311, 184 / 288 / 293 / 311 / 324, 184 / 332 / 353, 205, 223 / 232 / 287 / 288 / 332 / 353, 223 / 287 / 288, 223 / 287 / 288 / 293 / 311, 223 / 287 / 311 / 353, 223 / 288 / 293, 232 / 287 / 288 / 293, 232 / 287 / 288 / 293 / 311, 232 / 287 / 288 / 293 / 321, 232 / 287 / 311, 232 / 288 / 293, 232 / 288 / 293 / 332, 234 / 287 / 288 / 293 / 311, 287 / 288 / 293, 287 / 288 / 311 / 324, 287 / 293, 287 / 311, 287 / 311 / 332, 287 / 324, 288 / 293, 288 / 293 / 311 / 324 / 353, 288 / 293 / 324, 293 / 311 and 311 / 332.

[0188] In some embodiments, the polynucleotide is capable of hybridizing under high stringency conditions to a reference polynucleotide sequence selected from the odd-numbered sequences within the range of SEQ ID NO: 5 - 801 or its complementary sequence, and encodes a polypeptide having imine reductase activity with one or more improved properties as described herein. In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an imine reductase polypeptide 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 higher identity to SEQ ID NO: 50, and the imine reductase polypeptide has an amino acid sequence comprising one or more residue differences at residue positions selected from the following compared to SEQ ID NO: 50: 29 / 57 / 94 / 153 / 184 / 205 / 261 / 265 / 287, 29 / 57 / 94 / 153 / 205 / 265, 29 / 57 / 94 / 184 / 205 / 277 / 353, 29 / 57 / 94 / 205 / 259 / 261 / 265, 29 / 57 / 94 / 205 / 261 / 353, 29 / 57 / 153 / 184 / 205 / 259 / 261 / 265 / 277, 29 / 57 / 153 / 184 / 205 / 353, 29 / 57 / 153 / 205 / 261 / 265, 29 / 57 / 153 / 205 / 261 / 265 / 277, 29 / 57 / 153 / 205 / 265 / 277, 29 / 57 / 153 / 205 / 277 / 353, 29 / 57 / 184 / 205 / 259 / 261 / 265 / 277 / 287 / 357, 29 / 57 / 184 / 205 / 259 / 261 / 265 / 287, 29 / 57 / 205 / 259 / 261 / 265, 29 / 57 / 205 / 259 / 261 / 265 / 287, 29 / 57 / 205 / 261 / 265 / 353, 29 / 94 / 153 / 184 / 205, 29 / 94 / 153 / 205 / 259 / 261 / 265, 29 / 94 / 153 / 205 / 259 / 261 / 287, 29 / 94 / 153 / 205 / 259 / 261 / 287 / 353, 29 / 94 / 184 / 205 / 259 / 261, 29 / 94 / 184 / 205 / 261, 29 / 94 / 205, 29 / 94 / 205 / 259 / 261 / 287, 29 / 94 / 205 / 261, 29 / 94 / 261 / 265 / 287 / 353, 29 / 153 / 184 / 205, 29 / 153 / 205, 29 / 153 / 205 / 259 / 261 / 265 / 277 / 287, 29 / 153 / 261 / 265 / 287 / 353,29 / 184 / 205、29 / 184 / 205 / 259 / 261 / 277、29 / 184 / 205 / 353、29 / 184 / 261 / 265、29 / 184 / 265、29 / 205、29 / 205 / 259 / 261、29 / 205 / 259 / 261 / 265 / 287、29 / 205 / 259 / 261 / 287、29 / 205 / 261、29 / 205 / 261 / 265、29 / 205 / 261 / 265 / 353、29 / 205 / 261 / 353、29 / 205 / 277、29 / 205 / 287、29 / 205 / 287 / 353、29 / 259 / 261 / 265、29 / 261 / 353、57 / 94 / 153 / 184 / 205 / 259 / 261、57 / 94 / 153 / 205 / 261 / 265、57 / 94 / 153 / 259 / 261 / 265、57 / 94 / 153 / 261 / 265、57 / 94 / 184 / 205 / 261 / 265 / 287、57 / 94 / 184 / 261 / 265 / 287、57 / 94 / 205 / 259、57 / 94 / 205 / 259 / 261 / 265 / 277 / 287、57 / 94 / 205 / 261 / 265、57 / 94 / 205 / 277、57 / 153 / 205 / 259 / 261 / 265 / 287、57 / 153 / 205 / 259 / 265 / 277、57 / 153 / 205 / 261、57 / 153 / 205 / 261 / 265、57 / 153 / 205 / 265、57 / 153 / 261 / 265、57 / 153 / 261 / 265 / 287 / 353、57 / 205、57 / 205 / 259 / 261、57 / 205 / 259 / 261 / 265 / 277 / 287 / 353、57 / 205 / 259 / 261 / 287 / 353、57 / 205 / 259 / 265、57 / 205 / 261、57 / 205 / 261 / 265 / 277 / 287 / 353、57 / 205 / 261 / 277、57 / 205 / 261 / 277 / 353、57 / 261 / 265 / 353、94 / 126 / 184 / 205 / 259 / 261 / 265、94 / 153 / 184 / 205、94 / 153 / 184 / 259 / 261 / 265、94 / 153 / 205 / 259 / 261 / 265 / 353、94 / 153 / 205 / 287、94 / 153 / 205 / 287 / 353、94 / 153 / 205 / 353、94 / 184 / 205 / 259 / 261、94 / 184 / 205 / 259 / 261 / 265 / 287、94 / 184 / 205 / 259 / 261 / 353、94 / 184 / 205 / 261 / 265 / 287 / 353, 94 / 184 / 353, 94 / 205, 94 / 205 / 259 / 261 / 265, 94 / 205 / 259 / 261 / 265 / 277 / 287, 94 / 205 / 259 / 261 / 265 / 353, 94 / 205 / 259 / 261 / 287 / 353, 94 / 205 / 261 / 265 / 277, 94 / 205 / 261 / 265 / 353, 94 / 205 / 261 / 353, 94 / 205 / 277, 94 / 259 / 261 / 265, 153 / 184 / 205 / 261 / 277 / 287, 153 / 184 / 261 / 265 / 287, 153 / 205, 153 / 205 / 261, 153 / 205 / 261 / 265 / 287, 153 / 259 / 261 / 265, 153 / 259 / 265 / 287, 153 / 261 / 265, 153 / 261 / 265 / 287 / 353, 153 / 265, 153 / 265 / 277 / 287 / 353, 184 / 205, 184 / 205 / 259 / 261 / 287, 184 / 205 / 261, 184 / 205 / 261 / 265, 184 / 205 / 261 / 287, 184 / 205 / 277, 184 / 205 / 287 / 353, 184 / 261 / 265 / 287, 202, 205, 205 / 259 / 261, 205 / 259 / 261 / 265, 205 / 259 / 261 / 265 / 277 / 287 / 353, 205 / 259 / 261 / 277 / 287, 205 / 259 / 265, 205 / 261, 205 / 261 / 265, 205 / 261 / 265 / 274, 205 / 261 / 265 / 287, 205 / 261 / 265 / 287 / 353, 205 / 261 / 277, 205 / 261 / 353, 205 / 277 / 287, 205 / 287, 223, 225, 256, 259 / 261 / 265, 261 / 265 / 287, 265 / 287, 283 and 294.,

[0189] In some embodiments, the polynucleotide is capable of hybridizing under high stringency conditions to a reference polynucleotide sequence selected from the odd-numbered sequences within the range of SEQ ID NO: 5 - 801 or its complementary sequence, and encodes a polypeptide having imine reductase activity with one or more improved properties as described herein. In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an imine reductase polypeptide 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 higher identity to SEQ ID NO: 306, the imine reductase polypeptide having an amino acid sequence comprising one or more residue differences at residue positions selected from the following compared to SEQ ID NO: 306: 197 / 198 / 201 / 259, 198 / 201 / 259 / 280, 202 / 205 / 221 / 223 / 225 / 261, 202 / 205 / 221 / 223 / 261 / 294, 202 / 221 / 222 / 225 / 256 / 261 / 294, 202 / 221 / 223 / 225 / 283 / 294, 205 / 221 / 222, 205 / 221 / 223 / 225 / 256 / 261 / 283, 219, 221 / 222 / 223 / 225, 221 / 223, 221 / 223 / 225, 221 / 223 / 225 / 256 / 261, 221 / 223 / 225 / 261, 221 / 223 / 225 / 294, 221 / 225, 221 / 225 / 256, 221 / 225 / 256 / 261, 221 / 225 / 256 / 261 / 283 / 294, 223 / 225 / 256 / 261, 225 / 256 / 261, 280 and 280 / 306.

[0190] In some embodiments, the polynucleotide is capable of hybridizing under high stringency conditions to a reference polynucleotide sequence of an odd-numbered sequence selected from within the range of SEQ ID NOs: 5 - 801 or its complementary sequence, and encodes a polypeptide having imine reductase activity with one or more improved properties as described herein. In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an imine reductase polypeptide 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 higher identity to SEQ ID NO: 648, the imine reductase polypeptide having an amino acid sequence comprising one or more residue differences at residue positions selected from the following compared to SEQ ID NO: 648: 17 / 198 / 259 / 280, 197, 197 / 198, 197 / 198 / 223 / 259, 197 / 223 / 277 / 280, 197 / 259, 197 / 277, 198, 198 / 223, 198 / 223 / 259 / 277 / 280, 198 / 259, 198 / 259 / 277, 198 / 259 / 277 / 280, 198 / 277 / 280, 223 / 259, 223 / 259 / 280, 258, 259, 259 / 268 / 277 / 280, 259 / 277, 259 / 280, 263, 277 and 280.

[0191] In some embodiments, the polynucleotide is capable of hybridizing under high stringency conditions to a reference polynucleotide sequence of an odd-numbered sequence selected from within the range of SEQ ID NOs: 5 - 801 or its complementary sequence, and encodes a polypeptide having imine reductase activity with one or more improved properties as described herein. In some embodiments, the polynucleotide capable of hybridizing under high stringency conditions encodes an imine reductase polypeptide 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 higher identity to SEQ ID NO: 708, the imine reductase polypeptide having an amino acid sequence comprising one or more residue differences at residue positions selected from the following compared to SEQ ID NO: 708: 141, 154, 197, 197 / 198 and 278.

[0192] In some embodiments, a polynucleotide capable of hybridizing under high stringency conditions encodes an engineered polypeptide having imine reductase activity with improved properties, the engineered polypeptide 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 higher identity to SEQ ID NO: 4, 16, 50, 306, 648, and / or 708. 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 imine reductase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs: 5 - 801.

[0193] In some embodiments, a polynucleotide capable of hybridizing under high stringency conditions encodes an engineered polypeptide having imine reductase activity with improved properties, the engineered polypeptide 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 higher identity to SEQ ID NO: 4. 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 imine reductase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs: 5 - 35.

[0194] In some embodiments, a polynucleotide capable of hybridizing under high stringency conditions encodes an engineered polypeptide having imine reductase activity with improved properties, the engineered polypeptide 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 higher identity to SEQ ID NO:16. 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 imine reductase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs: 37-301.

[0195] In some embodiments, a polynucleotide capable of hybridizing under high stringency conditions encodes an engineered polypeptide having imine reductase activity with improved properties, the engineered polypeptide 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 higher identity to SEQ ID NO:50. 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 imine reductase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs: 301-645.

[0196] In some embodiments, a polynucleotide capable of hybridizing under high stringency conditions encodes an engineered polypeptide having imine reductase activity with improved properties, the engineered polypeptide 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 higher identity to SEQ ID NO:306. 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 imine reductase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs: 647-703.

[0197] In some embodiments, a polynucleotide capable of hybridizing under high stringency conditions encodes an engineered polypeptide having imine reductase activity with improved properties, the engineered polypeptide 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 higher identity to SEQ ID NO: 648. 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 imine reductase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs: 705 - 785.

[0198] In some embodiments, a polynucleotide capable of hybridizing under high stringency conditions encodes an engineered polypeptide having imine reductase activity with improved properties, the engineered polypeptide 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 higher identity to SEQ ID NO: 708. 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 imine reductase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs: 787 - 801.

[0199] In some embodiments, an isolated polynucleotide encoding any of the engineered imine reductase polypeptides provided herein is manipulated in a variety of ways to provide for expression of the polypeptide. In some embodiments, the polynucleotide encoding the polypeptide is provided as an expression vector, wherein one or more control sequences are present to regulate the expression of the polynucleotide and / or polypeptide. Depending on the expression vector, manipulation of the isolated polynucleotide prior to its insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art.

[0200] In some embodiments, the 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, but are not limited to, promoters obtained from the following: the Escherichia coli lac operon, the agarase gene (dagA) of Streptomyces coelicolor, the levansucrase gene (sacB) of Bacillus subtilis, the α-amylase gene (amyL) of Bacillus licheniformis, the maltogenic amylase gene (amyM) of Bacillus stearothermophilus, the α-amylase gene (amyQ) of Bacillus amyloliquefaciens, the penicillinase gene (penP) of Bacillus licheniformis, the xylA and xylB genes of Bacillus subtilis, and the prokaryotic β-lactamase gene (see, e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75:3727-3731

[1978] ), as well as 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 following genes: the TAKA amylase of Aspergillus oryzae, the aspartic proteinase of Rhizomucor miehei, the neutral α-amylase of Aspergillus niger, the acid-stable α-amylase of Aspergillus niger, the glucoamylase (glaA) of Aspergillus niger or Aspergillus awamori, the lipase of Rhizomucor miehei, the alkaline protease of Aspergillus oryzae, the triose phosphate isomerase of Aspergillus oryzae, the acetamidase of Aspergillus nidulans, and the trypsin-like protease of Fusarium oxysporum (see, e.g., WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoter from the Aspergillus niger neutral α-amylase gene and the Aspergillus oryzae triose phosphate isomerase gene), and its mutant, truncated, and hybrid promoters.Exemplary yeast cell promoters can be from the following genes: Saccharomyces cerevisiae enolase (ENO-1), 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, e.g., Romanos et al., Yeast 8:423-488

[1992] ).

[0201] In some embodiments, the control sequence is a suitable transcription terminator sequence, which is a sequence recognized by the 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 functional in the selected host cell can be used in the present invention. For example, exemplary transcription terminators for filamentous fungal host cells can be obtained from the following genes: Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger α-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the following genes: 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).

[0202] In some embodiments, the control sequence is a suitable leader sequence, which is an untranslated 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 functions in the selected host cell can be used. Exemplary leader sequences for filamentous fungal host cells are obtained from the genes: Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase. Suitable leader sequences for yeast host cells include, but are not limited to, those obtained from the genes: 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). The control sequence can also be a polyadenylation sequence, which is a sequence that is operably linked to the 3' end of the nucleic acid sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to the transcribed mRNA. Any polyadenylation sequence that functions in the selected host cell can be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, those from the genes: 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, for example, Guo and Sherman, Mol. Cell. Bio., 15:5983-5990

[1995] ).

[0203] 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 may inherently contain a signal peptide coding region that is naturally linked in translation reading frame to a segment of the coding region encoding a secreted polypeptide. Optionally, the 5' end of the coding sequence may 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 selected host cell can be used for the expression of the engineered imine reductase polypeptides provided herein. Effective signal peptide coding regions for bacterial host cells include, but are not limited to, signal peptide coding regions obtained from the genes of: Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus α-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutral proteases (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] ). Effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, signal peptide coding regions obtained from the genes of: 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, those from the genes of: Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase.

[0204] 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 referred to as a "proenzyme," "propolypeptide," or in some cases as a "zymogen." The propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Propeptide coding regions include, but are not limited to, the genes of: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae α-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 positioned immediately adjacent to the amino terminus of the polypeptide and the signal peptide region is positioned immediately adjacent to the amino terminus of the propeptide region.

[0205] In some embodiments, regulatory sequences are also utilized. These sequences facilitate regulation of polypeptide expression relative to host cell growth. Examples of regulatory systems are those that cause gene expression to be switched on or off in response to chemical or physical stimuli, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to, the lac, tac, and trp operon 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 α-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter.

[0206] The present invention also provides recombinant expression vectors that contain a polynucleotide encoding an engineered imine reductase polypeptide and one or more expression regulatory regions, such as promoters and terminators, origins of replication, etc., depending on the type of host into which they are to be introduced. In some embodiments, the various nucleic acids and control sequences described above can be combined to produce a recombinant expression vector that includes one or more convenient restriction sites to allow insertion or substitution of a nucleic acid sequence encoding a variant imine reductase polypeptide at such sites. Optionally, the polynucleotide sequences of the present invention are expressed by inserting the polynucleotide sequence or a nucleic acid construct containing the polynucleotide into an appropriate expression vector. When producing an expression vector, the coding sequence is positioned in the vector such that the coding sequence is operably linked to appropriate control sequences for expression.

[0207] The recombinant expression vector can be any vector (e.g., a plasmid or a virus) that can conveniently undergo recombinant DNA procedures and can result in the expression of the variant imine reductase polynucleotide sequence. The choice of vector will generally 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 plasmid or a closed circular plasmid.

[0208] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity and whose replication 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, when introduced into a host cell, is integrated into the genome and replicated along with the chromosome into which it is integrated. In addition, a single vector or plasmid or two or more vectors or plasmids, or a transposon, that together contain the total DNA to be introduced into the genome of the host cell can be used.

[0209] In some embodiments, the expression vector preferably comprises one or more selectable markers that allow for easy selection of transformed cells. A "selectable marker" is a gene whose product confers biocide or viral resistance, resistance to heavy metals, prototrophy for auxotrophs, etc. Examples of selectable markers for bacteria include, but are not limited to, the dal gene 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 adenylyltransferase), and trpC (anthranilate synthase) and their equivalents. In another aspect, the invention provides a host cell comprising a polynucleotide encoding at least one engineered imine reductase polypeptide of the invention, the polynucleotide being operably linked to one or more control sequences for expression of the engineered imine reductase in the host cell. Host cells for use in expressing polypeptides encoded by the expression vectors of the invention are well known in the art and include, but are not limited to, bacterial cells such as Escherichia coli, Vibrio fluvialis, Streptomyces, and Salmonella typhimurium cells; fungal cells such as yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris [ATCC Deposit 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 such as W3110 (ΔfhuA) and BL21.

[0210] Accordingly, in another aspect, the invention provides a method for producing an engineered imine reductase polypeptide, the method comprising culturing a host cell capable of expressing a polynucleotide encoding the engineered imine reductase polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the method further comprises the step of isolating and / or purifying the imine reductase polypeptide as described herein.

[0211] Suitable culture media and growth conditions for the host cells described above are well known in the art. Polynucleotides encoding imine reductase polypeptides can be introduced into cells by a variety of methods known in the art. Techniques include, among others, electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0212] Engineered imine reductases having the properties disclosed herein can be obtained by subjecting polynucleotides encoding naturally occurring or engineered imine reductase polypeptides to mutagenesis and / or directed evolution methods known in the art and as described herein. Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling (see, for example, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751

[1994] ; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and U.S. Patent 6,537,746). Other directed evolution procedures that can be used include, among others, the staggered extension process (StEP), in vitro recombination (see, for example, Zhao et al., Nat. Biotechnol., 16:258–261

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

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

[1996] ).

[0213] For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides to generate variant libraries that can be expressed, screened, and assayed. Mutagenesis and directed evolution methods are well known in the art (see, for example, U.S. Patent 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; 6,180,406; 6,251,674; 6,265,201; 6,277,638; 6,287,861; 6,287,862; 6,291,242; 6,297,053; 6,303,344; 6,309,883; 6,319,713; 6,319,714; 6,323,030; 6,326,204; 6,335,160; 6,335,198; 6,344,356; 6,352,859; 6,355,484; 6,358,740; 6,358,742; 6,365,377; 6,365,408; 6,368,861; 6,372,497; 6,337,186; 6,376,246; 6,379,964; 6,387,702; 6,391,552; 6,391,640; 6,395,547; 6,406,855; 6,406,910; 6,413,745; 6,413,774; 6,420,175; 6,423,542; 6,426,224; 6,436,675; 6,444,468; 6,455,253; 6,479,652; 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; 6,521,453; 6,528,311; 6,537,746; 6,573,098; 6,576,467; 6,579,678; 6,586,182; 6,602,986; 6,605,430; 6,613,514; 6,653,072; 6,686,515; 6,703,240; 6,716,631; 6,825,No. 001, No. 6,902,922, No. 6,917,882, No. 6,946,296, No. 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. 7,430,477, No. 7,462,469, No. 7,534,564, No. 7,620,500, No. 7,620,502, No. 7,629,170, No. 7,702,464, No. 7,747,391, No. 7,747,393, No. 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, No. 7,981,614, No. 8,014,961, No. 8,029,988, No. 8,048,674, No. 8,058,001, No. 8,076,138, No. 8,108,150, No. 8,170,806, No. 8,224,580, No. 8,377,681, No. 8,383,346, No. 8,457,903, No. 8,504,498, No. 8,589,085, No. 8,762,066, No. 8,768,871, No. 9,593,326, and all related U.S. and PCT and non-U.S. corresponding patents; Ling et al., Anal. Biochem., 254:157-78

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

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

[1994] ; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and WO 2009 / 152336, which are hereby incorporated by reference in their entirety).

[0214] In some embodiments, enzyme clones obtained after mutagenesis treatment are screened by subjecting the enzyme to a specified temperature (or other assay conditions, such as testing the enzyme's activity against a wide range of substrates), and measuring the amount of enzyme activity remaining after heat treatment or other assay conditions. Clones containing polynucleotides encoding imine reductase polypeptides are then sequenced to identify nucleotide sequence changes (if any), and used to express the enzyme in host cells. Measuring enzyme activity from an expression library can be carried out using any suitable method known in the art (e.g., standard biochemical techniques such as HPLC analysis).

[0215] In some embodiments, clones obtained after mutagenesis treatment can be screened for engineered imine reductases having one or more desired improved enzyme properties (e.g., improved regioselectivity). Measuring enzyme activity from an expression library can be carried out using standard biochemical techniques such as HPLC analysis and / or derivatization of the product (before or after separation), e.g., using dansyl chloride or OPA (see, e.g., Yaegaki et al., J Chromatogr. 356(1):163-70

[1986] ).

[0216] When the sequence of an engineered polypeptide is known, the polynucleotide encoding the enzyme can be prepared by standard solid-phase methods according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be synthesized individually and then ligated (e.g., by enzymatic ligation methods or chemical ligation methods or polymerase-mediated methods) to form any desired contiguous sequence. For example, polynucleotides and oligonucleotides encoding portions of imine reductase can be prepared by chemical synthesis as known in the art (e.g., the classic 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 it is typically carried out by 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. Additionally, substantially any nucleic acid can be obtained from any of a variety of commercial sources. In some embodiments, additional variations can be generated by synthesizing oligonucleotides containing deletions, insertions, and / or substitutions and combining the oligonucleotides in various permutations to produce engineered imine reductases with improved properties.

[0217] Thus, in some embodiments, a method for preparing an engineered imine reductase polypeptide comprises: (a) synthesizing a polynucleotide encoding a polypeptide that comprises 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 even-numbered sequence selected from SEQ ID NOs: 6-802 and having one or more residue differences compared to SEQ ID NOs: 4, 16, 50, 306, 648, and / or 708, wherein the residue difference is selected from any of the residue differences in Table 5.1, Table 6.1, Table 7.1, Table 8.1, Table 9.1, and Table 10.1; and (b) expressing the imine reductase polypeptide encoded by the polynucleotide.

[0218] In some embodiments of the method, the polynucleotide encodes an engineered imine reductase 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 - 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 can be conservative substitutions or non - conservative substitutions.

[0219] In some embodiments, any one or more of the well - known techniques for protein purification can be used to recover any of the engineered imine reductases expressed in a host cell from the cells and / or the culture medium. Well - known techniques for protein purification include, among others, lysozyme treatment, sonication, filtration, salting - out, ultra - centrifugation, and chromatography. Suitable solutions for lysing and efficiently extracting proteins from bacteria such as Escherichia coli are commercially available (e.g., CelLytic B TM , Sigma - Aldrich, St. Louis MO).

[0220] Chromatographic techniques for separating imine reductase polypeptides include, among others, reverse - phase chromatography, high - performance liquid chromatography, ion - exchange chromatography, gel electrophoresis, and affinity chromatography. The 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.

[0221] In some embodiments, affinity techniques can be used to isolate the improved imine reductase. For affinity chromatography purification, any antibody that specifically binds to the imine reductase polypeptide can be used. To generate the antibody, various host animals, including but not limited to rabbits, mice, rats, etc., can be immunized by injecting the imine reductase polypeptide or a fragment thereof. The imine reductase polypeptide or fragment can be attached to a suitable carrier such as BSA by way of side chain functional groups or a linker attached to the side chain functional groups. In some embodiments, affinity purification can use a specific ligand or dye affinity column that binds to the imine reductase (see, for example, EP0641862; Stellwagen, “Dye Affinity Chromatography,” in Current Protocols in Protein Science , Unit 9.2-9.2.16

[2001] ).

[0222] Methods of using engineered imine reductase

[0223] In some embodiments, the imine reductase described herein can be used in methods for converting one or more suitable substrates into products.

[0224] On the other hand, the engineered polypeptides disclosed herein can be used in methods for converting a substrate compound (2) or a structural analogue thereof and a substrate compound (3) or a structural analogue thereof into a product of compound (1) or a corresponding structural analogue. Generally, the structural analogues of compound (1) are included within structural formula (IV).

[0225] In some embodiments, the present disclosure provides a method for preparing a compound of structural formula (IV):

[0226]

[0227] wherein

[0228] R 1 is selected from a hydrogen atom, or an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, arylalkoxy, hydroxyalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocyclic hydrocarbyl, heteroaryl, and heteroarylalkyl; and

[0229] R 2 is independently selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, carboxyl, aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, alkylamino, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocyclic hydrocarbyl, heteroaryl, and heteroarylalkyl; and

[0230] R 3 is independently selected from methyl, d3-methyl, and ethyl;

[0231] The method includes the step of contacting a ketone substrate of structural formula (V) and an amine substrate of formula (VI) with an engineered polypeptide disclosed herein under suitable reaction conditions:

[0232]

[0233] Thereby preparing a product of formula (IV).

[0234] In some embodiments, the present disclosure provides a method for preparing compound (1);

[0235]

[0236] The method includes the step of contacting a substrate of compound (2)

[0237]

[0238] and a substrate of compound (3)

[0239]

[0240] with an engineered polypeptide disclosed herein, thereby preparing a product of compound (1).

[0241] An engineered imine reductase polypeptide derived from the wild-type enzyme of Arthrobacter sp. strain 1C can effectively convert substrate compound (2) and substrate compound (3) into product compound (1), but can also effectively convert a series of ketone substrate compounds of formula (I) and amine substrate compounds of formula (II) into secondary and tertiary amine product compounds of formula (III), as shown by the conversion reactions (A) to (E) listed in Table 2 below.

[0242]

[0243]

[0244] The present invention provides a number of exemplary engineered polypeptides having imine reductase activity. These exemplary polypeptides are evolved from previously engineered polypeptides SEQ ID NO: 4, 16, 50, 306, 648, and / or 708 (which were obtained by directed evolution from the wild-type CENDH of SEQ ID NO: 2) and exhibit improved properties, particularly increased activity and stability in the conversion of various ketone and amine substrates, including the conversion of compound (2a) and compound (3a) to amine product compound (1a), the conversion of compound (2) and compound (3a) to amine product compound (1b), the conversion of compound (2a) and compound (3b) to amine product compound (1c), the conversion of compound (2) and compound (3c) to amine product compound (1d), and the conversion of compound (2) and compound (3b) to amine product compound (1e). The results in Tables 5.1, 6.1, 7.1, 8.1, 9.1, and 10.1 provide variants having increased imine reductase activity and / or stability towards a range of ketone and amine substrates.

[0245] The structural and functional information of the exemplary non-naturally occurring (or engineered) imine reductase polypeptides of the present invention is based on five different high-throughput (HTP) screening assays used in the directed evolution of these enzymes: the conversion of compound (2a) and compound (3a) to amine product compound (1a), the conversion of compound (2) and compound (3a) to amine product compound (1b), the conversion of compound (2a) and compound (3b) to amine product compound (1c), the conversion of compound (2) and compound (3c) to amine product compound (1d), and the conversion of compound (2) and compound (3b) to amine product compound (1e).

[0246] Thus, in some embodiments, the engineered polypeptides having imine reductase activity of the present invention (which have 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 a reference sequence selected from the even-numbered sequence identifiers SEQ ID NO: 4 - 802 and having one or more residue differences at residue positions selected from those provided herein compared to SEQ ID NO: 2) are capable of performing one or more of the following conversion reactions under suitable reaction conditions and have improved activity and / or improved stereoselectivity relative to the reference polypeptides of the even-numbered sequence identifiers SEQ ID NO: 4 - 802:

[0247] (a) The conversion of substrate compound (2a) and substrate compound (3a) to product compound (1a);

[0248] (b) Conversion of substrate compound (2) and substrate compound (3a) to product compound (1b);

[0249] (c) Conversion of substrate compound (2a) and substrate compound (3b) to product compound (1c);

[0250] (d) Conversion of substrate compound (2) and substrate compound (3c) to product compound (1d); and

[0251] (e) Conversion of substrate compound (2) and substrate compound (3b) to product compound (1e).

[0252] In some embodiments, an engineered polypeptide having imine reductase activity and capable of catalyzing one or more of the above-described conversion reactions (A)-(E) with improved activity and / or stereoselectivity under suitable reaction conditions comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to one of the even-numbered sequence identifiers SEQ ID NOs: 4-802, and amino acid residue differences compared to SEQ ID NO: 2 as present in any of the even-numbered sequence identifiers SEQ ID NOs: 4-802 provided in Tables 5.1, 6.1, 7.1, 8.1, 9.1 and 10.1.

[0253] In some embodiments, an engineered polypeptide having imine reductase activity and capable of catalyzing one or more of the above-described conversion reactions (A)-(E) with improved activity and / or stereoselectivity under suitable reaction conditions has an amino acid sequence comprising a sequence selected from the even-numbered sequence identifiers SEQ ID NOs: 4-802. The wild-type octopine dehydrogenase SEQ ID NO: 2 (CENDH) of Arthrobacter sp. strain C1 from which the engineered polypeptides of the present invention are derived does not have detectable activity in converting the ketone substrate of compound (2) and the amine substrate of compound (3) to the secondary amine product compound (1). However, in some embodiments, an engineered polypeptide having imine reductase activity is capable of converting the ketone and amine substrates provided herein to the secondary amine product compounds described herein.

[0254] In the embodiments provided herein and illustrated in the examples, various ranges of suitable reaction conditions that can be used in the method include, but are not limited to, substrate loading, cosubstrate loading, pH, temperature, buffer, solvent system, polypeptide loading, and reaction time. Additional suitable reaction conditions for the biocatalytic conversion of substrate compounds to product compounds using the engineered imine reductases described herein can be readily optimized by routine experimentation in accordance with the guidance provided herein, which includes, but is not limited to, contacting the engineered imine reductase polypeptide and one or more substrate compounds under experimental reaction conditions of concentration, pH, temperature, and solvent conditions, and detecting the product compounds.

[0255] The substrate compounds in the reaction mixture can vary, taking into account, for example, the desired amount of product compound, the effect of each substrate concentration on enzyme activity, the stability of the enzyme under the reaction conditions, and the percentage of conversion of each substrate to product. In some embodiments, suitable reaction conditions include a substrate compound loading of at least about 0.5 g / L to about 25 g / L, 1 g / L to about 25 g / L, 5 g / L to about 25 g / L, about 10 g / L to about 25 g / L, or 20 g / L to about 25 g / L for each of one or more substrates. In some embodiments, suitable reaction conditions include a substrate compound loading 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, or at least about 30 g / L or even greater for each of one or more substrates.

[0256] When performing the imine reductase-mediated methods described herein, the engineered polypeptide can be added to the reaction mixture in the form of a purified enzyme, a partially purified enzyme, intact cells transformed with the gene encoding the enzyme, extracts and / or lysates of such cells, and / or an enzyme immobilized on a solid support. Intact cells or cell extracts transformed with the gene encoding the engineered imine reductase, their lysates, and isolated enzymes can be used in a variety of different forms, including solid (e.g., freeze-dried, spray-dried, etc.) or semi-solid (e.g., crude paste). Cell extracts or cell lysates can be partially purified by precipitation (ammonium sulfate, polyethyleneimine, heat treatment, etc.) and then subjected to a desalting procedure (e.g., ultrafiltration, dialysis, etc.) prior to freeze-drying. Any enzyme preparation (including intact cell preparations) can be stabilized by crosslinking or immobilizing on a solid phase (e.g., Eupergit C, etc.) using known crosslinking agents such as, for example, glutaraldehyde.

[0257] Genes encoding engineered imine reductase polypeptides can be transformed into host cells individually or together into the same host cell. For example, in some embodiments, one set of host cells can be transformed with a gene encoding one engineered imine reductase polypeptide, and another set of host cells can be transformed with a gene encoding another engineered imine reductase polypeptide. The two sets of transformed cells can be used together in a reaction mixture in the form of intact cells or in the form of lysates or extracts derived therefrom. In other embodiments, host cells can be transformed with genes encoding more than one engineered imine reductase polypeptide. 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 imine reductase reaction.

[0258] In some embodiments, the improved activity and / or regioselectivity and / or stereoselectivity of the engineered imine reductase polypeptides disclosed herein provides methods in which higher conversion percentages can be achieved with lower concentrations of the engineered polypeptides. In some embodiments of the method, suitable reaction conditions include amounts of the 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 higher of the substrate compound load.

[0259] 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 imine reductase 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 g / L, 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.

[0260] During the reaction process, the pH of the reaction mixture can vary. The pH of the reaction mixture can be maintained at a desired pH or within a desired pH range. This can be accomplished by adding an acid or a base before and / or during the reaction process. Optionally, 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, for example, but are not limited to, borates, phosphates, 2-(N-morpholino)ethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), acetates, triethanolamine, and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris), among others. In some embodiments, the reaction conditions include water as a suitable solvent and no buffer.

[0261] In embodiments of the method, the reaction conditions include a suitable pH. The desired pH or desired pH range can be maintained by using an acid or a 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 reaction process. In some embodiments, suitable reaction conditions include a solution having a pH of from about 4 to about 10, from about 5 to about 10, from about 5 to about 9, from about 6 to about 9, or from about 6 to about 8. In some embodiments, the reaction conditions include a solution having a pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10.

[0262] In embodiments of the methods herein, a suitable temperature is used for the reaction conditions, such as taking into account the increase in reaction rate at higher temperatures and the activity of the enzyme during the reaction period. Thus, in some embodiments, suitable reaction conditions include a temperature of from about 10°C to about 60°C, from about 10°C to about 55°C, from about 15°C to about 60°C, from about 20°C to about 60°C, from about 20°C to about 55°C, from about 25°C to about 55°C, or from about 30°C to about 50°C. In some embodiments, suitable reaction conditions include a temperature 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 reaction process. In some embodiments, the temperature during the enzymatic reaction can be adjusted during the reaction process with changes in the temperature profile.

[0263] In some embodiments, the methods of the invention are carried out in a solvent. Suitable solvents include water, aqueous buffer solutions, organic solvents, polymeric solvents, and / or cosolvent systems, which typically comprise an aqueous solvent, an organic solvent, and / or a polymeric solvent. The aqueous solvent (water or an aqueous cosolvent system) can be pH-buffered or unbuffered. In some embodiments, the method of using an engineered imine reductase polypeptide can be carried out in an aqueous cosolvent system comprising: an organic solvent (such as ethanol, isopropyl alcohol (IPA), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl tert-butyl ether (MTBE), toluene, etc.), an ionic or polar solvent (such as 1-ethyl-4-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, glycerol, polyethylene glycol, etc.). In some embodiments, the cosolvent can be a polar solvent such as a polyol, dimethyl sulfoxide (DMSO), or a lower alcohol. The non-aqueous cosolvent component of the aqueous cosolvent system can be miscible with the aqueous component, providing a single liquid phase, or can be partially miscible or immiscible with the aqueous component, providing two liquid phases. Exemplary aqueous cosolvent systems can comprise water and one or more cosolvents selected from organic solvents, polar solvents, and polyol solvents. Generally, the cosolvent components of the aqueous cosolvent system are selected such that they do not inactivate the imine reductase unfavorably under the reaction conditions. Suitable cosolvent systems can be readily identified by measuring the enzyme activity of a particular engineered imine reductase with a defined substrate of interest in a candidate solvent system using an enzyme activity assay (such as those described herein).

[0264] In some embodiments of the method, suitable reaction conditions include an aqueous cosolvent, wherein the cosolvent comprises from about 1% to about 50% (v / v), from about 1% to about 40% (v / v), from about 2% to about 40% (v / v), from about 5% to about 30% (v / v), from about 10% to about 30% (v / v), or from about 10% to about 20% (v / v) DMSO. In some embodiments of the method, suitable reaction conditions can include an aqueous cosolvent comprising from 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) ethanol.

[0265] In some embodiments, the reaction conditions include surfactants for stabilizing or enhancing the reaction. The surfactants can include nonionic surfactants, cationic surfactants, anionic surfactants, and / or amphiphilic surfactants. Exemplary surfactants include, for example but not limited to, nonylphenoxypolyethoxyethanol (NP40), TRITON TM X-100 polyethylene glycol tert-octylphenyl ether, polyoxyethylene-stearamide, cetyltrimethylammonium bromide, oleyl amidosulfate, polyoxyethylene sorbitan monostearate, cetyl dimethylamine, etc. Any surfactant that can stabilize or enhance the reaction can be used. The concentration of the surfactant used in the reaction can generally be from 0.1 mg / ml to 50 mg / ml, particularly 1 mg / ml to 20 mg / ml.

[0266] In some embodiments, the reaction conditions include antifoaming agents, which help reduce or prevent the formation of foam in the reaction solution, such as when the reaction solution is mixed or sprayed. Antifoaming agents include nonpolar oils (such as mineral oil, silicone, etc.), polar oils (such as fatty acids, alkylamines, alkylamides, alkyl sulfates, etc.), and hydrophobic substances (such as treated silica, polypropylene, etc.), some of which also function as surfactants. Exemplary antifoaming agents include (Dow Corning), polyglycol copolymers, oxy / ethoxylated alcohols, and polydimethylsiloxane. In some embodiments, the antifoaming agent can be present in an amount of 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 can be present in an amount of 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 higher, as desired to facilitate the reaction.

[0267] The amount of reactants used in the imine reductase reaction will generally vary depending on the amount of the desired product and the amount of the imine reductase substrate used therewith. Those of ordinary skill in the art will readily understand how to vary these amounts to tailor them to the desired productivity and production scale levels.

[0268] In some embodiments, the order of addition of reactants is not critical. The reactants can be added simultaneously together to a solvent (e.g., a single-phase solvent, a biphasic aqueous cosolvent system, etc.), or alternatively, some reactants can be added separately and some reactants can be added together at different time points. For example, cofactors, cosubstrates, and substrates can be added to the solvent first.

[0269] Solid reactants (e.g., enzymes, salts, etc.) can be provided to the reaction in a variety of different forms, including powders (e.g., lyophilized powders, spray-dried powders, etc.), solutions, emulsions, suspensions, etc. Using methods and equipment known to those of ordinary skill in the art, the reactants can be easily lyophilized or spray-dried. For example, a protein solution can be frozen in small aliquots at -80 °C and then added to a pre-cooled lyophilization chamber, followed by application of a vacuum.

[0270] When using a biphasic aqueous cosolvent system, to improve mixing efficiency, the imine reductase and cosubstrate can be added and mixed into the aqueous phase first. The imine reductase substrate can be added and mixed in, followed by the organic phase, or the substrate can be dissolved in the organic phase and mixed in. Alternatively, the imine reductase substrate can be premixed in the organic phase and then added to the aqueous phase.

[0271] Generally, the method of the present invention is allowed to proceed until further conversion of the substrate to the product does not vary 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 the substrate is completely or nearly completely converted to the product. Conversion of the substrate to the product can be monitored using known methods by detecting the substrate and / or the product (with or without derivatization). Suitable analytical methods include gas chromatography, HPLC, MS, etc.

[0272] In some embodiments of the method, suitable reaction conditions include a substrate loading of at least about 5 g / L, 10 g / L, 20 g / L, or higher for each of one or more substrates, and wherein the method produces at least about 50%, 60%, 70%, 80%, 90%, 95%, or higher conversion of the substrate compound to the product compound in about 24 h or less, in about 12 h or less, in about 6 h or less, or in about 4 h or less.

[0273] When used in the method under suitable reaction conditions, the engineered imine reductase polypeptide of the present invention produces an excess of the desired product that is at least 90%, 95%, 96%, 97%, 98%, 99%, or higher enantiomeric excess compared to the undesired product.

[0274] In some additional embodiments of methods of using engineered imine reductase polypeptides to convert one or more compounds to product compounds, suitable reaction conditions can include an initial substrate loading for each of one or more substrates in a reaction solution, followed by contact with the polypeptide. The reaction solution is then further supplemented with additional substrate compounds added continuously or in batches, for each of one or more substrates, 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 higher over time. Thus, according to these suitable reaction conditions, for each of one or more substrates, the polypeptide is added to a solution having an initial substrate loading of at least about 1 g / L, 5 g / L or 10 g / L. After such addition of the polypeptide, then for each of one or more substrates, additional substrate is added continuously 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 or higher is reached for each of one or more substrates. Thus, in some embodiments of the method, suitable reaction conditions include adding the polypeptide to a solution having an initial substrate loading of at least about 1 g / L, 5 g / L or 10 g / L, and then adding 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 or higher is reached for each of one or more substrates. These substrate supplementation reaction conditions allow for higher substrate loadings to be reached while maintaining a high conversion of substrate to product of at least about 5%, 25%, 50%, 75%, 90% or higher for each of one or more substrates or both.

[0275] Any method disclosed herein for preparing a compound of formula (IV) or compound (1) using an engineered polypeptide can be carried out under a suitable range of reaction conditions, including but not limited to amine substrate range, ketone substrate range, temperature, pH, solvent system, substrate loading, polypeptide loading, cofactor loading, and reaction time. In one example, in some embodiments, the preparation of a compound of formula (IV) or compound (1) can be carried out, where the suitable reaction conditions include: (a) an amine substrate loading of about 1 g / L to 50 g / L of the substrate compound; (b) a ketone substrate loading of about 1 g / L to 50 g / L of the substrate compound; (c) about 0.5 g / L to 10 g / L of the engineered polypeptide; (d) 0 - 20% DMSO; (e) 1 g / L of PDH cofactor recycling enzyme, 0.1 g / L of NAD+ cofactor, and up to 20 g / L of phosphite; (f) a temperature of about 30 °C to 60 °C. In some embodiments, the suitable reaction conditions include: (a) about 2 g / L of the amine substrate compound; (b) about 2 g / L of the ketone substrate compound; (c) about 5 g / L of the engineered polypeptide; (d) 5% DMSO; (e) 1 g / L PDH cofactor recycling enzyme, 0.1 g / L NAD+ cofactor, and 20 g / L phosphite; and (f) about 30 °C.

[0276] In some embodiments, additional reaction components or additional techniques are carried out to supplement the reaction conditions. These can include taking measures to stabilize the enzyme or prevent enzyme inactivation, reduce product inhibition, and shift the reaction equilibrium towards the formation of the desired product.

[0277] In additional embodiments, any of the methods described above for converting one or more substrate compounds into a product compound can further include one or more steps selected from: extraction, separation, purification, and crystallization of the product compound. Methods, techniques, and protocols for extracting, separating, purifying, and / or crystallizing the product from the biocatalytic reaction mixture produced by the methods disclosed above are known to those of ordinary skill in the art and / or obtained by routine experimentation. Additionally, illustrative methods are provided in the examples below.

[0278] The various features and embodiments of the present invention are illustrated in the following representative examples, which are intended to be illustrative and not limiting.

[0279] Experiments

[0280] The following examples are provided, including the experiments and the results obtained, for illustrative purposes only and should not be construed as limiting the present invention.

[0281] In the following examples, the following abbreviations are applicable: ppm (parts per million); M (moles per liter); mM (millimoles per liter), uM and μM (micromoles per liter); nM (nanomoles per liter); mol (mole); gm and g (gram); mg (milligram); ug and μg (microgram); L and l (liter); ml and mL (milliliter); cm (centimeter); mm (millimeter); um and μm (micrometer); sec. (second); min(s) (minute); h(s) and hr(s) (hour); U (unit); MW (molecular weight); rpm (revolutions per minute); psi and PSI (pounds per square inch); °C (degrees Celsius); RT and rt (room temperature); CAM and cam (chloramphenicol); DMSO (dimethyl sulfoxide); PMBS (polymyxin B sulfate); IPTG (isopropyl β-D-1-thiogalactopyranoside); LB (Luria-Bertani broth); TB (Terrific broth; 12 g / L tryptone for bacteria, 24 g / L yeast extract, 4 mL / L glycerol, 65 mM potassium phosphate, pH 7.0, 1 mM MgSO 4 ); HEPES (HEPES zwitterionic buffer; 4-(2-hydroxyethyl)-piperazineethanesulfonic acid); SFP (shake flask powder); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); Escherichia coli W3110 (a commonly used laboratory Escherichia coli strain, available from Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput); HPLC (high performance liquid chromatography); FIOPC (fold improvement over positive control); Microfluidics (Microfluidics, Corp., Westwood, MA); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO; Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, MI); Agilent (Agilent Technologies, Inc., Santa Clara, CA); Corning (Corning, Inc., Palo Alto, CA); Dow Corning (Dow Corning, Corp., Midland, MI) and Gene Oracle (Gene Oracle, Inc., Mountain View, CA).

[0282] Example 1

[0283] Escherichia coli expression host containing recombinant CENDH gene

[0284] The wild-type opine dehydrogenase polypeptide (CENDH) from Arthrobacter sp. strain C1 reported was codon-optimized using the GeneIOS synthesis platform (GeneOracle) and synthesized into the gene of SEQ ID NO:2. The wild-type CENDH-encoding gene was cloned into the expression vector pCK110900 (see Figure 3 of US Patent Application Publication No. 2006 / 0195947), operably linked to the lac promoter under the control of the lacI repressor. The expression vector also contains the P15a origin of replication and the chloramphenicol resistance gene. The resulting plasmid was transformed into Escherichia coli W3110fhuA using standard methods known in the art. As known in the art, transformants were isolated by subjecting the cells to chloramphenicol selection (see, for example, US Patent No. 8,383,346 and WO2010 / 144103). As described in US Patent Nos. 9,487,760 and 9,695,451, the initial CENDH enzyme (shown as SEQ ID NO:4) used to generate the variants of the present invention was engineered based on the gene of SEQ ID NO:2.

[0285] Example 2

[0286] Preparation of high-throughput CENDH-containing wet cell pellets

[0287] Escherichia coli cells containing the recombinant CENDH-encoding gene from monoclonal colonies were inoculated into 180 μl of LB containing 1% glucose and 30 μg / mL chloramphenicol in the wells of a 96-well shallow-well microtiter plate. The plate was sealed with an 2 O-permeable seal, and the culture was grown overnight at 30 °C, 200 rpm, and 85% humidity. Then, 20 μl of each cell culture was transferred to the wells of a 96-well deep-well plate containing 380 μL of TB and 30 μg / mL CAM. The deep-well plate was sealed with an 2 O-permeable seal and cultured at 30 °C, 250 rpm, and 85% humidity until an OD 600 of 0.6 - 0.8 was reached. Then the cell culture was induced with IPTG to a final concentration of 1 mM and cultured overnight under the same conditions as initially used. Then the cells were pelleted by centrifugation at 4000 rpm for 10 minutes. In some cases, the overnight cultures were combined from two plates, then centrifuged, and duplicate pellets were obtained. The supernatant was discarded, and the pellet was frozen at -80 °C before lysis.

[0288] Example 3

[0289] Preparation of high-throughput CENDH-containing cell lysates

[0290] First, 200 μl of lysis buffer containing 200 mM triethanolamine (TEoA) buffer pH 8.5, 1 mg / mL lysozyme, and 0.5 mg / mL PMBS was added to the cell paste in each well generated as described in Example 2. The cells were lysed for 2 hours at room temperature with shaking on a bench top shaker. The plate was then centrifuged at 4000 rpm and 4 °C for 15 min. The clarified supernatant was used for the biocatalytic reaction to determine its activity level.

[0291] Example 4

[0292] Preparation of lyophilized lysates from shake flask (SF) cultures

[0293] The selected HTP cultures grown as described above were plated onto LB agar plates containing 1% glucose and 30 μg / ml CAM and grown overnight at 37 °C. Single colonies from each culture were transferred to 6 ml of LB containing 1% glucose and 30 μg / ml CAM. The cultures were grown at 30 °C, 250 rpm for 18 h and subcultured at approximately 1:50 into 250 ml of TB containing 30 μg / ml CAM to a final OD of 0.05 600 . The cultures were grown at 30 °C, 250 rpm for approximately 195 minutes to an OD between 0.6 - 0.8 600 , and induced with 1 mM IPTG. The cultures were then grown at 30 °C, 250 rpm for 20 h. The cultures were centrifuged at 4000 rpm for 20 min. The supernatant was discarded and the pellet was resuspended in 30 ml of 200 mM TEoA buffer, pH 8.5. The cell pellet (4000 rpm × 20 min) was frozen at -80 °C for 120 minutes. The frozen pellet was resuspended in 30 ml of 200 mM TEoA buffer, pH 8.5, and lysed using a Microfluidizer system (Microfluidics) at 18,000 psi. The lysate was pelleted (10,000 rpm × 60 min) and the supernatant was frozen and lyophilized to produce shake flask (SF) enzyme.

[0294] Example 5

[0295] Activity improvement of CENDH relative to SEQ ID NO:4 in high throughput screening

[0296] SEQ ID NO:4 was chosen as the parental enzyme, which has evolved towards improved imine reductase activity for the conversion of cyclohexanone and norvaline to the secondary amine product. As disclosed in U.S. Patent Nos. 9,487,760 and 9,695,451, it has one amino acid difference (N198H) relative to SEQ ID NO:2. The polypeptide of SEQ ID NO:4 has no detectable activity to convert chlorobiphenylpyruvic acid (Compound (2)) and L-alanine ester (Compound (3)) to its amine product (Compound (1)) or phenylpyruvate (Compound (2a)) and L-alanine ester (Compound (3)) to its amine product. Libraries of engineered genes were generated using well-known techniques (such as saturation mutagenesis, recombination of previously identified beneficial mutations) to improve its imine reductase activity towards the reaction product (Compound (1a)) of phenylpyruvate (Compound (2a)) and L-alanine (Compound (3a)). Each gene-encoded polypeptide was produced in HTP as described in Example 2, duplicate precipitates were obtained, and soluble lysates were produced as described in Example 3.

[0297] To lyse the cells, 200 μl of lysis buffer containing 200 mM TEoA buffer pH 8.5, 1 mg / mL lysozyme, and 0.5 mg / mL PMBS was added to the cell pellet. The cells were incubated at room temperature for 2 hours with shaking on a benchtop oscillator. The plate was then centrifuged at 4000 rpm and 4 °C for 15 min, and the clarified supernatant was used for subsequent biocatalytic reactions.

[0298] The HTP reaction was carried out in a 96-well deep-well plate containing 200 μL of 0.2 M TEoA pH 8.5, 2.5 g / L sodium phenylpyruvate, 6 g / L L-alanine (∼5x molar excess), 1 g / L glucose dehydrogenase (GDH) 105 (U.S. Patent Nos. 7,816,111 and 7,939,309), 10 g / L glucose, 3 g / L NAD+, and 5 μL or 10 μL of the above HTP supernatant. The HTP plate was incubated in a Thermotron (3 mm swing, mode #AJ185, Infors) at 30 °C and 400 rpm for 12 hours. The reaction was quenched with 200 μl of acetonitrile and mixed using a benchtop oscillator for 5 minutes. The plate was then centrifuged at 4000 rpm for 10 minutes. 4 μL of the supernatant was further diluted to 3996 μL of water and loaded onto a RapidFire for analysis as described in Example 11.

[0299] Activity relative to SEQ ID NO:4 was calculated as fold improvement over the positive control (FIOPC). It was determined by dividing the mass spectrometric signal of the product in each sample under the specified reaction conditions by the mass spectrometric signal of phenylpyruvic acid product (compound (1a)) in the parental variant (positive control) present in the same plate, and is shown in Table 5.1.

[0300]

[0301] Example 6

[0302] Activity improvement of CENDH relative to SEQ ID NO:16 in high throughput screening

[0303] After screening variants described in Example 5, SEQ ID NO:16 was selected as the parental enzyme. A library of engineered genes was generated using well-known techniques (such as saturation mutagenesis, recombination of previously identified beneficial mutations). Each gene-encoded polypeptide was produced in HTP as described in Example 2, duplicate precipitates were obtained, and soluble lysates were produced as described in Example 3.

[0304] To lyse the cells, 200 μl of lysis buffer containing 200 mM TEoA buffer pH 8.5, 1 mg / mL lysozyme and 0.5 mg / mL PMBS was added to the cell pellet. The cells were incubated for 2 hours at room temperature with shaking on a benchtop shaker. The plate was then centrifuged at 4000 rpm and 4 °C for 15 minutes, and the clarified supernatant was used for subsequent biocatalytic reactions.

[0305] The HTP reaction was carried out in a 96-well deep well plate containing 200 μL of 0.2 M TEoA pH 8.5, 2.5 g / L 3-[4-(3-chlorophenyl)phenyl]-2-oxopropanoic acid (referred to herein as chlorobiphenylpyruvic acid or (compound (2)), 4 g / L L-alanine (compound (3a)) (~5x molar excess), 1 g / L GDH 105, 10 g / L glucose, 3 g / L NAD+, 5% DMSO and 100 μl of the above HTP supernatant. The HTP plate was incubated in a Thermotron (3 mm swing, mode #AJ185, Infors) at 30 °C and 400 rpm for 12 hours. In parallel, HTP reactions of phenylpyruvate (compound (2a)) and L-alanine reaction (compound (3a)) were also carried out as described in Example 5. The reaction was quenched with 0.1% formic acid in methanol at a ratio of 1:10 and mixed using a benchtop shaker for 5 minutes. The plate was then centrifuged at 4000 rpm for 10 minutes, the supernatant was further diluted with water at a ratio of 1:40, and then loaded onto RapidFire for analysis as described in Example 11.

[0306] The activity relative to SEQ ID NO:50 was calculated as the fold increase over the positive control (FIOPC). It was determined by dividing the mass spectrometry signal of the product in each sample under the specified reaction conditions by the mass spectrometry signal of the phenylpyruvic acid product (Compound (1a)) or chlorobiphenylpyruvic acid product (Compound (1b)) in the parental variant (positive control) present in the same plate, as shown in Table 6.1.

[0307]

[0308]

[0309]

[0310]

[0311]

[0312] Example 7

[0313] Activity improvement of CENDH relative to SEQ ID NO:50

[0314] After screening variants described in Example 6, SEQ ID NO:50 was selected as the parental enzyme. A library of engineered genes was generated using well-known techniques (such as saturation mutagenesis, recombination of previously identified beneficial mutations). Each gene-encoded polypeptide was produced in HTP as described in Example 2, duplicate precipitates were obtained, and soluble lysates were produced as described in Example 3.

[0315] To lyse the cells, 300 μl of lysis buffer containing 200 mM TEoA buffer pH 8.5, 1 mg / mL lysozyme, and 0.5 mg / mL PMBS was added to the cell pellet. The cells were incubated at room temperature for 2 hours with shaking on a bench-top shaker. The plate was then centrifuged at 4000 rpm and 4 °C for 15 minutes, and the clarified supernatant was used for subsequent biocatalytic reactions.

[0316] The HTP reaction was carried out in the same manner as in Example 6 to detect the reaction products of chlorobiphenylpyruvic acid (Compound (2)) and L-alanine (Compound (3a)) (Compound (1b)).

[0317] The activity relative to SEQ ID NO:50 was calculated as the fold increase over the positive control (FIOPC) and is shown in Table 7.1. It was determined by dividing the mass spectrometry signal of the product in each sample under the specified reaction conditions by the mass spectrometry signal of the chlorobiphenylpyruvic acid product (Compound (1b)) in the parental variant (positive control) present in the same plate.

[0318]

[0319]

[0320]

[0321]

[0322]

[0323] Example 8

[0324] Activity improvement of CENDH relative to SEQ ID NO:306

[0325] After screening for variants described in Example 7, SEQ ID NO:306 was selected as the parental enzyme. A library of engineered genes was generated using well-known techniques (such as saturation mutagenesis, recombination of previously identified beneficial mutations). Each gene-encoded polypeptide was produced by HTP as described in Example 2, duplicate precipitates were obtained, and soluble lysates were produced as described in Example 3.

[0326] To lyse the cells, 300 μl of lysis buffer containing 200 mM TEoA buffer pH 8.5, 1 mg / mL lysozyme, and 0.5 mg / mL PMBS was added to the cell pellet. The cells were incubated at room temperature for 2 hours with shaking on a bench-top shaker. The plate was then centrifuged at 4000 rpm and 4 °C for 15 minutes, and the clarified supernatant was used for subsequent biocatalytic reactions.

[0327] The HTP reaction was carried out in a 96-well deep-well plate containing 200 μL of 0.2 M TEoA pH 8.5, 1.25 g / L chlorobiphenylpyruvic acid (Compound (2)), 3.6 g / L 3,3,3-d3 L-alanine ester (Compound (3b)) (∼5x molar excess), 0.5 g / L PDH wt, 25 mM phosphite, 1.5 g / L NAD+, 5% DMSO, and 100 μl of the above HTP supernatant to produce the product of Compound (1e). The HTP plate was incubated in a Thermotron (3 mm swing, mode #AJ185, Infors) at 30 °C and 400 rpm for 12 hours. HTP reactions were also carried out to determine the reaction products of pyruvate (Compound (2a)) and 3,3,3-d3 L-alanine ester (Compound (3b)) (Compound (1c)). The reaction conditions were the same as those for the chlorobiphenylpyruvic acid reaction described above, except that 1.25 g / L sodium pyruvate and 2.6 g / L 3,3,3-d3-alanine ester (∼5x molar excess) were used.

[0328] The reaction was quenched with 0.1% formic acid in methanol at a ratio of 1:10 and mixed using a tabletop shaker for 5 minutes. The plate was then centrifuged at 4000 rpm for 10 minutes, and the supernatant was further diluted with water at a ratio of 1:40 and then loaded onto RapidFire for analysis as described in Example 11. The HTP reaction of chlorophenylpyruvic acid and 3,3,3-d3 L-alanine ester was also quenched with 200 μl of acetonitrile, 40 μL of the supernatant was further diluted into 160 μL of 50% acetonitrile, and loaded onto LC-MS for analysis as described in Example 12.

[0329] The HTP reaction was also carried out in a 96-well deep-well plate containing 200 μL of 0.2M TEA pH 8.5, 1.25 g / L chlorophenylpyruvic acid (Compound (2)), ~3.6 g / L ethylamidopropylalanine (Compound (3c)) (~5x molar excess), 0.5 g / L PDH wt, 25 mM phosphite, 1.5 g / L NAD+, 5% DMSO, and 100 μl of the above HTP supernatant to produce the product of Compound (1d). The HTP plate was incubated in a Thermotron (3 mm swing, mode #AJ185, Infors) at 30 °C and 400 rpm for 12 hours. The reaction was quenched with 200 μl of acetonitrile and mixed using a tabletop shaker for 5 minutes. The plate was then centrifuged at 4000 rpm for 10 minutes, 40 μL of the supernatant was further diluted into 160 μL of 50% acetonitrile, and loaded onto LC-MS for analysis as described in Example 12.

[0330] The activity relative to SEQ ID NO:306 was calculated as the fold increase over the positive control (FIOPC) and is shown in Table 8.1. It was determined by dividing the mass spectrometry signal of the product in each sample under the specified reaction conditions by the mass spectrometry signal of the D3-chlorophenylpyruvate ester product (Compound (1e)), D3-phenylpyruvate ester product (Compound (1c)), D3-chlorophenylpyruvic acid product (product of the undesired hydrolyzed ester substrate), D3-phenylpyruvic acid signal (product of the undesired hydrolyzed ester substrate), or chlorophenylpyruvic acid amide product (Compound (1d)) in the parental variant (positive control) present in the same plate.

[0331]

[0332]

[0333] Example 9

[0334] Activity improvement of CENDH relative to SEQ ID NO:648

[0335] After screening for variants as described in Example 8, SEQ ID NO:648 was selected as the parental enzyme. A library of engineered genes was generated using well-known techniques such as saturation mutagenesis, recombination of previously identified beneficial mutations. Each gene-encoded polypeptide was produced in HTP as described in Example 2, duplicate precipitates were obtained, and soluble lysates were produced as described in Example 3.

[0336] To lyse the cells, 200 μl of lysis buffer containing 200 mM TEoA buffer pH 8.5, 1 mg / mL lysozyme, and 0.5 mg / mL PMBS was added to the cell pellet. The cells were incubated for 2 hours at room temperature with shaking on a benchtop shaker. The plate was then centrifuged at 4000 rpm and 4 °C for 15 minutes, and the clarified supernatant was used for subsequent biocatalytic reactions.

[0337] The HTP reaction was carried out in a 96-well deep-well plate containing 200 μL of 0.2 M TEoA pH 8.5, 1.25 g / L chlorobiphenylpyruvic acid (Compound (2)), 3.6 g / L 3,3,3-d3 L-alanine ester (Compound (3b)) (∼5x molar excess), 0.5 g / L PDH wt, 25 mM phosphite, 1.5 g / L NAD+, 5% DMSO, and 100 μL of the above HTP supernatant. The HTP plate was incubated in a Thermotron (3 mm swing, mode #AJ185, Infors) at 30 °C and 400 rpm for 12 hours. The HTP plate was incubated in a Thermotron (3 mm swing, mode #AJ185, Infors) at 30 °C and 400 rpm for 12 hours. The reaction was quenched with 0.1% formic acid in methanol at a ratio of 1:10 and mixed using a benchtop shaker for 5 minutes. The plate was then centrifuged at 4000 rpm for 10 minutes, the supernatant was further diluted with water at a ratio of 1:40, and then loaded onto RapidFire for analysis as described in Example 11. All samples were also quenched with 200 μl of acetonitrile, 40 uL of the supernatant was further diluted to 160 uL of 50% acetonitrile, and loaded onto LC-MS for analysis as described in Example 12.

[0338] The activity relative to SEQ ID NO:648 was calculated as the fold increase over the positive control (FIOPC) and is shown in Table 9.1. It was determined by dividing the mass spectrometry signal of the product in each sample under the specified reaction conditions by the mass spectrometry signal of the chlorobiphenylpyruvate ester product (Compound (1e)) or D3-chloro-biphenylpyruvic acid product (product of the undesired hydrolyzed ester substrate) in the parental variant (positive control) present in the same plate.

[0339]

[0340]

[0341]

[0342] Example 10

[0343] Activity improvement of CENDH relative to SEQ ID NO:708

[0344] After screening for variants as described in Example 9, SEQ ID NO:708 was selected as the parental enzyme. A library of engineered genes was generated using well-known techniques such as saturation mutagenesis, recombination of previously identified beneficial mutations. Each gene-encoded polypeptide was produced in HTP as described in Example 2, a single pellet was obtained, and the soluble lysate was produced as described in Example 3.

[0345] To lyse the cells, 200 μl of lysis buffer containing 200 mM TEoA buffer pH 8.5, 1 mg / mL lysozyme, and 0.5 mg / mL PMBS was added to the cell pellet. The cells were incubated at room temperature for 2 hours with shaking on a benchtop shaker. The plate was then centrifuged at 4000 rpm and 4 °C for 15 minutes, and the clarified supernatant was used for subsequent biocatalytic reactions.

[0346] The HTP reaction was carried out in a 96-well deep-well plate containing 200 μL of 0.2 M TEoA pH 8.5, 1.25 g / L chlorobiphenylpyruvic acid (Compound (2)), 3.5 g / L L-alanine ester (Compound (3)) (∼5x molar excess), 0.5 g / L PDH wt, 20 mM phosphite, 1.5 g / L NAD+, 5% DMSO, and 50 μL of the above HTP supernatant. The HTP plate was incubated at 30 °C and 600 rpm in a Thermotron (3 mm swing, mode #AJ185, Infors) for 20 hours. The reaction was then quenched and further analyzed as described in Example 9.

[0347] The activity relative to SEQ ID NO:708 was calculated as the fold increase over the positive control (FIOPC) and is shown in Table 10.1. It was determined by dividing the mass spectrometry signal of the product in each sample under the specified reaction conditions by the mass spectrometry signal of the chlorobiphenylpyruvate ester product (Compound (1)) in the parental variant (positive control) present in the same plate.

[0348]

[0349] Example 11

[0350] RapidFire analysis detection of CENDH reaction products

[0351] Using the instruments and parameters described in Table 11.1 below, the formation of diastereomeric forms of phenylpyruvic acid phenylalanine product (Compound (1a)), chlorobiphenylalanine product (Compound (1b)), D3-labeled phenylpyruvic acid phenylalanine ester product (Compound (1c)), D3-labeled chlorobiphenylalanine ester product (Compound (1e)), and chlorobiphenylalanine ester product (Compound (1)) in the HTP assay mixtures prepared in Examples 5 - 10 was analyzed by RapidFire SPE-MS / MS.

[0352]

[0353]

[0354]

[0355] Example 12

[0356] LC-UV-MS analysis and detection of CENDH reaction products

[0357] Using the instrument parameters and conditions shown in Table 12.1, a portion of the samples in Examples 8 - 10 and all of the HTP chlorobiphenylpyruvic acid and ethylamidophenylalanine reaction mixtures in Example 8 were analyzed using an LC-MS method. As described in the examples, they were prepared as dilutions in water and acetonitrile. The mass of the products was used to determine the peaks of the product isomers. Based on LC-MS, all (D3)-chlorobiphenylpyruvic acid products were detected as diastereomeric forms. All (D3)-chlorobiphenylpyruvic acid ester products (Compound (1e)) were detected as the s,r product isomers eluting at 1.1 min. Four peaks were detected for the chlorobiphenylpyruvic acid ethylamide product (Compound (1d)), assuming each peak represents one of the four enantiomers, and all peaks were used to calculate the activity of the chlorobiphenylpyruvic acid ethylamide product.

[0358]

[0359]

[0360] For all purposes, all publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, patent application, or other document were specifically and individually indicated to be incorporated by reference for all purposes.

[0361] Although various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the invention.

Claims

1. An engineered imine reductase, wherein the amino acid residue difference between the polypeptide sequence of the engineered imine reductase and SEQ ID NO:4 is Y222F, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

4.

2. An engineered imine reductase, wherein the amino acid residue differences between the polypeptide sequence of the engineered imine reductase and SEQ ID NO:4 are Y222F, Y293F / H / Q, and 1 to 6 amino acid residue differences selected from N94K / R, V184R / Q, L223S / T, N288S, S29R, I287K / T, A311V, G353E, S232A, D324L, R355K, S356K, A357C, V358C, E359-, T332V, A234V, I99T, A96V, and A321V, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:4, and the engineered imine reductase has improved activity compared to SEQ ID NO:

4.

3. An engineered imine reductase, wherein the amino acid residue differences between the polypeptide sequence of the engineered imine reductase and SEQ ID NO:4 are Y222F, N94K / R, and 1 to 6 amino acid residue differences selected from V184R / Q, L223S / T, N288S, Y293F / H / Q, S29R, I287K / T, A311V, G353E, S232A, D324L, R355K, S356K, A357C, V358C, E359-, T332V, I99T, A96V, V298A, and A72V, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:4, and the engineered imine reductase has improved activity compared to SEQ ID NO:

4.

4. An engineered imine reductase, wherein the amino acid residue differences between the polypeptide sequence of the engineered imine reductase and SEQ ID NO:4 are Y222F, S29R, V184Q, L223S, N288S, Y293F, D324L, and 1 to 9 amino acid residue differences selected from A57G, P205C / A / T / S, E261L / V / S / R, Q265I, N277V, I287T / K, G353E, N94K, Q184R, C256V / L, Y259F, N153F / L, G225F / Y, A357S, H202M / L, V274A, G126C, F294I, I283L, and S223G, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:4, and the engineered imine reductase has improved activity compared to SEQ ID NO:

4.

5. An engineered imine reductase, wherein the amino acid residue differences between the polypeptide sequence of the engineered imine reductase and SEQ ID NO: 4 are Y222F, S29R, V184Q / R, L223S, N288S, Y293F, D324L, N94K, P205A, E261V, Q265I, I287T, G353E and 1 - 7 amino acid residue differences selected from H202M / L, Y221S / H / L, F222I, G225Y / F / L, C256L / T / V, E261R / T / Y, F294I, V197T, H198S / A, M201L, Y259V, S223G, I283M, F219L, A205T / S, Y280W / D / V / I / E / L and S306F, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 4, and wherein the engineered imine reductase has improved activity compared to SEQ ID NO:

4.

6. An engineered imine reductase, wherein the amino acid residue differences between the polypeptide sequence of the engineered imine reductase and SEQ ID NO: 4 are Y222I, S29R, V184Q / R, L223S, N288S, Y293F, D324L, N94K, P205A, E261V / R, Q265I, I287T, G353E, H202M, Y221S, G225Y, C256L, F294I and 1 - 5 amino acid residue differences selected from Y263E / D / V / Q / M / N / G / L / F / R / W, H198S / A, Y259W / V / N, N277T / W / F / G / P, Y280F / G / K / D / W, A268S, V197L, S223G, W258Y and H17P, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 4, and wherein the engineered imine reductase has improved activity compared to SEQ ID NO:

4.

7. An engineered imine reductase, wherein the amino acid residue differences between the polypeptide sequence of the engineered imine reductase and SEQ ID NO: 4 are Y222I, S29R, V184Q / R, L223S, N288S, Y293F, D324L, N94K, P205A, E261V / R, Q265I, I287T, G353E, H202M, Y221S, G225Y, C256L, F294I, H198S, Y259W, N277T, Y280F and 1 - 2 amino acid residue differences selected from T141D, A154G / H, P278V / E, V197A / P and S198A, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 4, and wherein the engineered imine reductase has improved activity compared to SEQ ID NO:

4.

8. An engineered imine reductase, wherein the amino acid sequence of the engineered imine reductase is any one of SEQ ID NO: 16, 50, 306, 648, and 708.

9. An engineered imine reductase, wherein the amino acid sequence of the engineered imine reductase is any even-numbered sequence of SEQ ID NO: 16 and 38 - 802.

10. The engineered imine reductase according to any one of claims 2 - 9, wherein the improved activity includes the improved production of the compound of structural formula (IV) wherein R 1 selected from a hydrogen atom, or an optionally substituted alkyl, alkenyl, alkynyl, alkoxy, arylalkoxy, hydroxyalkyl, heteroalkyl, heteroalkenyl, heteroalkynyl, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocyclic hydrocarbyl, heteroaryl, and heteroarylalkyl; and wherein R 2 independently selected from alkyl, alkenyl, alkynyl, hydroxyalkyl, carboxyl, aminocarbonyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carboxyalkyl, alkylamino, haloalkyl, alkylthioalkyl, cycloalkyl, aryl, arylalkyl, heterocyclic hydrocarbyl, heteroaryl and heteroarylalkyl; and wherein R 3 independently selected from methyl, d3-methyl and ethyl.

11. The engineered imine reductase according to claim 10, wherein the improved activity includes the improved production of compound (1). 。 12. The engineered imine reductase according to claim 10, wherein the improved activity includes the improved utilization of compound (2). 。 13. The engineered imine reductase according to claim 10, wherein the improved activity includes the improved utilization of compound (3). 。 14. The engineered imine reductase according to claim 10, wherein the improved activity includes compound (1) From compound (2) and compound (3) of improved production.

15. The engineered imine reductase according to claim 10, wherein the improved activity includes the improved conversion of one or more of the following transformation reactions (A) to (E): 。 16. The engineered imine reductase according to claim 10, wherein the improved activity includes improved enantioselectivity.

17. The engineered imine reductase according to any one of claims 1 - 9 and 11 - 16, wherein the engineered imine reductase is purified.

18. A composition, the composition comprising at least one engineered imine reductase provided according to any one of claims 1 - 17.

19. An engineered polynucleotide, the engineered polynucleotide encoding the engineered imine reductase according to any one of claims 1 - 16.

20. An engineered polynucleotide, the engineered polynucleotide being shown by any odd-numbered sequence of SEQ ID NO: 15 or 37 - 801.

21. A vector, the vector comprising the engineered polynucleotide according to claim 19 or 20.

22. The vector according to claim 21, the vector further comprising at least one control sequence.

23. A host cell, the host cell comprising the vector according to claim 21 or 22.

24. The host cell according to claim 23, wherein the host cell produces at least one engineered imine reductase according to any one of claims 1 - 16.

25. A method for producing an engineered imine reductase in a host cell, the method comprising culturing the host cell according to claim 23 or 24 in a medium under suitable conditions to produce at least one engineered imine reductase.

26. The method according to claim 25, the method further comprising the step of recovering the engineered imine reductase.

27. The method according to claim 25 or 26, further comprising the step of purifying the at least one engineered imine reductase.

Images