Engineered nitroaldolase

Abstract

The present invention provides engineered nitroaldolase polypeptides useful in the production of beta-nitroalcohols, as well as polynucleotides, compositions, and methods utilizing these engineered polypeptides.

Description

[0001] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 350,128, filed on June 8, 2022, which is hereby incorporated by reference in its entirety for all purposes. Technical Field

[0002] The present invention provides engineered nitroaldolase polypeptides useful for producing beta-nitroalcohols and tertiary amino alcohols, as well as compositions and methods utilizing these engineered polypeptides.

[0003] Reference to a sequence listing, table or computer program

[0004] An official copy of the sequence listing is submitted with the specification as an XML file named "CX2-230WO1ST26.xml", created on June 1, 2023, and is 2,633,728 bytes in size. The submitted sequence listing is part of the specification and is incorporated herein by reference in its entirety.

[0005] background

[0006] Chiral tertiary amino alcohols are important building blocks in the pharmaceutical industry. In particular, chiral tertiary amino alcohols are intermediates for compounds used in the treatment of cystic fibrosis and chronic obstructive pulmonary disease (COPD). However, traditional chemical synthesis methods for chiral tertiary amino alcohols may have disadvantages, such as requiring long synthetic routes, utilizing expensive substrates, providing poor yields and selectivity, and producing toxic by-products.

[0007] Therefore, more cost-effective and environmentally sensitive biocatalytic synthetic routes are of interest for the efficient production of these compounds. However, only a few biocatalytic routes are known to produce chiral tertiary amino alcohol compounds.

[0008] One approach may involve the use of a two-enzyme cascade to generate chiral β-amino alcohols via β-hydroxy-α-amino acid intermediates (Dückers et al. Appl Microbiol Biotechnol, 2010, 88: 409-424). However, in all reported literature examples, this approach is limited to the production of chiral secondary alcohols rather than the desired chiral tertiary alcohols.

[0009] Another potential pathway is the enzymatic enantioselective addition of nitromethane to trifluoroacetone to form a chiral tertiary nitroaldol product, followed by chemical reduction to a chiral amino alcohol. This is not a known natural reaction. However, there is literature precedent for some hydroxynitrile lyases and other enzymes that catalyze this reaction.

[0010] These include the (R)-selective nitroaldolase reaction catalyzed by hydroxynitrile lyases from Acidobacterium capsulatum and Granulicella tundricula (M. Bekerle-Bogner, M. Gruber-Khadjawi, H. Wiltsche, R. Wiedner, H. Schwab, K. Steiner, ChemCatChem 2016, 8, 2214). Also reported is the Henry (nitroaldol) reaction using a hydroxynitrile lyase from Hevea brasiliensis (Gruber-Khadjawi, M., Purkarthofer, T., Skranc, W. and Griengl, H. (2007) Adv. Synth. Catal., 349: 1445-1450) and a transglutaminase from Streptoverticillium griseoverticillatum (Tang R., Guan Z., He Y., Zhu W., J of Molec Catal B: Enzymatic, 63: 1-2, 2010, 62-67). Other examples of similar enzyme-catalyzed reactions are known in the literature.

[0011] However, there are currently no reports showing the activity of hydroxynitrile lyase / nitroaldolase on ketone substrates, and there are currently no reports showing the highly (S)-selective activity of hydroxynitrile lyase as a nitroaldolase. Nitroaldolases with (S)-selective activity on ketones would enable the synthesis of a range of useful chiral amino alcohol compounds.

[0012] Overview

[0013] The present invention provides novel biocatalysts for the synthesis of β-nitro alcohols and tertiary amino alcohols and related methods of use. The nitroaldolase biocatalysts of the present disclosure are engineered variants of a polypeptide (SEQ ID NO: 2) encoded by a homologous gene (SEQ ID NO: 1) from Baliospermum montanum. These engineered polypeptides are capable of catalyzing the conversion of trifluoroacetone and nitromethane to β-nitro alcohols, which are precursors to tertiary amino alcohol products.

[0014] 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 of SEQ ID NO: 2, comprising at least one substitution or set of substitutions at one or more positions selected from the group consisting of: 7 / 9, 7 / 9 / 33 / 68 / 72 / 158, 7 / 9 / 33 / 72 / 175 / 197 / 230, 7 / 9 / 33 / 72 / 230, 7 / 9 / 68 / 72 / 197, 7 / 9 / 158 / 175, 7 / 9 / 175, 7 / 9 / 197, 7 / 33 / 68 / 72 / 158, 7 / 9 / 33 / 72 / 175 / 197 / 230. / 11 / 33 / 72 / 197 / 230、9 / 33 / 68 / 158 / 197、9 / 33 / 72 / 230、9 / 33 / 97 / 230、9 / 68 / 72、9 / 97 / 175 / 202 / 218、9 / 175、9 / 202、11、13、14 / 33 / 68 / 72 / 97、18、18 / 33 / 68 / 97 / 158 / 175 / 239、18 / 33 / 72 / 97 / 158 / 175、18 / 33 / 72 / 97 / 158 / 239、18 / 33 / 72 / 97 / 175 / 202、18 / 33 / 72 / 175 / 202 / 237 / 239、18 / 33 / 72 / 197 / 237、18 / 68 / 72、18 / 68 / 72 / 158 / 175 / 237 / 239、18 / 68 / 72 / 175 / 202 / 239、18 / 68 / 72 / 239、18 / 68 / 175 / 223 / 237 / 239、18 / 68 / 239、18 / 72 / 97 / 175 / 237 / 239、18 / 72 / 175 / 197、18 / 72 / 237、18 / 72 / 239、18 / 97 / 175 / 237 / 239、18 / 97 / 223 / 237 / 239、18 / 175、18 / 197 / 239、18 / 239、20 / 124、33 / 68 / 72、33 / 68 / 72 / 175 / 230、33 / 68 / 223 / 239、33 / 72、33 / 72 / 97 / 197、33 / 72 / 175、33 / 175、39、44 / 97、52、68 / 72、68 / 72 / 97、68 / 72 / 97 / 197、68 / 72 / 197、68 / 72 / 197 / 239、68 / 97 / 175、68 / 97 / 175 / 197 / 223、72 / 97 / 158 / 223 / 237、72 / 175 / 197、97 / 158 / 175 / 197、97 / 175、103、121、122、124、125、128、133、148、175、197 and 197 / 202, wherein positions are numbered with reference to SEQ ID NO: 2. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of 7V / 9V, 7V / 9V / 33V / 68L / 72E / 158Y, 7V / 9V / 33V / 72E / 175P / 197V / 230I, 7V / 9V / 33V / 72E / 230I, 7V / 9V / 68L / 72E / 197V, 7V / 9V / 158Y / 175P, 7V / 9V / 175P, 7V / 9V / 197V, 7V / 33V, 7V / 68L / 72E / 175P, 7V / 72E, 7V / 72E / 97I / 175P / 230I, 7V / 9V / 33V / 72E / 197V / 230I, 9V / 33V / 68L / 158Y / 197V, 9V / 33V / 72E / 230I, 9V / 33V / 97I / 23 0I, 9V / 68L / 72E, 9V / 97I / 175P / 202I / 218M, 9V / 175P, 9V / 202I, 11S, 13G, 14Y / 33V / 68L / 72E / 97I, 18C / 33V / 68L / 97I / 158Y / 175P / 239F, 18C / 33V / 7 2E / 97I / 158Y / 175P, 18C / 33V / 72E / 97I / 158Y / 239F, 18C / 33V / 72E / 97I / 1 75P / 202I, 18C / 33V / 72E / 175P / 202I / 237P / 239F, 18C / 33V / 72E / 197V / 23 7P, 18C / 68L / 72E, 18C / 68L / 72E / 158Y / 175P / 237P / 239F, 18C / 68L / 72E / 1 75P / 202I / 239F, 18C / 68L / 72E / 239F, 18C / 68L / 175P / 223P / 237P / 239F, 1 8C / 68L / 239F, 18C / 72E / 97I / 175P / 237P / 239F, 18C / 72E / 175P / 197V, 18C / 72E / 237P, 18C / 72E / 239F, 18C / 97I / 175P / 237P / 239F, 18C / 97I / 223P / 2 37P / 239F, 18C / 175P, 18C / 197V / 239F, 18C / 239F, 18I, 20H / 124C, 33V / 68 L / 72E, 33V / 68L / 72E / 175P / 230I, 33V / 68L / 223P / 239F, 33V / 72E, 33V / 72 E / 97I / 197V, 33V / 72E / 175P, 33V / 175P, 39A, 39G, 44N / 97I, 52S, 68L / 72E,68L / 72E / 97I, 68L / 72E / 97I / 197V, 68L / 72E / 197V, 68L / 72E / 197V / 239F, 68L / 97 I / 175P, 68L / 97I / 175P / 197V / 223P, 72E / 97I / 158Y / 223P / 237P, 72E / 175P / 197V , 97I / 158Y / 175P / 197V, 97I / 175P, 103M, 103S, 121Y, 122C, 122Q, 124C, 124E, 124T, 124Y, 125R, 128H, 128Y, 133A, 133C, 148M, 175P, 197V and 197V / 202I, where the positions are numbered with reference to SEQ ID NO:2. In some embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of: I7V / I9V, I7V / I9V / A33V / I68L / K72E / F158Y, I7V / I9V / A33V / K72E / K175P / I197V / V230I, I7V / I9V / A33V / K72E / V230I, I7V / I9V / I68L / K72E / I197V, I7V / I9V / F158Y / K175P, I7V / I9V / A33V / K72E / K175P I9V / K175P, I7V / I9V / I197V, I7V / A33V, I7V / I68L / K72E / K175P, I7V / K72E, I7V / K72E / V97I / K175P / V230I, I9 V, I9V / T11G / A33V / K72E / I197V / V230I, I9V / A33V / I68L / F158Y / I197V, I9V / A33V / K72E / V230I, I9V / A33V / V9 7I / V230I, I9V / I68L / K72E, I9V / V97I / K175P / V202I / Q218M, I9V / K175P, I9V / V202I, T11S, C13G, H14Y / A33V / I68L / K72E / V97I、L18C / A33V / I68L / V97I / F158Y / K175P / I239F、L18C / A33V / K72E / V97I / F158Y / K175P、L18C / A33V / K72E / V97I / F158Y / I239F、L18C / A33V / K72E / V97I / K175P / V202I、L18C / A33V / K72E / K175P / V202I / I23 7P / I239F, L18C / A33V / K72E / I197V / I237P, L18C / I68L / K72E, L18C / I68L / K72E / F158Y / K175P / I237P / I239F,L18C / I68L / K72E / K175P / V202I / I239F, L18C / I68L / K72E / I239F, L18C / I68L / K175P / K223P / I237P / I239F, L18C / I68L / I239F、L18C / K72E / V97I / K175P / I237P / I239F、L18C / K72E / K175P / I197V、L18C / K72E / I237P、L18C / K72E / I239F、L18 C / V97I / K175P / I237P / I239F, L18C / V97I / K223P / I237P / I239F, L18C / K175P, L18C / I197V / I239F, L18C / I239F, L18I, Y20H / V124C, A33V / I68L / K72E, A33V / I68L / K72E / K175P / V230I, A33V / I68L / K223P / I239F, A33V / K72E, A33V / K72E / V97 I / I197V, A33V / K72E / K175P, A33V / K175P, V39A, V39G, D44N / V97I, G52S, I68L / K72E, I68L / K72E / V97I, I68L / K72E / V9 7I / I197V, I68L / K72E / I197V, I68L / K72E / I197V / I239F, I68L / V97I / K175P, I68L / V97I / K175P / I197V / K223P, K72E / V9 7I / F158Y / K223P / I237P, K72E / K175P / I197V, V97I / F158Y / K175P / I197V, V97I / K175P, H103M, H103S, F121Y, S122C, S122Q, V124C, V124E, V124T, V124Y, F125R, W128H, W128Y, F133A, F133C, L148M, K175P, I197V, and I197V / V202I, wherein the positions are numbered with reference to SEQ ID NO: 2. 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 NO: 4 to SEQ ID NO: 176. 、

[0015] The present invention also 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 of SEQ ID NO: 2, comprising at least one substitution or a set of substitutions selected from the group consisting of 7 / 11 / 33 / 72 / 97, 7 / 11 / 33 / 97, 7 / 11 / 72, 7 / 11 / 158, 7 / 11 / 237, 7 / 33 / 72 / 97 / 158 / 175 / 197 / 237 / 239, 9 / 11 / 33, 9 / 11 / 33 / 72, 9 / 11 / 72, 9 / 33 / 68 / 97 / 237 / 239, 9 / 72 / 97 / 158 / 237 / 239, 9 / 218 / 239, 10, 11, 12, 13, 14, 18, 33 / 68 / 72 / 97 / 237 / 239, 33 / 72 / 158 / 239, 39, 40, 41, 48, 49, 51, 54, 54 / 126, 57, 68 / 175 / 239, 82, 84, 85, 86, 103, 104, 105, 107, 115, 117, 119, 119 / 120, 120, 122, 123, 124, 127, 13 0, 131, 133, 134, 137 / 145, 144, 145, 146, 147, 148, 149, 150, 152, 157, 158, 159, 169, 173, 174, 175, 180, 181, 183, 203, 204, 208, 209, 210, 215, 218, 236 and 237, wherein the positions are numbered with reference to SEQ ID NO:2. In some embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of 7V / 11G / 33V / 72E / 97I, 7V / 11G / 33V / 97I, 7V / 11G / 72E, 7V / 11G / 158Y, 7V / 11G / 237P, 7V / 33V / 72E / 97I / 158Y / 175P / 197V / 237P / 239F, 9V / 11G / 33V, 9V / 11G / 33V / 72E, 9V / 11G / 72E, 9V / 33V / 68L / 97I / 237P / 239F, 9V / 72E / 97I / 158Y / 237P / 239F, 9V / 218M / 239F, 10G, 10P, 10R, 11R, 11S, 12K, 13L, 14R, 18S, 18T, 33V / 68L / 72E / 97I / 237P / 239F, 33V / 72E / 158Y / 239F, 39R, 39W, 40L, 40S, 40T, 40V, 41C, 48W, 49Q, 49R, 51A, 51Q, 51S, 51T, 54N / 126I, 54Y, 57N,68L / 175P / 239F, 82T, 84L, 85A, 85L, 85M, 85Q, 86W, 103P, 104A, 104G, 104S, 105D, 107G, 107W, 115C, 115K, 115L, 115V, 117C, 117L, 119L, 119N / 120F, 120C, 120L, 12 0Y, 122G, 122T, 123A, 123P, 123T, 124P, 127A, 127L, 127M, 130M, 130V, 131L, 133W , 134D, 134F, 134L, 134R, 134V, 134W, 137I / 145V, 144L, 145F, 145L, 145P, 146D, 14 6G, 146N, 147R, 148P, 148V, 149W, 150I, 150L, ​​150V, 152P, 157V, 158I, 158L, 158M , 158R, 158W, 158Y, 159G, 159H, 159L, 169G, 169I, 169S, 173E, 173Y, 174C, 174G, 1 75D, 180G, 180V, 181C, 183K, 203Q, 204E, 204R, 204V, 208A, 208C, 208E, 208G, 208L, 208T, 208V, 209C, 210V, 215L, 218T, 236G, 236L, 237L, 237S and 237W, wherein the positions are numbered with reference to SEQ ID NO:2. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of: I7V / T11G / A33V / K72E / V97I, I7V / T11G / A33V / V97I, I7V / T11G / K72E, I7V / T11G / F158Y, I7V / T11G / I237P, I7V / A33V / K72E / V97I / F158Y / K175P / I197V / I237P / I239F, I9V / T11G / A33V, I9V / T11G / A33V / K72E, I9V / T11G / K72E, 3V / I68L / V97I / I237P / I239F, I9V / K72E / V97I / F158Y / I237P / I239F, I9V / Q218M / I239F, H10G, H10P, H10R, T11R, T11S, I12K, C13L, H14R, L18S, L18T, A33V / I68L / K72E / V97I / I237P / I239F, A33V / K72E / F158Y / I239F, V39R, V39W, A40L, A40S, A40T, A40V, S41C, L48W, E49Q, E49R,I51A, I51Q, I51S, I51T, W54N / T126I, W54Y, Y57N, I68L / K175P / I239F, G82T, I84L, N85A, N85L, N85M, N8 5Q, I86W, H103P, N104A, N104G, N104S, A105D, M107G, M107W, A115C, A115K, A115L, A115V, V117C, V117L, K119L, K119N / K120F, K120C, K120L, K120Y, S122G, S122T, E123A, E123P, E123T, V124P, D127A, D127L, D 127M, D130M, D130V, S131L, F133W, S134D, S134F, S134L, S134R, S134V, S134W, T137I / A145V, T144L, A14 5F, A145L, A145P, V146D, V146G, V146N, E147R, L148P, L148V, G149W, D150I, D150L, ​​D150V, T152P, I157 V, F158I, F158L, F158M, F158R, F158W, F158Y, S159G, S159H, S159L, A169G, A169I, A169S, V173E, V173Y, R174C, R174G, K175D, E180G, E180V, Q181C, L183K, Y203Q, G204E, G204R, G204V, Q208A, Q208C, Q208E, Q208G, Q208L, Q208T, Q208V, I209C, F210V, Q215L, Q218T, K236G, K236L, I237L, I237S, and I237W, wherein the positions are numbered with reference to SEQ ID NO: 2. 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 NO: 178 to SEQ ID NO: 462.

[0016] The present invention also 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 of SEQ ID NO: 24, comprising at least one substitution or a set of substitutions selected from the group consisting of 9 / 39 / 122 / 157, 18, 18 / 33 / 39 / 230, 18 / 33 / 175, 18 / 39 / 44 / 118, 18 / 175, 20, 33 / 39, 33 / 39 / 44 / 118 / 121、33 / 39 / 44 / 121、33 / 39 / 44 / 152、33 / 39 / 44 / 175、33 / 39 / 44 / 197、33 / 39 / 44 / 210 / 230、33 / 39 / 118 / 121 / 175、33 / 39 / 121 / 175、33 / 39 / 197 / 210、33 / 39 / 210、33 / 39 / 210 / 230、33 / 118 / 121、33 / 118 / 121 / 175, 39, 39 / 44 / 103, 39 / 44 / 118, 39 / 44 / 152 / 230, 39 / 44 / 175 / 197 / 230, 39 / 103, 39 / 103 / 125, 39 / 103 / 125 / 127 / 146, 39 / 103 / 125 / 127 / 146 / 150, 39 / 103 / 125 / 146, 39 / 103 / 127 / 150, 39 / 103 / 150, 39 / 118 / 121, 39 / 197, 39 / 197 / 210, 43, 44 / 103, 81, 103, 103 / 125 / 127, 103 / 125 / 146, 103 / 127, 103 / 150, 118 / 121, 118 / 121 / 175, 118 / 121 / 197, 121, 152, 152 / 197, 154, 159, 163, 175, 210, 232, and 238, wherein the positions are numbered with reference to SEQ ID NO: 24. In some additional embodiments,The engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of 9V / 39W / 122T / 157V, 18I, 18I / 33V / 39W / 230I, 18I / 33V / 175P, 18I / 39W / 44N / 118L, 18I / 175P, 20H, 33V / 39W, 33V / 39W / 44N / 118L / 121Y, 33V / 39W / 44N / 121Y, 33V / 39W / 44N / 152F, 33V / 39W / 44N / 175P, 33V / 39W / 44N / 197V , 33V / 39W / 44N / 210M / 230I, 33V / 39W / 118L / 121Y / 175P, 33V / 39W / 121Y / 175P, 33V / 39W / 197V / 210M, 33V / 39W / 210M, 33V / 39W / 210M / 230I、33V / 118L / 121W / 175P、33V / 118L / 121Y、39W、39W / 44N / 118L、39W / 44N / 152F / 230I、39W / 44N / 175P / 197V / 230I、 39W / 44Y / 103S, 39W / 103M, 39W / 103M / 125R / 146L, 39W / 103M / 150P, 39W / 103Q, 39W / 103S, 39W / 103S / 125P / 127G / 146L / 150P, 39W / 103S / 125R, 39W / 103S / 125R / 127G / 146L, 39W / 103S / 127G / 150P, 39W / 118L / 121Y, 39W / 197V, 39W / 197V / 210M, 43M, 43Q, 43R, 43S, 43T, 44N / 103S, 81F, 103L, 103M, 103M / 125P / 146L, 103M / 127G, 103Q, 103S, 103S / 125P / 127G, 103S / 150P, 103V, 118L / 121Y, 118L / 121Y / 175P, 118L / 121Y / 197V, 121Y, 152A / 197V, 152F, 154I, 159Q, 163P, 175P, 210M, 232G, and 238M, wherein the positions are numbered with reference to SEQ ID NO: 24. In some additional embodiments,The engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of I9V / V39W / S122T / I157V, L18I, L18I / A33V / V39W / V230I, L18I / A33V / K175P, L18I / V39W / D44N / Y118L, L18I / K175P, Y20H, A33V / V39W, A33V / V39W / D44N / Y118L / F121Y, A33V / V39W / D44N / F121Y, A33V / V39W / D44N / T152F, A33V / V39W / D44N / K175P, A33V / V39W / D44N / I197 V, A33V / V39W / D44N / F210M / V230I, A33V / V39W / Y118L / F121Y / K175P, A33V / V39W / F121Y / K175P、A33V / V39W / I197V / F210M、A33V / V39W / F210M、A33V / V39W / F210M / V230I, A33V / Y118L / F121W / K175P, A33V / Y118L / F121Y, V39W , V39W / D44N / Y118L, V39W / D44N / T152F / V230I, V39W / D44N / K175P / I197V / V230I, V39W / D44Y / H103S, V39W / H103M, V39W / H103M / F125R / V146L, V39W / H103M / D150P, V39W / H103Q, V39W / H103S, V39W / H103S / F125P / D127G / V146 L / D150P, V39W / H103S / F125R, V39W / H103S / F125R / D127G / V146L, V39W / H1 03S / D127G / D150P, V39W / Y118L / F121Y, V39W / I197V, V39W / I197V / F210M, I43M, I43Q, I43R, I43S, I43T, D44N / H103S, G81F, H103L, H103M, H103M / F125P / V146L, H103M / D127G, H103Q, H103S, H103S / F125P / D127G, H103S / D150P, H103V, Y118L / F121Y, Y118L / F121Y / K175P, Y118L / F121Y / I197V, F121Y, T152A / I197V, T152F, A154I, S159Q, I163P, K175P, F210M, S232G, and Q238M,wherein positions are numbered with reference to SEQ ID NO: 24. 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 NO: 464 to SEQ ID NO: 596.

[0017] The present invention also 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 of SEQ ID NO: 24, comprising at least one substitution or a set of substitutions selected from the group consisting of: 7, 9 / 13, 9 / 13 / 39, 9 / 13 / 39 / 68 / 72 / 239, 9 / 13 / 39 / 68 / 239, 9 / 13 / 39 / 72 / 122, 9 / 13 / 39 / 97 / 239, 9 / 13 / 68 / 72 / 97 / 239, 9 / 13 / 68 / 97 / 239, 9 / 13 / 72 / 122. 4, 11, 12 / 42 / 125 / 127, 12 / 103, 12 / 103 / 150, 13, 13 / 39, 13 / 39 / 68, 13 / 39 / 68 / 72 / 97, 13 / 39 / 72 / 97 / 122 / 134 / 223, 13 / 39 / 97 / 239, 13 / 39 / 239, 13 / 68, 13 / 68 / 72 / 97 / 122 / 239, 16, 18 / 33 / 39 / 44 / 118 / 121 / 175 / 197 / 210, 18 / 33 / 39 / 44 / 121, 18 / 33 / 39 / 112 / 118 / 121 / 197, 18 / 33 / 39 / 121, 18 / 33 / 39 9 / 121 / 175, 18 / 33 / 39 / 121 / 175 / 197, 18 / 33 / 39 / 121 / 175 / 210, 18 / 33 / 39 / 121 / 210, 18 / 33 / 118 / 121, 18 / 33 / 121, 18 / 33 / 121 / 197, 18 / 33 / 121 / 210 / 230, 18 / 39 / 44 / 118 / 121 / 230, 18 / 39 / 44 / 121 / 175 / 210, 18 / 39 / 118 / 121, 18 / 39 / 118 / 121 / 210, 18 / 39 / 121 / 175, 18 / 39 / 121 / 175 / 210, 18 / 39 / 12 1 / 197 / 210、18 / 118 / 121、18 / 118 / 121 / 175、18 / 118 / 121 / 210、18 / 118 / 121 / 230、18 / 121、18 / 121 / 175、18 / 121 / 210、18 / 121 / 230、20、21、33 / 39 / 44 / 118 / 121、33 / 39 / 44 / 118 / 121 / 210 / 230、33 / 39 / 44 / 121、33 / 39 / 44 / 121 / 152、33 / 39 / 44 / 121 / 175 / 210、33 / 39 / 44 / 121 / 230、33 / 39 / 73 / 121 / 197、33 / 39 / 118 / 121 / 175、33 / 39 / 118 / 121 / 175 / 230、33 / 39 / 121、33 / 39 / 121 / 175、33 / 39 / 121 / 210 / 230、33 / 118 / 121、33 / 118 / 121 / 175、33 / 118 / 121 / 197、33 / 121、33 / 121 / 230、37 / 91、39 / 44 / 118 / 121 / 175 / 210、39 / 68 / 72 / 239, 39 / 118 / 121, 39 / 118 / 121 / 210, 39 / 121, 39 / 121 / 210 / 230, 43, 44, 76, 79, 101, 118 / 121, 118 / 121 / 175, 118 / 121 / 197, 121, 121 / 175 / 230, 121 / 210, 148, 153, 159, 161, 172, 200, 207, 233, 245 and 248, wherein the positions are numbered with reference to SEQ ID NO:24. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of 7E, 9V / 13G, 9V / 13G / 39W, 9V / 13G / 39W / 68L / 72E / 239F, 9V / 13G / 39W / 68L / 239F, 9V / 13G / 39W / 72E / 122T, 9V / 13G / 39W / 97I / 239F, 9V / 13G / 68L / 72E / 239F 2E / 97I / 239F, 9V / 13G / 68L / 97I / 134D / 239F, 9V / 13G / 68L / 97I / 239F, 9V / 13G / 72E, 9V / 13G / 13 4D, 11N, 12A / 42W / 125R / 127G, 12A / 103S, 12A / 103S / 150P, 13F, 13G / 39W, 13G / 39W / 68L, 13G / 39 W / 68L / 72E / 97I, 13G / 39W / 72E / 97I / 122T / 134D / 223P, 13G / 39W / 97I / 239F, 13G / 39W / 239F, 13 G / 68L, 13G / 68L / 72E / 97I / 122T / 239F, 16M, 16V, 18C / 33V / 39W / 112R / 118L / 121W / 197V, 18C / 33 V / 39W / 121W / 175P / 210M, 18C / 33V / 39W / 121W / 210M, 18C / 33V / 39W / 121Y / 175P, 18C / 33V / 118L / 121W、18C / 33V / 118L / 121Y、18C / 33V / 121Y、18C / 33V / 121Y / 197V、18C / 33V / 121Y / 210M / 230I、18C / 39W / 44N / 121Y / 175P / 210M、18C / 39W / 118L / 121W、18C / 39W / 121W / 175P / 210M、18C / 39W / 121W / 197V / 210M、18C / 118L / 121W、18C / 118L / 121Y、18C / 121V、18C / 121V / 175P、18C / 121W、18C / 121W / 175P、18C / 121W / 210M、18C / 121Y、18C / 121Y / 230I、18I / 33V / 39W / 44N / 118L / 121W / 175P / 197V / 210M、18I / 33V / 39W / 44N / 121T、18I / 33V / 39W / 121T / 175P、18I / 33V / 39W / 121V / 175P / 197V、18I / 33V / 39W / 121W、18I / 33V / 118L / 121W、18I / 39W / 44N / 118L / 121W / 230I、18I / 39W / 44N / 121V / 175P / 210M、18I / 39W / 118L / 121W / 210M、18I / 39W / 121V / 175P、18I / 118L / 121T、18I / 118L / 121W、18I / 118L / 121W / 175P、18I / 118L / 121W / 210M、18I / 118L / 121Y、18I / 118L / 121Y / 230I、18I / 121T / 175P、18I / 121W、18I / 121Y / 210M、20G、20K、20R、20T、21V、33V / 39G / 44N / 121W / 152A、33V / 39W / 44N / 118L / 121W / 210M / 230I、33V / 39W / 44N / 118L / 121Y、33V / 39W / 44N / 121V、33V / 39W / 44N / 121Y、33V / 39W / 44N / 121Y / 175P / 210M、33V / 39W / 44N / 121Y / 230I、33V / 39W / 73N / 121Y / 197V、33V / 39W / 118L / 121W / 175P、33V / 39W / 118L / 121Y / 175P、33V / 39W / 118L / 121Y / 175P / 230I、33V / 39W / 121V、33V / 39W / 121W、33V / 39W / 121W / 210M / 230I、33V / 39W / 121Y / 175P、33V / 118L / 121W / 175P、33V / 118L / 121W / 197V、33V / 118L / 121Y、33V / 121W、33V / 121Y / 230I、37L / 91G, 39G / 68L / 72E / 239F, 39W / 44N / 118L / 121W / 175P / 210M, 39W / 118L / 121W / 210M, 39W / 118L / 121Y, 3 9W / 118L / 121Y / 210M, 39W / 121W, 39W / 121W / 210M / 230I, 43E, 43Q, 43S, 43W, 44V, 76P, 79F, 101L, 118L / 121W , 118L / 121Y, 118L / 121Y / 175P, 118L / 121Y / 197V, 121V, 121W, 121Y, 121Y / 175P / 230I, 121Y / 210M, 148F, 153W, 159Q, 161M, 172A, 172E, 172G, 172Q, 172R, 172V, 200G, 207N, 233R, 245D, 245G, 248G and 248S, wherein the positions are numbered with reference to SEQ ID NO:24. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of I7E, I9V / C13G, I9V / C13G / V39W, I9V / C13G / V39W / I68L / K72E / I239F, I9V / C13G / V39W / I68L / I239F, I9V / C13G / V39W / K72E / S122T, I9V / C13G / V39W / I68L / K72E / I239F, 9W / V97I / I239F, I9V / C13G / I68L / K72E / V97I / I239F, I9V / C13G / I68L / V97I / S134D / I239F, I9 V / C13G / I68L / V97I / I239F, I9V / C13G / K72E, I9V / C13G / S134D, S11N, I12A / G42W / F125R / D127G , I12A / H103S, I12A / H103S / D150P, C13F, C13G / V39W, C13G / V39W / I68L, C13G / V39W / I68L / K72 E / V97I, C13G / V39W / K72E / V97I / S122T / S134D / K223P, C13G / V39W / V97I / I239F, C13G / V39W / I2 39F, C13G / I68L, C13G / I68L / K72E / V97I / S122T / I239F, A16M, A16V, L18C / A33V / V39W / H112R / Y118L / F121W / I197V, L18C / A33V / V39W / F121W / K175P / F210M, L18C / A33V / V39W / F121W / F210M,L18C / A33V / V39W / F121Y / K175P、L18C / A33V / Y118L / F121W、L18C / A33V / Y118L / F121Y、L18C / A33V / F121Y、L18C / A33V / F121Y / I197V、L18C / A33V / F121Y / F210M / V230I、L18C / V39W / D44N / F121Y / K175P / F210M、L18C / V39W / Y118L / F121W、L18C / V39W / F121W / K175P / F210M、L18C / V39W / F121W / I197V / F210M、L18C / Y118L / F121W、L18C / Y118L / F121Y、L18C / F121V、L18C / F121V / K175P、L18C / F121W、L18C / F121W / K175P、L18C / F121W / F210M、L18C / F121Y、L18C / F121Y / V230I、L18I / A33V / V39W / D44N / Y118L / F121W / K175P / I197V / F210M、L18I / A33V / V39W / D44N / F121T、L18I / A33V / V39W / F121T / K175P、L18I / A33V / V39W / F121V / K175P / I197V、L18I / A33V / V39W / F121W、L18I / A33V / Y118L / F121W、L18I / V39W / D44N / Y118L / F121W / V230I、L18I / V39W / D44N / F121V / K175P / F210M、L18I / V39W / Y118L / F121W / F210M、L18I / V39W / F121V / K175P、L18I / Y118L / F121T、L18I / Y118L / F121W、L18I / Y118L / F121W / K175P、L18I / Y118L / F121W / F210M、L18I / Y118L / F121Y、L18I / Y118L / F121Y / V230I、L18I / F121T / K175P、L18I / F121W、L18I / F121Y / F210M、Y20G、Y20K、Y20R、Y20T、K21V、A33V / V39G / D44N / F121W / T152A、A33V / V39W / D44N / Y118L / F121W / F210M / V230I、A33V / V39W / D44N / Y118L / F121Y、A33V / V39W / D44N / F121V、A33V / V39W / D44N / F121Y, A33V / V39W / D44N / F121Y / K175P / F210M, A33V / V39W / D44N / F121Y / V230I, A33V / V39W / K73N / F12 1Y / I197V、A33V / V39W / Y118L / F121W / K175P、A33V / V39W / Y118L / F121Y / K175P、A33V / V39W / Y118L / F121Y / K175P / V230I、 A33V / V39W / F121V, A33V / V39W / F121W, A33V / V39W / F121W / F210M / V230I, A33V / V39W / F121Y / K175P, A33V / Y118L / F121W / K175P, A33V / Y118L / F121W / I197V, A33V / Y118L / F121Y, A33V / F121W, A33V / F121Y / V230I, D37L / E91G, V39G / I68L / K72E / I239F、V39W / D44N / Y118L / F121W / K175P / F210M、V39W / Y118L / F121W / F210M、V39W / Y118L / F121Y、V39W / Y118L / F121Y / F2 10M, V39W / F121W, V39W / F121W / F210M / V230I, I43E, I43Q, I43S, I43W, D44V, L76P, E79F, V101L, Y118L / F121W, Y118L / F1 21Y, Y118L / F121Y / K175P, Y118L / F121Y / I197V, F121V, F121W, F121Y, F121Y / K175P / V230I, F121Y / F210M, L148F, L153W, S159Q, S161M, L172A, L172E, L172G, L172Q, L172R, L172V, V200G, D207N, A233R, L245D, L245G, I248G, and I248S, wherein positions are numbered with reference to SEQ ID NO: 24. 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 NO: 598 to SEQ ID NO: 842. ,

[0018] The present invention also 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 of SEQ ID NO:496, comprising at least one substitution or a set of substitutions selected from the following: 43 / 103, 43 / 103 / 172 / 238, 43 / 103 / 238 / 241, 43 / 238, 81 / 232, 103, 103 / 159 / 238, 103 / 194 / 238, 103 / 238, 122, 232, 238 and 238 / 241, wherein the positions are numbered with reference to SEQ ID NO:496. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of 43E / 103S / 238M / 241R, 43M / 103L, 43Q / 103S / 238M / 241R, 43Q / 238M, 43S / 103S / 172R / 238M, 43S / 238M, 81F / 232G, 103L, 103S, 103S / 159Q / 238M, 103S / 194L / 238M, 103S / 238M, 103V, 122C, 122I, 122K, 122L, 122M, 122Q, 122R, 122T, 122V, 232G, 238M, and 238M / 241R, wherein positions are numbered with reference to SEQ ID NO:496. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of I43E / H103S / Q238M / K241R, I43M / H103L, I43Q / H103S / Q238M / K241R, I43Q / Q238M, I43S / H103S / L172R / Q238M, I43S / Q238M, G81F / S232G, H 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 NO: 844 to SEQ ID NO: 892.

[0019] The present invention also 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 of SEQ ID NO: 496, comprising at least one substitution or a set of substitutions selected from the group consisting of: 12, 43, 43 / 47 / 103, 43 / 47 / 103 / 232, 43 / 103, 43 / 103 / 121 / 172 / 241, 43 / 103 / 172, 43 / 103 / 172 / 238, 43 / 103 / 238 / 238. 194 / 238, 103 / 238, 122, 131, 146, 178, 180 and 221, wherein the positions are numbered with reference to SEQ ID NO:496.In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of: 12V, 43E / 103S / 121W / 172R / 241R, 43E / 103S / 238M, 43E / 103S / 238M / 241R, 43M / 47G / 103L, 43M / 47G / 103V, 43M / 103L, 43M / 103S, 43M / 103S, 3V, 43Q / 47G / 103S / 232G, 43Q / 103L / 172R, 43Q / 103S, 43Q / 103S / 238M / 241R, 43Q / 121W, 43 Q / 238M, 43S, 43S / 103L, 43S / 103S, 43S / 103S / 172R, 43S / 103S / 172R / 238M, 43S / 103S / 238M / 241R、43S / 194L / 238M、43S / 238M、44V / 103S / 121W / 238M、44V / 103S / 238M、44V / 103S / 238 M / 241R, 44V / 121W / 238M / 241R, 47G / 103L, 63P, 103L, 103S, 103S / 121W, 103S / 121W / 172R / 178A, 180L, 180V and 221G, wherein the positions are numbered with reference to SEQ ID NO:496.In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of: I12V, I43E / H103S / Y121W / L172R / K241R, I43E / H103S / Q238M, I43E / H103S / Q238M / K241R, I43M / Q47G / H103L, I43M / Q47G / H103V, I43M / H103L, I43M / H103S, I43M / H103V, I43M / H103V 43Q / Q47G / H103S / S232G, I43Q / H103L / L172R, I43Q / H103S, I43Q / H103S / Q238M / K241R, I43Q / Y121W, I43Q / Q 238M, I43S, I43S / H103L, I43S / H103S, I43S / H103S / L172R, I43S / H103S / L172R / Q238M, I43S / H103S / Q238M / K241R, I43S / Y194L / Q238M, I43S / Q238M, N44V / H103S / Y121W / Q238M, N44V / H103S / Q238M, N44V / H103S / Q23 8M / K241R, N44V / Y121W / Q238M / K241R, Q47G / H103L, T63P, H103L, H103S, H103S / Y121W, H103S / Y121W / L172R / Q238M, H103S / S159Q / Q238M, H103S / L172R / Q238M, H103S / Y194L / Q238M, H103S / Q238M, H103V, S122H, S122I, S122K, S122L, S122M, S122N, S122R, S122T, S122V, S131Y, V146I, F178A, E180L, E180V, and N221G, wherein positions are numbered with reference to SEQ ID NO: 496. 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 NO: 894 to SEQ ID NO: 980.

[0020] The present invention also 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 of SEQ ID NO: 860, comprising at least one substitution or a set of substitutions selected from the group consisting of: 12, 12 / 43 / 63 / 103 / 146 / 180 / 232 / 241, 12 / 43 / 63 / 103 / 146 / 241, 12 / 43 / 81 / 103 / 241, 12 / 43 / 81 / 146 / 232 / 241, 12 / 43 / 81 / 146 / 232 / 241. 1 / 180 / 241、12 / 43 / 81 / 232 / 241、12 / 43 / 81 / 241、12 / 43 / 103、12 / 43 / 103 / 146 / 180 / 232 / 241、12 / 43 / 103 / 146 / 180 / 241、12 / 43 / 103 / 180 / 241、12 / 43 / 103 / 241、12 / 43 / 146 / 180 / 232 / 241、12 / 43 / 180 / 241、12 / 63 / 81 / 103 / 241、12 / 81、12 / 8 1 / 180 / 241, 12 / 103 / 146 / 241, 12 / 103 / 180 / 241, 12 / 146, 12 / 146 / 180 / 241, 12 / 180 / 232 / 241, 12 / 241, 18, 43 / 81 / 232 / 241, 43 / 103 / 146 / 180, 43 / 103 / 180 / 241, 43 / 103 / 241, 43 / 180 / 232, 43 / 232 / 241, 63 / 103 / 180 / 232, 78, 81, 81 / 103 / 146, 81 / 146 / 180 / 241, 81 / 146 / 241, 81 / 180 / 241, 84, 103, 103 / 146, 103 / 146 / 180, 103 / 146 / 180 / 241, 103 / 180, 103 / 180 / 241, 103 / 261, 144, 145, 146 / 180 / 232, 146 / 180 / 241, 146 / 241, 150, 166, 172, 180 / 232, 232 / 241, and 241, wherein the positions are numbered with reference to SEQ ID NO:860. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of 12V, 12V / 43M / 63P / 103L / 146I / 180L / 232G / 241R, 12V / 43M / 81F / 103L / 241R, 12V / 43M / 81F / 146I / 232G / 241R, 12V / 43M / 81F / 180V / 241R, 12V / 43M / 81F / 232G / 241R,12V / 43M / 81F / 241R, 12V / 43M / 103L / 180L / 241R, 12V / 43M / 103L / 241R, 12V / 43M / 103V / 146I / 180V / 241R, 12V / 43M / 103V / 241R, 12V / 43M / 1 46I / 180L / 232G / 241R, 12V / 43M / 180L / 241R, 12V / 43M / 180V / 241R, 12V / 43Q / 63P / 103L / 146I / 241R, 12V / 43Q / 103L / 146I / 180V / 232G / 241R , 12V / 43Q / 103L / 146I / 180V / 241R, 12V / 43Q / 103L / 180V / 241R, 12V / 43Q / 103V, 12V / 63P / 81F / 103L / 241R, 12V / 81F, 12V / 81F / 180V / 241R, 12V / 103L / 146I / 241R, 12V / 103L / 180L / 241R, 12V / 103V / 180V / 241R, 12V / 146I, 12V / 146I / 180L / 241R, 12V / 180L / 232G / 241R, 12V / 180V / 2 32G / 241R, 12V / 241R, 18V, 43M / 81F / 232G / 241R, 43M / 103L / 146I / 180V, 43M / 103L / 180V / 241R, 43M / 103L / 241R, 43M / 103V / 146I / 180V, 43 M / 180L / 232G, 43M / 232G / 241R, 43Q / 103V / 146I / 180L, 63P / 103L / 180L / 232G, 78A, 81F / 103L / 146I, 81F / 146I / 180L / 241R, 81F / 146I / 241R , 81F / 180L / 241R, 81Q, 84Y, 103A, 103L, 103L / 146I, 103L / 146I / 180V, 103L / 180L, 103L / 180V / 241R, 103N, 103T, 103T / 261V, 103V / 146I / 180L / 241R, 144V, 145V, 146I / 180L / 241R, 146I / 180V / 232G, 146I / 241R, 150H, 166L, 172L, 172V, 180L / 232G, 232G / 241R and 241R, wherein the positions are numbered with reference to SEQ ID NO:860. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from: I12V,I12V / S43M / T63P / S103L / V146I / E180L / S232G / K241R、I12V / S43M / G81F / S103L / K241R、I12V / S43M / G81F / V146I / S232G / K241R、I12V / S43M / G81F / E180V / K241R、I12V / S43M / G81F / S232G / K241R、I12V / S43M / G81F / K241R、I12V / S43M / S103L / E180L / K241R、I12V / S43M / S103L / K241R、I12V / S43M / S103V / V146I / E180V / K241R、I12V / S43M / S103V / K241R、I12V / S43M / V146I / E180L / S232G / K241R、I12V / S43M / E180L / K241R、I12V / S43M / E180V / K241R、I12V / S43Q / T63P / S103L / V146I / K241R、I12V / S43Q / S103L / V146I / E180V / S232G / K241R、I12V / S43Q / S103L / V146I / E180V / K241R、I12V / S43Q / S103L / E180V / K241R、I12V / S43Q / S103V、I12V / T63P / G81F / S103L / K241R、I12V / G81F、I12V / G81F / E180V / K241R、I12V / S103L / V146I / K241R、I12V / S103L / E180L / K241R、I12V / S103V / E180V / K241R、I12V / V146I、I12V / V146I / E180L / K241R、I12V / E180L / S232G / K241R、I12V / E180V / S232G / K241R、I12V / K241R、L18V、S43M / G81F / S232G / K241R、S43M / S103L / V146I / E180V、S43M / S103L / E180V / K241R、S43M / S103L / K241R、S43M / S103V / V146I / E180V、S43M / E180L / S232G、S43M / S232G / K241R、S43Q / S103V / V146I / E180L、T63P / S103L / E180L / S232G、G78A、G81F / S103L / V146I、G81F / V146I / E180L / K241R、G81F / V146I / K241R、G81F / E180L / K241R, G81Q, I84Y, S103A, S103L, S103L / V146I, S103L / V146I / E180 V, S103L / E180L, S103L / E180V / K241R, S103N, S103T, S103T / A261V, S103V / V146I / E180L / K241R, T144V, A145V, V146I / E180L / K241R, V146I / E180V / S232G, V146I / K241R, D150H, V166L, R172L, R172V, E180L / S232G, S232G / K241R, and K241R, wherein the positions are numbered with reference to SEQ ID NO: 860. 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 NO: 982 to SEQ ID NO: 1118.

[0021] The present invention also 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 of SEQ ID NO: 860, comprising at least one substitution or a set of substitutions selected from the group consisting of 9, 12, 12 / 43 / 63 / 103 / 146 / 180 / 232 / 241, 12 / 43 / 63 / 103 / 146 / 241, 12 / 43 / 103 / 146 / 180 / 232 / 241, 12 / 43 / 63 / 103 / 146 / 180 / 232 / 241. / 103 / 146 / 180 / 241、12 / 43 / 103 / 146 / 232 / 241、12 / 43 / 103 / 146 / 241、12 / 43 / 103 / 180 / 241、12 / 43 / 146 / 180 / 232 / 241、12 / 43 / 146 / 241、12 / 43 / 180 / 232、12 / 43 / 180 / 241、12 / 63 / 103 / 146 / 232 / 241、12 / 103 / 146 / 241、12 / 10 3 / 180 / 241, 12 / 146, 12 / 146 / 180 / 241, 12 / 146 / 232 / 241, 12 / 146 / 241, 12 / 180, 12 / 180 / 232 / 241, 12 / 180 / 241, 12 / 241, 18, 43 / 63 / 103 / 180 / 232 / 241, 43 / 103 / 146 / 180, 43 / 103 / 180, 43 / 103 / 180 / 232 / 241, 43 / 103 / 180 / 24 1, 43 / 180 / 232, 63 / 103 / 180 / 232, 103, 103 / 146 / 180, 103 / 146 / 180 / 241, 103 / 180, 103 / 180 / 241, 103 / 241, 118, 119, 122, 144, 145, 146 / 180 / 241, 154, 160, 172, 180 / 232, 180 / 232 / 241, 180 / 241, 208, 211 and 212, wherein the positions are numbered with reference to SEQ ID NO:860. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of 9V, 12V, 12V / 43M / 63P / 103L / 146I / 180L / 232G / 241R, 12V / 43M / 103L / 146I / 241R, 12V / 43M / 103L / 180L / 241R, 12V / 43M / 103V / 146I / 180V / 241R, 12V / 43M / 103V / 146I / 232G / 241R, 12V / 43M / 146I / 180L / 232G / 241R, 12V / 43M / 180L / 232G,12V / 43M / 180L / 241R, 12V / 43Q / 63P / 103V / 146I / 241R, 12V / 43Q / 103L / 146I / 180V / 232G / 241R, 12V / 43Q / 103L / 146I / 180V / 241R, 12V / 43Q / 103 L / 146I / 241R, 12V / 43Q / 103L / 180V / 241R, 12V / 43Q / 146I / 241R, 12V / 6 3P / 103L / 146I / 232G / 241R, 12V / 103L / 180L / 241R, 12V / 103L / 180V / 241 R、12V / 103V / 146I / 241R、12V / 103V / 180L / 241R、12V / 103V / 180V / 241R , 12V / 146I, 12V / 146I / 180L / 241R, 12V / 146I / 232G / 241R, 12V / 146I / 24 1R, 12V / 180L, 12V / 180L / 232G / 241R, 12V / 180L / 241R, 12V / 180V, 12V / 1 80V / 232G / 241R, 12V / 180V / 241R, 12V / 241R, 18V, 43M / 63P / 103L / 180V / 232G / 241R, 43M / 103L / 146I / 180V, 43M / 103L / 180V, 43M / 103L / 180V / 2 41R、43M / 103V / 146I / 180V、43M / 180L / 232G、43Q / 103L / 180V / 232G / 241 R, 43Q / 103V / 146I / 180L, 43Q / 103V / 180L / 241R, 43Q / 103V / 180V / 232G / 241R, 63P / 103L / 180L / 232G, 103A, 103L, 103L / 146I / 180V, 103L / 146I 241R, 103N, 103T, 103V / 146I / 180L / 241R, 103V / 180V / 241R, 118L, 118V, 119H, 122H, 144H, 144V, 145V, 146I / 180L / 241R, 154R, 160L, 172C, 172L, 172V, 180L / 232G, 180L / 241R, 180V / 232G, 180V / 232G / 241R, 208F, 211F and 212P, wherein the positions are numbered with reference to SEQ ID NO:860. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from: I9V, I12V,I12V / S43M / T63P / S103L / V146I / E180L / S232G / K241R、I12V / S43M / S103L / V146I / K241R、I12V / S43M / S103L / E180L / K241R、I12V / S43M / S103V / V146I / E180V / K241R、I12V / S43M / S103V / V146I / S232G / K241R、I12V / S43M / V146I / E180L / S232G / K241R、I12V / S43M / E180L / S232G、I12V / S43M / E180L / K241R、I12V / S43Q / T63P / S103V / V146I / K241R、I12V / S43Q / S103L / V146I / E180V / S232G / K241R、I12V / S43Q / S103L / V146I / E180V / K241R、I12V / S43Q / S103L / V146I / K241R、I12V / S43Q / S103L / E180V / K241R、I12V / S43Q / V146I / K241R、I12V / T63P / S103L / V146I / S232G / K241R、I12V / S103L / E180L / K241R、I12V / S103L / E180V / K241R、I12V / S103V / V146I / K241R、I12V / S103V / E180L / K241R、I12V / S103V / E180V / K241R、I12V / V146I、I12V / V146I / E180L / K241R、I12V / V146I / S232G / K241R、I12V / V146I / K241R、I12V / E180L、I12V / E180L / S232G / K241R、I12V / E180L / K241R、I12V / E180V、I12V / E180V / S232G / K241R、I12V / E180V / K241R、I12V / K241R、L18V、S43M / T63P / S103L / E180V / S232G / K241R、S43M / S103L / V146I / E180V、S43M / S103L / E180V、S43M / S103L / E180V / K241R、S43M / S103V / V146I / E180V、S43M / E180L / S232G、S43Q / S103L / E180V / S232G / K241R、S43Q / S103V / V146I / E180L、S43Q / S103V / E180L / K241R、S43Q / S103V / E180V / S232G / K241R, T63P / S103L / E180L / S232G, S103A, S103L, S103L / V146I / E180V, S103L / V146I / E180V / K241R, S103L / E180L, S103L / E180V / K241R, S103L / K241R, S103N, S103T, S103V / V146I / E180L / K241R, S1 03V / E180V / K241R, Y118L, Y118V, K119H, S122H, T144H, T144V, A145V, V146I / E180L / K241R, A154R, N160L, R172C, R172L, R172V, E180L / S232G, E180L / K241R, E180V / S232G, E180V / S232G / K241R, Q208F, S211F, and R212P, wherein the positions are numbered with reference to SEQ ID NO: 860. 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 NO: 1120 to SEQ ID NO: 1212.

[0022] The present invention also 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 of SEQ ID NO: 1120, comprising at least one substitution or a set of substitutions selected from the group consisting of: 3, 7, 7 / 158, 7 / 158 / 175, 7 / 158 / 197 / 202, 27 / 43 / 115 / 172, 27 / 43 / 172, 29, 32, 43 / 110 / 115 / 172, 44, 46, 48 / 172, 55, 59, 6 7, 71, 72, 73, 94 / 103 / 118 / 146, 103, 103 / 144 / 146 / 172 / 180, 103 / 146 / 154 / 172 / 180 / 232, 103 / 154, 118, 118 / 144 / 215, 118 / 172 / 180, 137, 142, 146, 146 / 154, 154, 158, 172 / 190, 184, 198, 205, 216, 223, 226, 229, 247, 261 and 263, wherein the positions are numbered with reference to SEQ ID NO:1120. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of 3D, 7V, 7V / 158Y, 7V / 158Y / 175P, 7V / 158Y / 197V / 202I, 27E / 43I / 115S / 172L, 27E / 43I / 172L, 29G, 29R, 32G, 32R, 43I / 110T / 115S / 172L, 44S, 46H, 46V, 48I / 172L, 55A, 55G, 55R, 55S, 59G, 59T, 67N, 71S, 72G, 73R, 94Q / 103L / 118V / 146I, 103L, 103L / 144V / 146I / 172L / 180 V, 103L / 146I / 154R / 172L / 180V / 232G, 103L / 154R, 118V, 118V / 144V / 215H, 11 8V / 172L / 180V, 137M, 137R, 142V, 146I, 146I / 154R, 154R, 158Y, 172L / 190S, 1 84S, 198L, 205G, 205L, 216M, 216V, 223A, 223C, 223E, 223L, 223M, 223S, 226L, 226V, 229L, 229V, 229W, 247V, 261G, 261H, 263E, 263G, 263L and 263S, wherein the positions are numbered with reference to SEQ ID NO:1120.In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of S3D, I7V, I7V / F158Y, I7V / F158Y / K175P, I7V / F158Y / I197V / V202I, Q27E / S43I / A115S / R172L, Q27E / S43I / R172L, A29G, A29R, N32G, N32R, S4 3I / I110T / A115S / R172L, N44S, R46H, R46V, L48I / R172L, E55A, E55G, E55R, E55S, E59G, E59T , S67N, G71S, K72G, K73R, P94Q / V103L / Y118V / V146I, V103L, V103L / T144V / V146I / R172L / L18 0V, V103L / V146I / A154R / R172L / L180V / S232G, V103L / A154R, Y118V, Y118V / T144V / Q215H, Y 118V / R172L / L180V, T137M, T137R, T142V, V146I, V146I / A154R, A154R, F158Y, R172L / T190S, In some additional embodiments, the engineered polypeptide comprises an amino acid sequence having at least 80% sequence identity to any even-numbered sequence set forth in SEQ ID NO: 1214 to SEQ ID NO: 1344.

[0023] The present invention also 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 of SEQ ID NO:1120, comprising at least one substitution or a set of substitutions selected from the following: 73, 80, 103 / 146 / 209, 103 / 172 / 209, 118 / 119 / 172 / 209, 135, 144 / 209 / 232, 172 / 180 / 209, 181 and 223, wherein the positions are numbered with reference to SEQ ID NO:1120. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of 73R, 80A, 80G, 103L / 146I / 209C, 103L / 172C / 209C, 118V / 119H / 172C / 209C, 135V, 144V / 209C / 232G, 172C / 180V / 209C, 181S and 223E, wherein the positions are numbered with reference to SEQ ID NO:1120. In some additional embodiments, the engineered polypeptide comprises at least one substitution or set of substitutions selected from the group consisting of K73R, S80A, S80G, V103L / V146I / I209C, V103L / R172C / I209C, Y118V / K119H / R172C / I209C, N135V, T144V / I209C / S232G, R172C / L180V / I209C, Q181S, and K223E, wherein positions are numbered with reference to SEQ ID NO: 1120. In some additional embodiments, the engineered polypeptide comprises an amino acid sequence having at least 80% sequence identity to any even-numbered sequence set forth in SEQ ID NO: 1346 to SEQ ID NO: 1366.

[0024] The present invention also 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 of SEQ ID NO: 1348, comprising at least one substitution or a set of substitutions selected from the group consisting of: 2, 7 / 46, 39, 46, 46 / 55, 46 / 55 / 72, 46 / 55 / 92, 46 / 55 / 118, 46 / 55 / 226 / 257 / 263, 46 / 55 / 263, 46 / 70, 46 / 70 / 92 / 118 / 261 / 263, 46 / 70 / 175 / 263, 46 / 72, 46 / 118, 55 / 257 / 261, 55 / 261 / 263, 55 / 263, 105, 118, 118 / 226 / 261 / 263 and 263, wherein the positions are numbered with reference to SEQ ID NO:1348. In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of 2G, 7V / 46H, 39F, 39G, 39M, 39Q, 39S, 39T, 39V, 46H, 46H / 55R, 46H / 55R / 72C, 46H / 55R / 92A, 46H / 55R / 118V, 46H / 55R / 226V / 257Q / 263L, 46H / 55R / 263L, 46H / 70N, 46H / 70N / 92A / 118V / 261G / 263L, 46H / 70N / 175P / 263G, 46H / 72C, 46H / 118V, 55R / 257Q / 261G, 55R / 261G / 263I, 55R / 263G, 105G, 118V, 118V / 226V / 261G / 263I and 263G, wherein the positions are numbered with reference to SEQ ID NO:1348.In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of V2G, I7V / R46H, W39F, W39G, W39M, W39Q, W39S, W39T, W39V, R46H, R46H / E55R, R46H / E55R / K72C, R46H / E55R / K92A, R46H / E55R / Y118V, R46H / E55R / K226V / S257Q / A263L, R46H / E55R / A2 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 NO: 1368 to SEQ ID NO: 1422.

[0025] The present invention also 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 of SEQ ID NO: 1348, comprising at least one substitution or a set of substitutions selected from the group consisting of: 2, 7 / 46, 7 / 46 / 175, 22 / 46 / 55 / 118, 27 / 46 / 72, 27 / 4 6 / 146 / 158 / 202、27 / 46 / 175、27 / 118、29 / 70 / 118、39、46、46 / 55、46 / 55 / 59 / 261 / 263、46 / 55 / 72、46 / 55 / 92、46 / 55 / 118、46 / 55 / 118 / 263、46 / 55 / 146、46 / 55 / 146 / 202、46 / 55 / 226 / 257 / 263、46 / 55 / 263、46 / 70、46 / 70 / 92 / 118 / 261 / 263、46 / 70 / 175 / 263、46 / 72、46 / 92、46 / 92 / 175、46 / 118、46 / 146、46 / 146 / 257 / 263、55 / 70 / 118、55 / 257 / 26 1, 55 / 257 / 263, 55 / 261 / 263, 55 / 263, 70, 92 / 118, 105, 118, 118 / 226 / 261 / 263, 146 / 257 / 263, 146 / 261 / 263, 192 / 257 / 261, 202, 257 / 261 / 263, 261 / 263 and 263, wherein the positions are numbered with reference to SEQ ID NO:1348.In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of 2G, 7V / 46H, 7V / 46H / 175P, 22V / 46H / 55R / 118V, 27G / 46H / 72C, 27G / 46H / 146I / 158Y / 202I, 27G / 46H / 175P, 27G / 118V, 29V / 70N / 118V, 39M, 39Q, 39S, 39 ... 9T, 39V, 46H, 46H / 55R, 46H / 55R / 59T / 261G / 263L, 46H / 55R / 72C, 46H / 55R / 92A, 46H / 55R / 118V, 4 6H / 55R / 118V / 263S, 46H / 55R / 146I, 46H / 55R / 146I / 202I, 46H / 55R / 226V / 257Q / 263L, 46H / 55R / 2 63L, 46H / 70N, 46H / 70N / 92A / 118V / 261G / 263L, 46H / 70N / 175P / 263G, 46H / 72C, 46H / 92A, 46H / 92 A / 175P, 46H / 118V, 46H / 146I, 46H / 146I / 257Q / 263I, 55R / 70N / 118V, 55R / 257Q / 261G, 55R / 257Q / 263L, 55R / 261G / 263I, 55R / 263G, 70N, 92A / 118V, 105G, 118V, 118V / 226V / 261G / 263I, 146I / 257Q / 263S, 146I / 261G / 263L, 192V / 257Q / 261G, 202I, 257Q / 261G / 263G, 261G / 263I and 263G, wherein the positions are numbered with reference to SEQ ID NO:1348.In some additional embodiments, the engineered polypeptide comprises at least one substitution or a set of substitutions selected from the group consisting of V2G, I7V / R46H, I7V / R46H / K175P, L22V / R46H / E55R / Y118V, Q27G / R46H / K72C, Q27G / R46H / V146I / F158Y / V202I, Q27G / R46H / K175P, Q27G / Y118V, A29V / E70N / Y118V, W39M, W39Q, W39S, W39T, W39V, R46H, R46H / E55R, R46H / E55R / E59T / A261G / A263L, R46H / E55R / K72C, R46H / E55R / K92A, R46H / E55R / Y118V, R46 H / E55R / Y118V / A263S, R46H / E55R / V146I, R46H / E55R / V146I / V202I, R46H / E55R / K226V / S257Q / A263L, R46H / E55R / A2 63L, R46H / E70N, R46H / E70N / K92A / Y118V / A261G / A263L, R46H / E70N / K175P / A263G, R46H / K72C, R46H / K92A, R46H / K9 2A / K175P, R46H / Y118V, R46H / V146I, R46H / V146I / S257Q / A263I, E55R / E70N / Y118V, E55R / S257Q / A261G, E55R / S257 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 NO: 1424 to SEQ ID NO: 1484.

[0026] The present invention also provides engineered polynucleotides encoding at least one engineered polypeptide described in the above paragraphs. In some embodiments, the engineered polynucleotide comprises the odd-numbered sequences listed in SEQ ID NO:3 to SEQ ID NO:1483.

[0027] The present invention also provides a vector comprising at least one engineered polynucleotide described above. In some embodiments, the vector further comprises at least one control sequence.

[0028] The present invention also provides a host cell comprising a vector provided herein. In some embodiments, the host cell produces at least one engineered polypeptide provided herein.

[0029] The present invention also provides a method for producing an engineered nitroaldolase polypeptide, the method comprising the step of culturing a host cell 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.

[0030] Description of the invention

[0031] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry and nucleic acid chemistry described below are those well known in the art and generally adopted. Such technology is well known and described in many textbooks and reference works well known to those skilled in the art. For chemical synthesis and chemical analysis, standard techniques or their modified forms are used. All patents, patent applications, articles and publications mentioned herein (both above and below) are hereby expressly incorporated herein by reference.

[0032] 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 the circumstances in which they are used by those skilled in the art. Therefore, the terms that will be defined below are more fully described by reference to the present invention as a whole.

[0033] It should be understood that the general description above and the detailed description below are only exemplary and illustrative, rather than limiting the present invention. The section titles used herein are only for organizational purposes and are not to be construed as limiting the subject matter described. Numerical ranges include numbers that limit the range. Therefore, each numerical range disclosed herein is intended to include each narrower numerical range that falls within such a wider numerical range, as such a narrower numerical range is clearly written out in full herein. It is also intended that each maximum (or minimum) numerical limit disclosed herein includes each lower (or higher) numerical limit, as such lower (or higher) numerical limits are clearly written out herein.

[0034] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polypeptide" includes more than one polypeptide. Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting.

[0035] It should be understood that where the term "comprising" is used in the description of various embodiments, those skilled in the art will understand that in some specific cases, the embodiment may be alternatively described using the language of "consisting essentially of" or "consisting of". It should also be understood that where the term "optional" or "optionally" is used in the description of various embodiments, the event or circumstance described subsequently may or may not occur, and the description includes instances where the event or circumstance occurs and instances where it does not occur. It should be understood that the general description above and the detailed description below are exemplary and illustrative only and are not intended to 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.

[0036] abbreviation:

[0037] The abbreviations used to encode the amino acids are conventional and are as follows:

[0038] Amino Acids Three letter abbreviation Single letter abbreviation Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamate Glu E Glutamine Gln Q Glycine Gly G Histidine HIs H Isoleucine I1e 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 ryr Y Valine Val V

[0039] When a three-letter abbreviation is used, unless specifically preceded by "L" or "D" or clear from the context in which the abbreviation is used, the amino acid may be about the α-carbon (C α ) is the L-configuration or the D-configuration of α-alanine. For example, "Ala" represents alanine without specifying the configuration about the α-carbon, while "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively.

[0040] When single-letter abbreviations are used, capital letters represent amino acids in the L-configuration about the α-carbon, and lowercase letters represent amino acids in the D-configuration about 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 mixtures thereof), the sequence is presented in the amino (N) to carboxyl (C) direction according to common convention.

[0041] Abbreviations for genetically encoded nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically described, abbreviated nucleotides may be ribonucleosides or 2'-deoxyribonucleosides. Nucleosides may be designated individually or collectively as ribonucleosides or 2'-deoxyribonucleosides. When a nucleic acid sequence is represented by a string of single-letter abbreviations, the sequence is presented in a 5' to 3' direction according to conventional convention, and the phosphate is not shown.

[0042] definition:

[0043] With reference to the present invention, technical and scientific terms used in the description herein shall have the meanings commonly understood by one of ordinary skill in the art, unless specifically defined otherwise. Accordingly, the following terms are intended to have the following meanings.

[0044] "EC" numbers refer to the enzyme nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions that the enzymes catalyze.

[0045] "ATCC" refers to the American Type Culture Collection, whose biological collection includes genes and strains.

[0046] "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0047] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modifications (e.g., glycosylation, phosphorylation, lipidation, myristylation, ubiquitination, etc.). Included in this definition are D- and L-amino acids, as well as mixtures of D- and L-amino acids, and polymers comprising mixtures of D- and L-amino acids and D- and L-amino acids.

[0048] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Likewise, nucleotides may be referred to by their commonly accepted single-letter codes.

[0049] As used herein, "polynucleotide" and "nucleic acid" refer to two or more nucleosides covalently linked together. A polynucleotide may be completely comprised of ribonucleotides (i.e., RNA), completely comprised of 2' deoxyribonucleotides (i.e., DNA), or a mixture of ribonucleotides and 2' deoxyribonucleotides. Although nucleosides are usually linked together via standard phosphodiester bonds, a polynucleotide may include one or more non-standard bonds. A polynucleotide may be single-stranded or double-stranded, or may include both single-stranded and double-stranded regions. In addition, although a polynucleotide typically includes naturally occurring coding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it may include one or more modified and / or synthesized nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthesized nucleobases are nucleobases encoding amino acid sequences.

[0050] "Nitroaldolase", "hydroxynitrile lyase" or "oxynitrilase" refers to an enzyme classified according to EC 4.1.2.47 that has natural activity in the interconversion of cyanohydrins to cyanide and the corresponding aldehydes and ketones. These enzymes are also known to have activity as nitroaldolases in the conversion of nitroalkanes and aldehydes to β-nitroalcohols. Although the nitroaldolase of the present invention is derived from Schizonepeta japonica, the present invention is not so limited and the nitroaldolase may be derived from any suitable organism or produced synthetically. As used herein, nitroaldolase includes naturally occurring (wild type) nitroaldolase as well as non-naturally occurring engineered polypeptides produced by artificial manipulation.

[0051] "Coding sequence" refers to a portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

[0052] "Naturally occurring" or "wild-type" refers to a form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence that is present in an organism, can be isolated from a source in nature, and has not been intentionally modified by human manipulation.

[0053] As used herein, "recombinant," "engineered," and "non-naturally occurring" when used with respect to a cell, nucleic acid, or polypeptide, refers to a material, or a material corresponding to the native or original form of the material, that has been modified in a manner not originally found in nature. In some embodiments, the cell, nucleic acid, or polypeptide is identical to a naturally occurring cell, nucleic acid, or polypeptide, but is produced or derived from synthetic materials and / or manipulated through the use of recombinant techniques. Non-limiting examples include, among others, recombinant cells that express genes not found in the natural (non-recombinant) form of the cell or that express natural genes that are otherwise expressed at different levels.

[0054] "Percentage of sequence identity" and "percentage of 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) compared to the reference sequence for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions at which the same nucleic acid base or amino acid residue appears in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions at which the same nucleic acid base or amino acid residue appears in the two sequences or the nucleic acid base or amino acid residue is aligned with a gap to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will appreciate that there are many established algorithms that can be used to compare two sequences. Optimal alignment of sequences for comparison can be performed, 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 search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444

[1988] ), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin software package), or by visual inspection as is known in the art. Examples of algorithms suitable for determining percent sequence identity and sequence similarity include, but are not limited to, BLAST and BLAST 2.0 algorithms, 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 analysis is publicly available through the website of the National Center for Biotechnology Information. 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 certain positive threshold score T when aligned with a word of the same length in the database sequence. T is referred to as the neighborhood word score threshold (see, Altschul et al., supra).These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence to the point where the cumulative alignment score cannot be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for matching residue pairs; always >0) and N (penalty score for mismatched residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when: the cumulative alignment score drops by an 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 (E) of 10, M=5, N=-4, and comparison of both chains. For amino acid sequences, the BLASTP program uses the following as defaults: a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (See, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

[1989] ). Exemplary determinations of sequence alignments 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.

[0055] "Reference sequence" refers to a designated sequence used as the basis for sequence comparison. A reference sequence can be a subset of a larger sequence, for example, a segment of a full-length gene or polypeptide sequence. Typically, a reference sequence is a length of at least 20 nucleotides or amino acid residues, a length of at least 25 residues, a length of at least 50 residues, or the full length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides can each (1) contain a sequence similar to the two sequences (i.e., a portion of the complete sequence), and (2) can also contain a different (divergent) sequence between the two sequences, a sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides in a "comparison window" to identify and compare the sequence similarity of a local region. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, wherein the reference sequence is a sequence that can have one or more changes in the primary sequence. For example, "a reference sequence based on SEQ ID NO:4 with valine at a residue corresponding to X14" or X14V refers to a reference sequence in which the corresponding residue (tyrosine) at X14 in SEQ ID NO:4 has been changed to valine.

[0056] "Comparison window" refers to a conceptual segment of at least about 20 consecutive nucleotide positions or amino acid residues, wherein a sequence can be compared to a reference sequence of at least 20 consecutive nucleotides or amino acids, and wherein for optimal alignment of the two sequences, a portion of the sequence in the comparison window may include 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions). The comparison window may be longer than 20 consecutive residues, and optionally includes windows of 30, 40, 50, 100 or more.

[0057] As used herein, "substantially identical" refers to a polynucleotide or polypeptide sequence having at least 80% sequence identity, at least 85% identity, at least 89% to 95% sequence identity, or more generally at least 99% sequence identity in a comparison window of at least 20 residue positions, usually in a window of at least 30-50 residues, compared to a reference sequence, wherein the percentage of sequence identity is calculated by comparing the reference sequence and a sequence comprising a deletion or addition totaling 20% ​​or less of the reference sequence over the comparison window. In some specific embodiments applied to polypeptides, the term "substantially identical" means that when the best alignment is performed using the default gap weights such as by the program GAP or BESTFIT, 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 compared sequences differ due to conservative amino acid substitutions.

[0058] When used in the context of numbering a given amino acid or polynucleotide sequence, "corresponding to," "reference to," and "relative to" refer to the numbering of the residues of a given amino acid or polynucleotide sequence when the given amino acid or polynucleotide sequence is compared to a specified reference sequence. In other words, the residue numbers or residue positions of a given polymer are specified with respect to a reference sequence, rather than being specified 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 an engineered nitroaldolase, can be optimized for residue matching between the two sequences by introducing spaces to align with the reference sequence. In these cases, the residues in a given amino acid or polynucleotide sequence are numbered with respect to the reference sequence to which it is aligned, despite the presence of spaces.

[0059] "Amino acid difference" or "residue difference" refers to the change of the amino acid residue at one position of a polypeptide sequence relative to the amino acid residue at the corresponding position in the reference sequence. The position of the amino acid difference is generally referred to as "Xn" herein, where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, "the residue difference at position X25 compared to SEQ ID NO:2" refers to the change of the amino acid residue at the polypeptide position corresponding to position 25 of SEQ ID NO:2. Therefore, if the reference polypeptide SEQ ID NO:2 has valine at position 25, "the residue difference at position X25 compared to SEQ ID NO:2" refers to the amino acid substitution of any residue except valine at the position of the polypeptide corresponding to position 25 of SEQ ID NO:2. In most examples herein, the specific amino acid residue difference at one position is indicated as "XnY", where "Xn" specifies 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 the reference polypeptide). In some embodiments, more than one amino acid may appear at a specified residue position (i.e., the substituted amino acids may be listed in the form of XnY / Z, where Y and Z represent substituted amino acid residues). In some examples (e.g., Table 2-1, Table 2-2, Table 3-1, Table 3-2, Table 4-1, Table 4-2, Table 5-1, Table 5-2, Table 6-1, Table 6-2, Table 7-1, and Table 7-2), the present invention also provides specific amino acid differences represented by the conventional symbol "AnB", where A is a single letter identifier for a residue in a reference sequence, "n" is the number of a residue position in a reference sequence, and B is a single letter identifier for a residue substitution in the sequence of an engineered polypeptide. In addition, in some cases, a polypeptide of the present invention may include one or more amino acid residue differences relative to a reference sequence, indicated by a list of designated positions that are changed relative to the reference sequence. In some additional embodiments, the present invention provides engineered polypeptide sequences comprising both conservative amino acid substitutions and non-conservative amino acid substitutions.

[0060] 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 similar amino acid definition class. For example, but not limited to, an amino acid with an aliphatic side chain is replaced by another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain is replaced by another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain is replaced by another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is replaced by another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is replaced by another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and / or a hydrophobic amino acid or a hydrophilic amino acid is replaced by another hydrophobic amino acid or a hydrophilic amino acid, respectively. Exemplary conservative substitutions are provided in Table 1 below.

[0061]

[0062] "Non-conservative substitutions" refer to substitutions of amino acids in a polypeptide with amino acids having significantly different side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affect (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., proline for glycine), (b) charge or hydrophobicity, or (c) side chain bulk. For example, but not limited to, exemplary non-conservative substitutions may be substitutions of basic or aliphatic amino acids for acidic amino acids; substitutions of small amino acids for aromatic amino acids; and substitutions of hydrophobic amino acids for hydrophilic amino acids.

[0063] "Deletion" refers to a modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletion can include removal of 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 that make up the reference enzyme, or up to 20% of the total number of amino acids, while retaining enzymatic activity and / or retaining the improved properties of the engineered nitroaldolase. Deletion can involve internal portions and / or terminal portions of a polypeptide. In various embodiments, deletion can include continuous segments or can be discontinuous.

[0064] "Insertion" refers to the modification of a polypeptide by adding one or more amino acids to a reference polypeptide. In some embodiments, the improved engineered nitroaldolase comprises one or more amino acids inserted into a naturally occurring nitroaldolase polypeptide, and one or more amino acids inserted into other improved nitroaldolase polypeptides. The insertion may be in an internal portion of the polypeptide or may be an insertion into the carboxyl or amino terminus. Insertion as used herein includes fusion proteins as known in the art. The insertion may be a continuous segment of amino acids or separated by one or more amino acids in a naturally occurring polypeptide.

[0065] As used herein, "fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion but the remaining amino acid sequence is identical to the corresponding positions in the sequence. A 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 70%, 80%, 90%, 95%, 98% and 99% of a full-length nitroaldolase polypeptide, such as the polypeptide of SEQ ID NO: 4 or the nitroaldolase polypeptide provided in the even-numbered sequences of SEQ ID NO: 4-1484.

[0066] "Isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that are naturally associated with it. The term includes polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., host cells or in vitro synthesis). Engineered nitroaldolase can be present in cells, in cell culture media, or prepared in various forms, such as lysates or isolated preparations. Like this, in some embodiments, the engineered nitroaldolase can be an isolated polypeptide.

[0067] "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 generally a substantially purified composition when the target species constitutes at least about 50% of the macromolecular species present by mole or % weight. Generally speaking, a substantially pure nitroaldolase composition will constitute about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species present in the composition by mole or % weight. In some embodiments, the target species is purified to substantially homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods), wherein the composition is substantially composed of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ionic species are not considered macromolecular species. In some embodiments, the separated engineered nitroaldolase polypeptide is a substantially pure polypeptide composition.

[0068] "Stereoselectivity" refers to the preferential formation of one stereoisomer relative to another stereoisomer in a chemical or enzymatic reaction. Stereoselectivity can be partial, where one stereoisomer is formed in preference to the other, or it can be complete, where only one stereoisomer is formed. When the stereoisomers are enantiomers, stereoselectivity is referred to as enantioselectivity, i.e., the fraction (usually reported as a percentage) of one enantiomer in the sum of the two enantiomers. The art typically reports it as enantiomeric excess ("ee") (usually as a percentage) calculated therefrom according to the following formula: [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer]. When the stereoisomers are diastereomers, stereoselectivity is referred to as diastereoselectivity, i.e., the fraction (usually reported as a percentage) of one diastereomer in a mixture of two diastereomers, typically reported as diastereomeric excess ("de"). Enantiomeric excess and diastereomeric excess are types of stereoisomeric excess.

[0069] "Highly stereoselective" refers to a chemical or enzymatic reaction that is capable of converting one or more substrates (eg, substrate compounds (2) and (3)) to the corresponding amine product (eg, compound (1)) with at least about 85% stereoisomeric excess.

[0070] As used herein, "improved enzyme properties" refers to at least one improved property of an enzyme. In some embodiments, the invention provides engineered nitroaldolase polypeptides that show improvements on any enzyme property compared to reference nitroaldolase polypeptides and / or nitroaldolase polypeptides and / or another engineered nitroaldolase polypeptides. For engineered nitroaldolase polypeptides described herein, comparisons are usually made for the parent enzyme in the nitroaldolase source, although in some embodiments, the reference enzyme can be another improved engineered nitroaldolase. Therefore, various nitroaldolase polypeptides can be determined and compared, including the level of "improvement" between wild-type and engineered nitroaldolase. Improved properties include, but are not limited to, such properties as enzyme activity (which can be represented by the percentage of substrate conversion), thermostability, solvent stability, pH activity spectrum, cofactor requirements, tolerance to inhibitors (refractoriness) (e.g., substrate or product inhibition), stereospecificity and stereoselectivity (including enantioselectivity).

[0071] "Enhanced enzyme activity" refers to an improved property of an engineered nitroaldolase polypeptide, which can be expressed as an increase in specific activity (e.g., product produced / time / weight protein) or an increase in the percent conversion of substrate to product (e.g., the percent conversion of a starting amount of substrate to product using a specified amount of nitroaldolase over a specified period of time) compared to a reference nitroaldolase. Exemplary methods for determining enzyme activity are provided in the Examples. Any property associated with enzyme activity can be affected, including typical enzyme properties. m 、V max or k cat , its variation can cause the increase of enzyme activity.The improvement of enzyme activity can be up to 2 times, 5 times, 10 times, 20 times, 25 times, 50 times or more of the enzyme activity of the naturally occurring or another engineered nitroaldolase activity in the nitroaldolase polypeptide source from about 1.2 times of the corresponding parent enzyme.Nitroaldolase activity can be measured by any standard assay, such as by monitoring the change of the property of substrate, cofactor or product.In some embodiments, the amount of the product generated can be measured by gas chromatography (GC).The comparison of enzyme activity is carried out using defined enzyme preparations, defined determinations under set conditions and one or more defined substrates, as described in further detail herein.Usually, when comparing lysates, determine the number of cells and the amount of the protein measured, and use the same expression system and the same host cell to minimize the change of the amount of the enzyme produced by the host cell and present in the lysate.

[0072] "Conversion" refers to the enzymatic conversion of one or more substrates into one or more corresponding products. "Conversion percentage" refers to the percentage of substrates converted into products within a certain period of time under specified conditions. Therefore, the "enzyme activity" or "activity" of a nitroaldolase polypeptide can be expressed as the "conversion percentage" of substrate to product.

[0073] "Thermostable" refers to a nitroaldolase polypeptide that retains similar activity (e.g., greater than 60% to 80%) after exposure to the same elevated temperature for a certain period of time (e.g., 0.5 hours to 24 hours) compared to a parent enzyme exposed to the same elevated temperature (e.g., 40°C-80°C).

[0074] "Solvent stable" refers to a nitroaldolase polypeptide that retains similar activity (e.g., more than 60% to 80%) after exposure to the same solvent at the same concentration for a certain period of time (e.g., 0.5 hour to 24 hours) compared to the parent enzyme exposed to a different concentration (e.g., 5%-99%) of the solvent (ethanol, isopropanol, dimethyl sulfoxide (DMSO), tetrahydrofuran, 2-methyltetrahydrofuran, acetone, toluene, butyl acetate, methyl tert-butyl ether, etc.).

[0075] "Thermo- and solvent-stable" refers to a nitroaldolase polypeptide that is thermostable and solvent-stable.

[0076] The term "stringent hybridization conditions" is used herein to refer to conditions under which nucleic acid hybrids are stable. As known to those skilled in the art, the stability of the hybrid is reflected in the melting temperature (T m ). In general, the stability of the hybrid varies with ionic strength, temperature, G / C content, and the presence of chaotropic agents. m Values ​​can be calculated using known methods for predicting melting temperatures (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., 26:6413

[1990] (erratum, Nucl. Acids Res., 26:6421

[1990] (erratum, Nucl. Acids Res., 26:6431

[1990] (erratum, Nucl. Acids Res., 26:6431

[1990] (erratum, Nucl. Acids Res., 26:6431

[1990] (erratum, Nucl. Acids Res., 26:6431

[1990] (erratum, Nucl. Acids Res., 26:6431

[1990] (erratum, Nucl. Acids Res., 26:6431

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

[1991] ); Sambrook et al., supra); Suggs et al., 1981, supra Developmental Biology UsingPurified 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 the complement of a sequence encoding an engineered nitroaldolase of the invention.

[0077] "Hybridization stringency" refers to hybridization conditions, such as washing conditions, in nucleic acid hybridization. Typically, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency. The term "moderately stringent hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids that have about 60% identity, preferably about 75% identity, about 85% identity, and greater than about 90% identity with the target polynucleotide. Exemplary moderately stringent conditions are conditions equivalent to hybridization at 42°C in 50% formamide, 5×Denhart 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 are consistent with a thermal melting temperature T as determined for a defined polynucleotide sequence under solution conditions. m Conditions that differ by about 10°C or less. In some embodiments, high stringency conditions refer to conditions that allow only hybridization of those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C (i.e., if the hybrid is unstable in 0.018M NaCl at 65°C, it will be unstable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridizing under conditions equivalent to 50% formamide, 5×Denhart solution, 5×SSPE, 0.2% SDS at 42°C, followed by washing in 0.1×SSPE and 0.1% SDS at 65°C. Another high stringency condition is hybridization under 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.

[0078] A "heterologous" polynucleotide refers to any polynucleotide that is introduced into a host cell by laboratory techniques and includes polynucleotides that are removed from a host cell, subjected to laboratory manipulations, and then reintroduced into the host cell.

[0079] "Codon optimized" refers to that the codons of the polynucleotides encoding proteins are changed to those codons preferentially used in a particular organism so that the encoded protein is effectively expressed in the organism of interest. Although the genetic code is degenerate, i.e., most amino acids are represented by several codons referred to as "synonyms" or "synonymous" codons, it is well known that the codon usage of a particular organism is non-random and biased for specific codon triplets. With respect to the aggregated protein coding regions of a given gene, a gene with a common function or ancestral origin, a highly expressed protein contrast low copy number protein, and an organism's genome, this codon usage bias may be higher. In some embodiments, the polynucleotides encoding nitroaldolase can be codon optimized for optimal production from a selected host organism for expression.

[0080] As used herein, "preferred, optimal, high codon usage bias codons" interchangeably refer to codons that are used more frequently in protein coding regions than other codons encoding the same amino acid. Preferred codons can be determined based on codon usage in a single gene, a group of genes with a common function or origin, highly expressed genes, codon frequencies in aggregating protein coding regions of an entire organism, codon frequencies in aggregating protein coding regions of related organisms, or a combination thereof. Codons whose frequency increases with the level of gene expression are generally the best codons for expression. Various methods are known for determining codon frequency (e.g., codon usage, relative synonymous codon usage) and codon preference in a particular organism, as well as the effective number of codons used in a gene, including multivariate analysis, such as using cluster analysis or correlation analysis (see, e.g., GCG Codon Preference, Genetics Computer Group Wisconsin Package; Codon W, Peden, University of Nottingham; McInerney, Bioinform., 14:372-73

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

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

[1990] ). Codon usage tables for many different organisms are available (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., supra; Henaut and Danchin, supra). Escherichia coli and Salmonella(eds.), ASM Press, Washington DC, pp. 2047-2066

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

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

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

[1997] ).

[0081] "Control sequences" are 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 may 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, control sequences include promoters and transcription and translation termination signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites that facilitate the connection of control sequences to the coding region of the nucleic acid sequence encoding the polypeptide.

[0082] "Operably linked" is defined herein as a configuration in which a control sequence is placed at an appropriate position (ie, in a functional relationship) relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide and / or polypeptide of interest.

[0083] "Promoter sequence" refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence includes transcriptional control sequences that mediate 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.

[0084] "Suitable reaction conditions" refer 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 nitroaldolase polypeptides of the invention are able to convert one or more substrate compounds into product compounds (e.g., converting compound (1) and compound (2) into compound (3), as shown in Scheme 1). Exemplary "suitable reaction conditions" are provided herein and illustrated by the Examples.

[0085] A "composition" refers to a mixture or combination of one or more substances, wherein each substance or component of the composition retains its individual properties. As used herein, a biocatalytic composition refers to a combination of one or more substances that can be used for biocatalysis.

[0086] "Loading" as in "compound loading" or "enzyme loading" or "cofactor loading" refers to the concentration or amount of a component in the reaction mixture at the start of the reaction.

[0087] In the context of a biocatalyst-mediated process, "substrate" refers to a compound or molecule on which the biocatalyst acts. For example, compound (1) and compound (2) are substrates for nitroaldolase.

[0088] In the context of a biocatalyst-mediated process, "product" refers to a compound or molecule produced by the action of the biocatalyst. For example, compound (3) is the product of the nitroaldolase-mediated conversion of compound (1) and compound (2).

[0089] "Alkyl" refers to a saturated hydrocarbon group having from 1 to 18 carbon atoms (inclusive), linear or branched, more preferably from 1 to 8 carbon atoms (inclusive), and most preferably 1 to 6 carbon atoms (inclusive). Alkyl groups having a specified number of carbon atoms are indicated in brackets (e.g., (C1-C6)alkyl refers to an alkyl group of 1 to 6 carbon atoms).

[0090] "Alkenyl" refers to a hydrocarbon group having from 2 to 12 carbon atoms (inclusive), straight or branched, containing at least one double bond but optionally containing more than one double bond.

[0091] "Alkynyl" refers to a hydrocarbon group having from 2 to 12 carbon atoms (inclusive), straight or branched, containing at least one triple bond but optionally more than one triple bond, and further optionally containing one or more double bonded moieties.

[0092] "Alkylene" refers to a linear or branched divalent hydrocarbon group having from 1 to 18 carbon atoms (inclusive), more preferably 1 to 8 carbon atoms (inclusive), and most preferably 1 to 6 carbon atoms (inclusive), optionally substituted with one or more suitable substituents. Exemplary "alkylene" include, but are not limited to, methylene, ethylene, propylene, butylene, etc.

[0093] "Alkenylene" refers to a straight or branched divalent hydrocarbon group having 2 to 12 carbon atoms (inclusive) and one or more carbon-carbon double bonds, more preferably 2 to 8 carbon atoms (inclusive), and most preferably 2 to 6 carbon atoms (inclusive), optionally substituted by one or more suitable substituents.

[0094] "Heteroalkyl", "heteroalkenyl" and "heteroalkynyl" refer to alkyl, alkenyl and alkynyl groups, respectively, 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 may replace carbon atoms include, but are not limited to, -O-, -S-, -SO-, -NR γ -, -PH-, -S(O)-, -S(O)2-, -S(O)NR γ -、-S(O)2NR γ - etc., including combinations thereof, where each R γ are independently selected from hydrogen, alkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl.

[0095] "Aryl" refers to an unsaturated aromatic carbocyclic group having from 6 to 12 carbon atoms (including endpoints) having a single ring (e.g., phenyl) or more than one condensed ring (e.g., naphthyl or anthracenyl). Exemplary aryl groups include phenyl, pyridyl, naphthyl, etc.

[0096] "Arylalkyl" refers to an alkyl group substituted with an aryl group (i.e., an "aryl-alkyl-" group), preferably having 1 to 6 carbon atoms (inclusive) in the alkyl portion and 6 to 12 carbon atoms (inclusive) in the aryl portion. Such arylalkyl groups are exemplified by benzyl, phenethyl, and the like.

[0097] "Aryloxy" refers to -OR λ Group, where R λ is an aryl group which may be optionally substituted.

[0098] "Cycloalkyl" refers to a cyclic alkyl group having 3 to 12 carbon atoms (inclusive), having a single cyclic ring or multiple condensed rings, which may be optionally substituted by 1 to 3 alkyl groups. Exemplary cycloalkyl groups include, but are not limited to, monocyclic structures and polycyclic 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.

[0099] "Cycloalkylalkyl" refers to an alkyl group substituted with a cycloalkyl group (i.e., a "cycloalkyl-alkyl-" group), preferably having from 1 to 6 carbon atoms (inclusive) in the alkyl portion and from 3 to 12 carbon atoms (inclusive) in the cycloalkyl portion. Such cycloalkylalkyl groups are exemplified by cyclopropylmethyl, cyclohexylethyl, and the like.

[0100] "Amino" refers to the group -NH2. Substituted amino refers to the following groups: -NHR η NR η R η and NR η R η R η , where each R η Independently selected from substituted or unsubstituted alkyl, cycloalkyl, cycloheteroalkyl, alkoxy, aryl, heteroaryl, heteroarylalkyl, acyl, alkoxycarbonyl, sulfhydryl, sulfinyl, sulfonyl, etc. Typical amino groups include, but are not limited to, dimethylamino, diethylamino, trimethylammonium, triethylammonium, methylsulfonylamino, furanyl-oxy-sulfonylamino, etc.

[0101] "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).

[0102] "Aminocarbonyl" refers to -C(O)NH2. Substituted aminocarbonyl refers to -C(O)NR η R η , where the amino group NR η R η As defined herein.

[0103] "Oxy" refers to a divalent group -O-, which may have various substituents to form different oxy groups including ethers and esters.

[0104] "Alkoxy" or "alkyloxy" are used interchangeably herein to refer to the group "-OR ζ ”, where R ζ is an alkyl group, including an optionally substituted alkyl group.

[0105] "Carboxyl" refers to -COOH.

[0106] "Carbonyl" refers to -C(O)-, which can have various substituents to form different carbonyl groups including acids, acid halides, aldehydes, amides, esters and ketones.

[0107] "Carboxyalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced with one or more carboxyl groups.

[0108] "Aminocarbonylalkyl" means an alkyl group substituted with an aminocarbonyl group as defined herein.

[0109] "Halogen" or "halo" refers to fluorine, chlorine, bromine and iodine.

[0110] "Haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by halogen. Therefore, the term "haloalkyl" is meant to include monohaloalkyl, dihaloalkyl, trihaloalkyl, etc. up to perhaloalkyl. For example, the expression "(C1-C2)haloalkyl" includes 1-fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, etc.

[0111] "Hydroxyl" refers to -OH.

[0112] "Hydroxyalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced with one or more hydroxy groups.

[0113] "Thiol" or "sulfanyl" refers to -SH. Substituted thiol or sulfanyl refers to -SR η , where R η is an alkyl group, an aryl group or other suitable substituents.

[0114] "Alkylthio" refers to -SR ζ , where R ζ is an alkyl group which may be optionally substituted. Typical alkylthio groups include, but are not limited to, methylthio, ethylthio, n-propylthio, and the like.

[0115] "Alkylthioalkyl" refers to an alkylthio group "-SR ζ "substituted alkyl, wherein R ζ is an alkyl group which may be optionally substituted.

[0116] "Sulfonyl" refers to -SO2-. Substituted sulfonyl refers to -SO2-R η , where R η is an alkyl group, an aryl group or other suitable substituents.

[0117] "Alkylsulfonyl" refers to -SO2-R ζ , where R ζ is an alkyl group which may be optionally substituted. Typical alkylsulfonyl groups include, but are not limited to, methylsulfonyl, ethylsulfonyl, n-propylsulfonyl, and the like.

[0118] "Alkylsulfonylalkyl" refers to an alkylsulfonyl group "-SO2-R ζ "substituted alkyl, wherein R ζ is an alkyl group which may be optionally substituted.

[0119] "Heteroaryl" refers to an aromatic heterocyclic group having 1 to 10 carbon atoms (inclusive) and 1 to 4 heteroatoms (inclusive) selected from oxygen, nitrogen and sulfur in the ring. Such heteroaryl groups can have a single ring (e.g., pyridyl or furanyl) or more than one condensed ring (e.g., indolizinyl or benzothienyl).

[0120] "Heteroarylalkyl" refers to an alkyl group substituted with a heteroaryl group (i.e., a "heteroaryl-alkyl-" group), preferably having 1 to 6 carbon atoms (inclusive) in the alkyl portion and 5 to 12 ring atoms (inclusive) in the heteroaryl portion. Examples of such heteroarylalkyl groups are pyridylmethyl and the like.

[0121] "Heterocycle", "heterocyclic" and interchangeably "heterocycloalkyl" refer to saturated or unsaturated groups having a single ring or more than one fused ring, having from 2 to 10 carbon ring atoms (inclusive) and from 1 to 4 heterocyclic atoms (inclusive) 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., indolinyl, dihydrobenzofuran or quinuclidinyl). Examples of heterocycles include, but are not limited to, furan, thiophene, thiazole, oxazole, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthylpyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, phenanthroline, isothiazole, phenazine, isoxazole, phenoxazine, phenothiazine, imidazolidine, imidazoline, piperidine, piperazine, pyrrolidine, indoline, and the like.

[0122] "Heterocycloalkylalkyl" refers to an alkyl group substituted with a heterocycloalkyl group (i.e., a "heterocycloalkyl-alkyl-" group), preferably having from 1 to 6 carbon atoms (inclusive) in the alkyl portion and from 3 to 12 ring atoms (inclusive) in the heterocycloalkyl portion.

[0123] "Membered ring" is intended to include any cyclic structure. The number before the term "membered" indicates the number of backbone atoms that make 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.

[0124] 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 atoms in common.

[0125] As used herein, "optionally substituted" with respect to the above chemical groups means that the position of the chemical group occupied by hydrogen may be replaced by another atom (unless otherwise specified) such as, but not limited to, carbon, oxygen, nitrogen or sulfur, or a 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, alkyl The invention also includes but is not limited to alkyl, alkylsulfonylamino, sulfonylamino, substituted sulfonylamino, cyano, amino, substituted amino, alkylamino, dialkylamino, aminoalkyl, acylamino, amidino, amidoximino, hydroxamoyl, phenyl, aryl, substituted aryl, aryloxy, arylalkyl, arylalkenyl, arylalkynyl, pyridinyl, imidazolyl, heteroaryl, substituted heteroaryl, heteroaryloxy, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloalkyl, cycloalkenyl, cycloalkylalkyl, substituted cycloalkyl, cycloalkyloxy, pyrrolidinyl, piperidinyl, morpholino, heterocyclyl, (heterocyclyl)oxy and (heterocyclyl)alkyl; wherein preferred heteroatoms are oxygen, nitrogen and sulfur. In addition, where there are open valences on these substituted chemical groups, they can be further substituted by alkyl, cycloalkyl, aryl, heteroaryl and / or heterocyclic groups, where these open valences are present on carbon, they can be further substituted by halogens and oxygen-bonded, nitrogen-bonded or sulfur-bonded substituents, and where there are more than one such open valences, these groups can be connected to form a ring by directly forming a bond or by forming a bond with a new heteroatom (preferably oxygen, nitrogen or sulfur). It is also envisioned that the above substitutions can be made, provided that replacing hydrogen with a substituent does not bring unacceptable instability to the molecules of the present invention, and is otherwise chemically reasonable. It will be understood by those of ordinary skill in the art that for any chemical group described as optionally substituted, only sterically achievable and / or synthetically feasible chemical groups are intended to be included. As used herein, "optionally substituted" refers to all subsequent modifiers in a term or a series of chemical groups. For example, in the term "optionally substituted arylalkyl", the "alkyl" portion and the "aryl" portion of the molecule may or may not be substituted, and for the series "optionally substituted alkyl, cycloalkyl, aryl and heteroaryl", the alkyl, cycloalkyl, aryl and heteroaryl groups independently of each other may or may not be substituted.

[0126] As used herein, "reaction" refers to one or more processes in which one or more substances or compounds or substrates are converted into one or more different substances, compounds.

[0127] Conversion of nitromethane and trifluoroacetone to β-nitroalcohols

[0128] Compound (4), (S)-3-amino-1,1,1-trifluoro-2-methylpropan-2-ol, is an intermediate for the synthesis of novel compounds for the treatment of cystic fibrosis.

[0129]

[0130] The enzymatic synthesis of compound (4), β-hydroxy-α-amino alcohol, provides an attractive alternative to traditional chemical synthesis. The use of enzyme biocatalysts can reduce chemical waste and allow for more efficient synthesis methods.

[0131] The use of a two-enzyme cascade to generate chiral β-amino alcohols via β-hydroxy-α-amino acid intermediates has been reported by others (Dückers et al. Appl Microbiol Biotechnol, 2010, 88:409-424). In the first step, threonine aldolase catalyzes the conversion of glycine (or another nucleophile) and acetaldehyde (or another electrophile) to threonine in a PLP-dependent mechanism. In the second step, an amino acid decarboxylase catalyzes the removal of the carboxyl group to produce an amino alcohol (this is also a PLP-dependent mechanism). We previously reported a two-enzyme cascade (US 63 / 285,377) that produced compound (4) using an engineered threonine aldolase and an engineered amino acid decarboxylase.

[0132] However, other biocatalytic pathways have been proposed and may provide additional utility and efficiency under industrial synthetic conditions. These include the use of nitrilase (also known as hydroxynitrile lyase) to produce geminal nitrile or β-nitro alcohol intermediates, followed by reduction to the β-hydroxy-α-amino alcohol of compound (4). The present disclosure details the use of nitrilase for nitroaldol conversion of nitromethane and trifluoroacetone to β-nitro alcohol intermediates. In the present disclosure, the term nitroaldolase is used to describe engineered nitrilase / hydroxynitrile lyase to highlight the improvements in enzyme functionality for the nitroaldol reaction.

[0133] Oxonitriles are mainly found in plants, typically drupes (e.g., almonds, apricots), where the conversion of naturally occurring cyanogenic glycosides or cyanolipid nitrile to cyanide is used as a defense mechanism against herbivores and microorganisms. Both (S) and (R) selective oxonitriles are known. As members of the α / β hydrolase fold family, oxonitriles have a catalytic triad similar to that of esterases. Natural substrates are believed to be acetone and benzaldehyde. Some oxonitriles contain non-catalytic FAD, which serves as a structural cofactor.

[0134] The proposed mechanism of oxonitrile enzymes shows that the activated serine in the catalytic triad acts as a general acid / base rather than a nucleophile (Gruber et al. J. Biol. Chem., 2004, 279: 20501-20510). This is unusual for members of the α / β hydrolase fold family, where serine usually acts as a nucleophile and forms an acyl enzyme intermediate.

[0135] The present disclosure provides novel biocatalysts for the synthesis of chiral β-amino alcohols via β-nitro alcohol intermediates followed by chemical reduction and related methods of use. The nitroaldolase biocatalysts of the present disclosure are engineered variants of a polypeptide (SEQ ID NO: 2) encoded by a homologous gene (SEQ ID NO: 1) from Psoralea corylifolia. These engineered polypeptides are capable of catalyzing the conversion of trifluoroacetone (compound (1)) and nitromethane (compound (2)) to compound (4) via intermediate compound (3), as depicted in Scheme 1 below.

[0136]

[0137] Solution 1.

[0138] In some embodiments, the disclosure provides a nitroaldolase having improved activity in the production of compound (3) compared to a reference polypeptide. In some embodiments, the engineered nitroaldolase has the activity of Scheme 1.

[0139] As described herein, the engineered polypeptides exhibit stereoselectivity; therefore, the nitroaldolase enzymes of Scheme 1 can be used to establish one or more chiral centers of a product. The nitroaldolase enzymes of the present disclosure produce either the (R) or (S) enantiomer.

[0140] In some embodiments, the disclosure provides an engineered nitroaldolase having improved stereoselectivity for the (S)-β-nitroalcohol product (compound (3)) compared to a reference polypeptide. In some embodiments, the disclosure provides an engineered polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid reference sequence of SEQ ID NO: 2, 24, 496, 860, 1120 or 1348, and further comprising one or more amino acid residue differences compared to the reference amino acid sequence, wherein the engineered nitroaldolase polypeptide has increased enantioselectivity for compound (3).

[0141] In some embodiments, the present disclosure provides an engineered polypeptide comprising an amino acid sequence having at least 80% sequence identity with an amino acid reference sequence of SEQ ID NO: 2, 24, 496, 860, 1120 or 1348, and further comprising one or more amino acid residue differences compared to the reference amino acid sequence, wherein the engineered nitroaldolase polypeptide has increased activity in the conversion of compound (1) and compound (2) to compound (3).

[0142] Engineered nitroaldolase peptide

[0143] The present invention provides nitroaldolase polypeptides, polynucleotides encoding the polypeptides, methods for preparing the polypeptides, and methods for using the polypeptides. Where the description relates to a polypeptide, it is understood that it may describe a polynucleotide encoding the polypeptide.

[0144] Suitable reaction conditions for carrying out the desired reaction with the above-described improved properties of the engineered polypeptide can be determined taking into account the concentration or amount of the polypeptide, substrate, co-substrate, buffer, solvent, pH, conditions including temperature and reaction time and / or conditions under which the polypeptide is immobilized on a solid support, as further described below and in the Examples.

[0145] In some embodiments, exemplary engineered nitroaldolases with improved properties, particularly in terms of stereoselective conversion of compound (1) and compound (2) to compound (3), comprise an amino acid sequence having one or more residue differences compared to SEQ ID NO: 2, 24, 496, 860, 1120 or 1348 at the residue positions indicated in Table 2-1, Table 2-2, Table 3-1, Table 3-2, Table 4-1, Table 4-2, Table 5-1, Table 5-2, Table 6-1, Table 6-2, Table 7-1, and Table 7-2.

[0146] The structural and functional information of exemplary non-naturally occurring (or engineered) polypeptides of the present invention is based on the conversion of compound (1) and compound (2) to compound (3), the results of which are shown in Table 2-1, Table 2-2, Table 3-1, Table 3-2, Table 4-1, Table 4-2, Table 5-1, Table 5-2, Table 6-1, Table 6-2, Table 7-1 and Table 7-2 below, as further described in the Examples. The odd-numbered sequence identifiers (i.e., SEQ ID NOs) in these tables refer to the nucleotide sequence encoding the amino acid sequence provided by the even-numbered SEQ ID NOs in these tables. Exemplary sequences are provided in the electronic sequence listing file accompanying the present invention, which is hereby incorporated by reference herein. As indicated, the amino acid residue differences are based on comparisons with the reference sequences SEQ ID NOs: 2, 24, 496, 860, 1120 and / or 1348.

[0147] Based on the conversion of compound (1) and compound (2) to compound (3), a homologue of nitroaldolase (SEQ ID NO: 2) from Semen maculatum was selected. SEQ ID NO: 1 is a codon-optimized polynucleotide synthesized based on the polypeptide sequence of SEQ ID NO: 2 for expression in Escherichia coli.

[0148] The activity and stereoselectivity of each engineered nitroaldolase polypeptide relative to the reference polypeptide of SEQ ID NO: 2, 24, 496, 860, 1120 and / or 1348 was determined as 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 nitroaldolase, the results of which are provided in the Examples. In some embodiments, the SFP format provides a purer powder preparation of the engineered polypeptide and can contain up to about 30% of the total protein of the engineered polypeptide.

[0149] In some embodiments, specific enzyme properties are associated with residue differences at residue positions indicated herein compared to SEQ ID NO: 2, 24, 496, 860, 1120, and / or 1348. In some embodiments, residue differences that affect polypeptide expression can be used to increase expression of an engineered nitroaldolase.

[0150] In view of the guidance provided herein, it is also contemplated that any of the exemplary engineered polypeptides comprising even-numbered sequences of SEQ ID NOs: 4-1484 can be used as a starting amino acid sequence for synthesizing other engineered nitroaldolase polypeptides by, for example, subsequent rounds of evolution incorporating new combinations of various amino acid differences from other polypeptides in Table 2-1, Table 2-2, Table 3-1, Table 3-2, Table 4-1, Table 4-2, Table 5-1, Table 5-2, Table 6-1, Table 6-2, Table 7-1, and Table 7-2 and other residue positions described herein. Additional improvements can be generated by including amino acid differences at residue positions that remain unchanged throughout earlier rounds of evolution.

[0151] In some embodiments, the engineered nitroaldolase 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 sequence SEQ ID NO:2 and 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: NO:2 differs from one or more residues at a residue position selected from the group consisting of: 7 / 9, 7 / 9 / 33 / 68 / 72 / 158, 7 / 9 / 33 / 72 / 175 / 197 / 230, 7 / 9 / 33 / 72 / 230, 7 / 9 / 68 / 72 / 197, 7 / 9 / 158 / 175, 7 / 9 / 175, 7 / 9 / 197, 7 / 33, 7 / 68 / 72 / 175, 7 / 72, 7 / 72 / 97 / 175 / 230, 9, 9 / 11 / 33 / 72 / 197 / 230, 9 / 33 / 68 / 158 / 197, 9 / 33 / 72 / 230, 9 / 33 / 72 / 230 3 / 97 / 230、9 / 68 / 72、9 / 97 / 175 / 202 / 218、9 / 175、9 / 202、11、13、14 / 33 / 68 / 72 / 97、18、18 / 33 / 68 / 97 / 158 / 175 / 239、18 / 33 / 72 / 97 / 158 / 175、18 / 33 / 72 / 97 / 158 / 239、18 / 33 / 72 / 97 / 175 / 202、18 / 33 / 72 / 175 / 202 / 237 / 239、18 / 33 / 72 / 197 / 237、18 / 68 / 72、18 / 68 / 72 / 158 / 175 / 237 / 239、18 / 68 / 72 / 175 / 202 / 239、18 / 68 / 72 / 239、18 / 68 / 175 / 223 / 237 / 239、18 / 68 / 239、18 / 72 / 97 / 175 / 237 / 239、18 / 72 / 175 / 197、18 / 72 / 237、18 / 72 / 239、18 / 97 / 175 / 237 / 239、18 / 97 / 223 / 237 / 239、18 / 175、18 / 197 / 239、18 / 239、20 / 124、33 / 68 / 72、33 / 68 / 72 / 175 / 230, 33 / 68 / 223 / 239, 33 / 72, 33 / 72 / 97 / 197, 33 / 72 / 175, 33 / 175, 39, 44 / 97, 52, 68 / 72, 68 / 72 / 97, 68 / 72 / 97 / 197, 68 / 72 / 197, 68 / 72 / 197 / 239, 68 / 97 / 175, 68 / 97 / 175 / 197 / 223, 72 / 97 / 158 / 223 / 237, 72 / 175 / 197, 97 / 158 / 175 / 197, 97 / 175, 103, 121, 122, 124,125, 128, 133, 148, 175, 197 and 197 / 202. In some embodiments, the engineered nitroaldolase 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 sequence SEQ ID NO: 2 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 7V / 9V / 33V / 68L / 72E / 158Y, 7V / 9V / 33V / 72E / 175P / 197V / 230I, 7V / 9V / 33V / 72E / 230I, 7V / 9V / 68L / 72E / 197V, 7V / 9V / 158Y / 175P, 7V / 9V / 175P, 7V / 9V / 197V, 7V / 33V, 7V / 68L / 72E / 175P, 7V / 72E, 7V / 72E / 97I / 175P / 230I, 9V, 9V / 11G / 33V / 72E / 197V / 230I, 9V / 33V / 68L / 158Y / 197V, 9V / 33V / 72E / 230I, 9V / 33V / 97I / 230I, 9V / 68L / 72E, 9V / 97I / 175P / 202I / 218M, 9V / 175P, 9V / 202I, 11S, 13G, 14Y / 33V / 68L / 72E / 97I, 18C / 33V / 68L / 97I / 158Y / 175P / 239F, 18C / 33V / 72E / 97I / 158 Y / 175P, 18C / 33V / 72E / 97I / 158Y / 239F, 18C / 33V / 72E / 97I / 175P / 202I, 18C / 33V / 72E / 175P / 202I / 237P / 239F, 18C / 33V / 72E / 1 97V / 237P, 18C / 68L / 72E, 18C / 68L / 72E / 158Y / 175P / 237P / 239F, 18C / 68L / 72E / 175P / 202I / 239F, 18C / 68L / 72E / 239F, 18C / 68L / 175P / 223P / 237P / 239F、18C / 68L / 239F、18C / 72E / 97I / 175P / 237P / 239F、18C / 72E / 175P / 197V、18C / 72E / 237P、18C / 72E / 239F , 18C / 97I / 175P / 237P / 239F, 18C / 97I / 223P / 237P / 239F, 18C / 175P, 18C / 197V / 239F, 18C / 239F, 18I, 20H / 124C, 33V / 68L / 72E,33V / 68L / 72E / 175P / 230I, 33V / 68L / 223P / 239F, 33V / 72E, 33V / 72E / 97I / 197V, 33V / 72E / 175P, 33V / 175P, 39A, 3 9G, 44N / 97I, 52S, 68L / 72E, 68L / 72E / 97I, 68L / 72E / 97I / 197V, 68L / 72E / 197V, 68L / 72E / 197V / 239F, 68L / 97I / 17 197V, and 197V / 202I. In some embodiments, the engineered nitroaldolase 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 sequence SEQ ID NO:2 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: ID NO: 2 differs from one or more residues selected from the group consisting of I7V / I9V, I7V / I9V / A33V / I68L / K72E / F158Y, I7V / I9V / A33V / K72E / K175P / I197V / V230I, I7V / I9V / A33V / K72E / V230I, I7V / I9V / I68L / K72E / I197V, I7V / I9V / F158Y / K175P, I7V / I9V / K175P, I7V / I9V / I197V, I7V / A33V, I7V / I68L / K72E / K175P, I7V / K72E, I7V / K72E / V175P, 175P / V230I, I9V, I9V / T11G / A33V / K72E / I197V / V230I, I9V / A33V / I68L / F 158Y / I197V, I9V / A33V / K72E / V230I, I9V / A33V / V97I / V230I, I9V / I68L / K7 2E, I9V / V97I / K175P / V202I / Q218M, I9V / K175P, I9V / V202I, T11S, C13G, H 14Y / A33V / I68L / K72E / V97I, L18C / A33V / I68L / V97I / F158Y / K175P / I239F,L18C / A33V / K72E / V97I / F158Y / K175P, L18C / A33V / K72E / V97I / F158Y / I23 9F、L18C / A33V / K72E / V97I / K175P / V202I、L18C / A33V / K72E / K175P / V202I / I237P / I239F, L18C / A33V / K72E / I197V / I237P, L18C / I68L / K72E, L18C / I6 8L / K72E / F158Y / K175P / I237P / I239F, L18C / I68L / K72E / K175P / V202I / I23 9F、L18C / I68L / K72E / I239F、L18C / I68L / K175P / K223P / I237P / I239F、L18 C / I68L / I239F, L18C / K72E / V97I / K175P / I237P / I239F, L18C / K72E / K175P / I197V, L18C / K72E / I237P, L18C / K72E / I239F, L18C / V97I / K175P / I237P / I2 39F, L18C / V97I / K223P / I237P / I239F, L18C / K175P, L18C / I197V / I239F, L1 8C / I239F, L18I, Y20H / V124C, A33V / I68L / K72E, A33V / I68L / K72E / K175P / V230I, A33V / I68L / K223P / I239F, A33V / K72E, A33V / K72E / V97I / I197V, A33 V / K72E / K175P, A33V / K175P, V39A, V39G, D44N / V97I, G52S, I68L / K72E, I6 8L / K72E / V97I, I68L / K72E / V97I / I197V, I68L / K72E / I197V, I68L / K72E / I1 97V / I239F, I68L / V97I / K175P, I68L / V97I / K175P / I197V / K223P, K72E / V9 7I / F158Y / K223P / I237P, K72E / K175P / I197V, V97I / F158Y / K175P / I197V, V 97I / K175P, H103M, H103S, F121Y, S122C, S122Q, V124C, V124E, V124T, V124Y, F125R, W128H, W128Y, F133A, F133C, L148M, K175P, I197V, and I197V / V202I.

[0152] In some embodiments, the engineered nitroaldolase 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 sequence SEQ ID NO:2 and 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: NO:2 differs from one or more residues at a residue position selected from the group consisting of: 7 / 11 / 33 / 72 / 97, 7 / 11 / 33 / 97, 7 / 11 / 72, 7 / 11 / 158, 7 / 11 / 237, 7 / 33 / 72 / 97 / 158 / 175 / 197 / 237 / 239, 9 / 11 / 33, 9 / 11 / 33 / 72, 9 / 11 / 72, 9 / 33 / 68 / 97 / 237 / 239, 9 / 72 / 97 / 158 / 237 / 239, 9 / 218 / 239, 10, 11, 12, 13, 14, 18, 33 / 68 / 72 / 97 / 237 / 239, 33 / 72 / 158 / 239, 33 / 72 / 158 / 239, 9, 40, 41, 48, 49, 51, 54, 54 / 126, 57, 68 / 175 / 239, 82, 84, 85, 86, 103, 104, 105, 107, 115, 117, 119, 119 / 120, 120, 122, 123, 124, 127, 130, 131, 133, 134, 137 / 145, 144, 145, 146, 147, 148, 149, 150, 152, 157, 158, 159, 169, 173, 174, 175, 180, 181, 183, 203, 204, 208, 209, 210, 215, 218, 236 and 237. In some embodiments, the engineered nitroaldolase 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:2 and one or more residue differences compared to SEQ ID NO:2 selected from the group consisting of: 7V / 11G / 33V / 72E / 97I, 7V / 11G / 33V / 97I, 7V / 11G / 72E, 7V / 11G / 158Y, 7V / 11G / 237P, 7V / 33V / 72E / 97I / 158Y / 175P / 197V / 237P / 239F, 9V / 11G / 33V / 72E / 97I / 158Y / 175P / 197V / 237P / 239F, 9V / 11G / 33V / 72E / 97I / 158Y / 175P / 197V / 237P / 239F, 9V / 11G / 33V / 72E / 97I V / 11G / 33V, 9V / 11G / 33V / 72E, 9V / 11G / 72E, 9V / 33V / 68L / 97I / 237P / 239F, 9V / 72 E / 97I / 158Y / 237P / 239F, 9V / 218M / 239F, 10G, 10P, 10R, 11R, 11S, 12K, 13L, 14R,18S, 18T, 33V / 68L / 72E / 97I / 237P / 239F, 33V / 72E / 158Y / 239F, 39R, 39W, 40L, 40S, 40T, 40V, 41C, 4 8W, 49Q, 49R, 51A, 51Q, 51S, 51T, 54N / 126I, 54Y, 57N, 68L / 175P / 239F, 82T, 84L, 85A, 85L, 85M, 85Q, 86W, 103P, 104A, 104G, 104S, 105D, 107G, 107W, 115C, 115K, 115L, 115V, 117C, 117L, 119L, 119N / 12 0F, 120C, 120L, 120Y, 122G, 122T, 123A, 123P, 123T, 124P, 127A, 127L, 127M, 130M, 130V, 131L, 133W , 134D, 134F, 134L, 134R, 134V, 134W, 137I / 145V, 144L, 145F, 145L, 145P, 146D, 146G, 146N, 147R, 148P, 148V, 149W, 150I, 150L, ​​150V, 152P, 157V, 158I, 158L, 158M, 158R, 158W, 158Y, 159G, 159H, 15 9L, 169G, 169I, 169S, 173E, 173Y, 174C, 174G, 175D, 180G, 180V, 181C, 183K, 203Q, 204E, 204R, 204V, 208A, 208C, 208E, 208G, 208L, 208T, 208V, 209C, 210V, 215L, 218T, 236G, 236L, 237L, 237S and 237W. In some embodiments, the engineered nitroaldolase 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:2 and 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:2. NO:2 has one or more residue differences selected from the group consisting of: I7V / T11G / A33V / K72E / V97I, I7V / T11G / A33V / V97I, I7V / T11G / K72E, I7V / T11G / F158Y, I7V / T11G / I237P, I7V / A33V / K72E / V97I / F158Y / K175P / I197V / I237P / I239F, I9V / T11G / A33V, I9V / T11G / A33V / K72E, I9V / T11G / K72E,I9V / A33V / I68L / V97I / I237P / I239F, I9V / K72E / V97I / F158Y / I237P / I239F, I9V / Q218M / I239F, H10G, H10P, H10R, T11R, T11S, I12K, C13L , H14R, L18S, L18T, A33V / I68L / K72E / V97I / I237P / I239F, A33V / K72E / F158Y / I239F, V39R, V39W, A40L, A40S, A40T, A40V, S41C, L48W, E49Q , E49R, I51A, I51Q, I51S, I51T, W54N / T126I, W54Y, Y57N, I68L / K175P / I239F, G82T, I84L, N85A, N85L, N85M, N85Q, I86W, H103P, N104A, N1 04G, N104S, A105D, M107G, M107W, A115C, A115K, A115L, A115V, V117C, V117L, K119L, K119N / K120F, K120C, K120L, K120Y, S122G, S122T, E1 23A, E123P, E123T, V124P, D127A, D127L, D127M, D130M, D130V, S131L, F133W, S134D, S134F, S134L, S134R, S134V, S134W, T137I / A145V, T 144L, A145F, A145L, A145P, V146D, V146G, V146N, E147R, L148P, L148V, G149W, D150I, D150L, ​​D150V, T152P, I157V, F158I, F158L, F158M, F 158R, F158W, F158Y, S159G, S159H, S159L, A169G, A169I, A169S, V173E, V173Y, R174C, R174G, K175D, E180G, E180V, Q181C, L183K, Y203Q, G204E, G204R, G204V, Q208A, Q208C, Q208E, Q208G, Q208L, Q208T, Q208V, I209C, F210V, Q215L, Q218T, K236G, K236L, I237L, I237S, and I237W.

[0153] In some embodiments, the engineered nitroaldolase 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:24 and one or more residue differences compared to SEQ ID NO:24 at residue positions selected from the group consisting of: 9 / 39 / 122 / 157, 18, 18 / 33 / 39 / 230, 18 / 33 / 175, 18 / 39 / 44 / 118, 18 / 175, 20, 33 / 39, 33 / 39 / 44 / 118 / 121, 33 / 39 / 44 / 121, 33 / 39 / 44 / 152, 33 / 39 / 44 / 153, 33 / 39 / 44 / 114, 33 / 39 / 44 / 155, 33 / 39 / 44 / 123, 33 / 39 / 44 / 157, 33 / 39 / 44 / 123 / 175, 33 / 39 / 44 / 197, 33 / 39 / 44 / 210 / 230, 33 / 39 / 118 / 121 / 175, 33 / 39 / 121 / 175, 33 / 39 / 197 / 210, 33 / 39 / 210, 33 / 39 / 210 / 230, 33 / 118 / 121, 33 / 118 / 121 / 175, 39, 39 / 44 / 103, 39 / 44 / 118、39 / 44 / 152 / 230、39 / 44 / 175 / 197 / 230、39 / 103、39 / 103 / 125、39 / 103 / 125 / 127 / 146、39 / 103 / 125 / 127 / 146 / 150、39 / 103 / 125 / 146、39 / 103 / 127 / 150、39 / 103 / 150、39 / 118 / 121、3 9 / 197, 39 / 197 / 210, 43, 44 / 103, 81, 103, 103 / 125 / 127, 103 / 125 / 146, 103 / 127, 103 / 150, 118 / 121, 118 / 121 / 175, 118 / 121 / 197, 121, 152, 152 / 197, 154, 159, 163, 175, 210, 232 and 238. In some embodiments, the engineered nitroaldolase 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: 24 and 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: 24.NO:24 has one or more residue differences selected from the group consisting of: 9V / 39W / 122T / 157V, 18I, 18I / 33V / 39W / 230I, 18I / 33V / 175P, 18I / 39W / 44N / 118L, 18I / 175P, 20H, 33V / 39W, 33V / 39W / 44N / 118L / 121Y, 33V / 39W / 44N / 121Y, 33V / 39W / 44N / 152F, 33V / 39W / 44N / 175P, 33V / 39W / 44N / 197V , 33V / 39W / 44N / 210M / 230I, 33V / 39W / 118L / 121Y / 175P, 33V / 39W / 121Y / 175P, 33V / 39W / 197V / 210M, 33V / 39W / 210M, 33V / 3 9W / 210M / 230I, 33V / 118L / 121W / 175P, 33V / 118L / 121Y, 39W, 39W / 44N / 118L, 39W / 44N / 152F / 230I, 39W / 44N / 175P / 197V / 23 0I、39W / 44Y / 103S、39W / 103M、39W / 103M / 125R / 146L、39W / 103M / 150P、39W / 103Q、39W / 103S、39W / 103S / 125P / 127G / 146L / 150P、39W / 103S / 125R、39W / 103S / 125R / 127G / 146L、39W / 103S / 127G / 150P、39W / 118L / 121Y、39W / 197V、39W / 197V / 210M、43 M, 43Q, 43R, 43S, 43T, 44N / 103S, 81F, 103L, 103M, 103M / 125P / 146L, 103M / 127G, 103Q, 103S, 103S / 125P / 127G, 103S / 150P, 103V, 118L / 121Y, 118L / 121Y / 175P, 118L / 121Y / 197V, 121Y, 152A / 197V, 152F, 154I, 159Q, 163P, 175P, 210M, 232G, and 238M. In some embodiments, the engineered nitroaldolase 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 sequence SEQ ID NO: 24 and 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: 24.NO:24 has one or more residue differences selected from the following: I9V / V39W / S122T / I157V, L18I, L18I / A33V / V39W / V230I, L18I / A33V / K175P, L18I / V39W / D44N / Y118L, L18I / K175P, Y20H, A33V / V39W, A33V / V39W / D44N / Y118L / F121Y, A33V / V39W / D44N / F121Y, A33V / V39W / D44N / T152F, A33V / V39W / D44N / K175P, A33V / V39W / D44N / I197V , A33V / V39W / D44N / F210M / V230I, A33V / V39W / Y118L / F121Y / K175P, A33V / V39W / F121Y / K175P, A33V / V39W / I197V / F210M, A33V / V39W / F210M, A33V / V 39W / F210M / V230I, A33V / Y118L / F121W / K175P, A33V / Y118L / F121Y, V39W, V39W / D44N / Y118L, V39W / D44N / T152F / V230I, V39W / D44N / K175P / I197V / V 230I, V39W / D44Y / H103S, V39W / H103M, V39W / H103M / F125R / V146L, V39W / H 103M / D150P, V39W / H103Q, V39W / H103S, V39W / H103S / F125P / D127G / V146L / D150P、V39W / H103S / F125R、V39W / H103S / F125R / D127G / V146L、V39W / H10 3S / D127G / D150P, V39W / Y118L / F121Y, V39W / I197V, V39W / I197V / F210M, I 154I, S159Q, I163P, K175P, F210M, S232G, and Q238M.

[0154] In some embodiments, the engineered nitroaldolase 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: 24 and 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: 24. NO:24 differs from one or more residues at a residue position selected from the group consisting of: 7, 9 / 13, 9 / 13 / 39, 9 / 13 / 39 / 68 / 72 / 239, 9 / 13 / 39 / 68 / 239, 9 / 13 / 39 / 72 / 122, 9 / 13 / 39 / 97 / 239, 9 / 13 / 68 / 72 / 97 / 239, 9 / 13 / 68 / 97 / 134 / 239, 9 / 13 / 68 / 97 / 239, 9 / 13 / 72, 9 / 13 / 134, 11, 12 / 42 / 125 / 127, 12 / 103, 12 / 103 / 150, 13 / 103 / 151, 13 / 103 / 152, 13 / 103 / 153, 13 / 103 / 154, 13 / 103 / 155, 13 / 103 / 156, 13 / 103 / 157, 13 / 103 / 158, 13 / 103 / 159, 13 / 103 / 159 , 13 / 39, 13 / 39 / 68, 13 / 39 / 68 / 72 / 97, 13 / 39 / 72 / 97 / 122 / 134 / 223, 13 / 39 / 97 / 239, 13 / 39 / 239, 13 / 68, 13 / 68 / 72 / 97 / 122 / 239, 16, 18 / 33 / 39 / 44 / 118 / 121 / 175 / 197 / 210, 18 / 33 / 39 / 44 / 121, 18 / 33 / 39 / 112 / 118 / 121 / 197, 18 / 33 / 39 / 121, 18 / 33 / 39 / 121 / 175, 18 / 33 / 39 / 121 / 175 / 197、18 / 33 / 39 / 121 / 175 / 210、18 / 33 / 39 / 121 / 210、18 / 33 / 118 / 121、18 / 33 / 121、18 / 33 / 121 / 197、18 / 33 / 121 / 210 / 230、18 / 39 / 44 / 118 / 121 / 230、18 / 39 / 44 / 121 / 175 / 210、18 / 39 / 118 / 121、18 / 39 / 118 / 121 / 210、18 / 39 / 121 / 175、18 / 39 / 121 / 175 / 21 0、18 / 39 / 121 / 197 / 210、18 / 118 / 121、18 / 118 / 121 / 175、18 / 118 / 121 / 210、18 / 118 / 121 / 230、18 / 121、18 / 121 / 175、18 / 121 / 210、18 / 121 / 230、20、21、33 / 39 / 44 / 118 / 121、33 / 39 / 44 / 118 / 121 / 210 / 230、33 / 39 / 44 / 121、33 / 39 / 44 / 121 / 152、33 / 39 / 44 / 121 / 175 / 210、33 / 39 / 44 / 121 / 230、33 / 39 / 73 / 121 / 197、33 / 39 / 118 / 121 / 175、33 / 39 / 118 / 121 / 175 / 230、33 / 39 / 121、33 / 39 / 121 / 175、33 / 39 / 121 / 210 / 230、33 / 118 / 121、33 / 118 / 121 / 175、33 / 118 / 121 / 197、33 / 121、33 / 121 / 230、37 / 91、39 / 44 / 1 : 18 / 121 / 175 / 210, 39 / 68 / 72 / 239, 39 / 118 / 121, 39 / 118 / 121 / 210, 39 / 121, 39 / 121 / 210 / 230, 43, 44, 76, 79, 101, 118 / 121, 118 / 121 / 175, 118 / 121 / 197, 121, 121 / 175 / 230, 121 / 210, 148, 153, 159, 161, 172, 200, 207, 233, 245 and 248. In some embodiments, the engineered nitroaldolase 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: 24 and 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: 24. NO:24 has one or more residue differences selected from the group consisting of: 7E, 9V / 13G, 9V / 13G / 39W, 9V / 13G / 39W / 68L / 72E / 239F, 9V / 13G / 39W / 68L / 239F, 9V / 13G / 39W / 72E / 122T, 9V / 13G / 39W / 97I / 239F, 9V / 13G / 68L / 72E / 97I / 239F, 9V / 13G / 68L / 97I / 134D / 239F, 9V / 13G / 68L / 97I / 239F, 9V / 13G / 72E, 9V / 13G / 134D, 11N, 12A / 42W / 125R / 127G, 12A / 103S, 12A / 103S / 150P, 13F, 13G / 39W, 13G / 39W / 68L, 13G / 39W / 68L / 72E / 97 I、13G / 39W / 72E / 97I / 122T / 134D / 223P、13G / 39W / 97I / 239F、13G / 39W / 239F , 13G / 68L, 13G / 68L / 72E / 97I / 122T / 239F, 16M, 16V, 18C / 33V / 39W / 112R / 11 8L / 121W / 197V, 18C / 33V / 39W / 121W / 175P / 210M, 18C / 33V / 39W / 121W / 210M,18C / 33V / 39W / 121Y / 175P、18C / 33V / 118L / 121W、18C / 33V / 118L / 121Y、18C / 33V / 121Y、18C / 33V / 121Y / 197V、18C / 33V / 121Y / 210M / 230I、18C / 39W / 44N / 121Y / 175P / 210M、18C / 39W / 118L / 121W、18C / 39W / 121W / 175P / 210M、18C / 39W / 121W / 197V / 210M、18C / 118L / 121W、18C / 118L / 121Y、18C / 121V、18C / 121V / 175P、18C / 121W、18C / 121W / 175P、18C / 121W / 210M、18C / 121Y、18C / 121Y / 230I、18I / 33V / 39W / 44N / 118L / 121W / 175P / 197V / 210M、18I / 33V / 39W / 44N / 121T、18I / 33V / 39W / 121T / 175P、18I / 33V / 39W / 121V / 175P / 197V、18I / 33V / 39W / 121W、18I / 33V / 118L / 121W、18I / 39W / 44N / 118L / 121W / 230I、18I / 39W / 44N / 121V / 175P / 210M、18I / 39W / 118L / 121W / 210M、18I / 39W / 121V / 175P、18I / 118L / 121T、18I / 118L / 121W、18I / 118L / 121W / 175P、18I / 118L / 121W / 210M、18I / 118L / 121Y、18I / 118L / 121Y / 230I、18I / 121T / 175P、18I / 121W、18I / 121Y / 210M、20G、20K、20R、20T、21V、33V / 39G / 44N / 121W / 152A、33V / 39W / 44N / 118L / 121W / 210M / 230I、33V / 39W / 44N / 118L / 121Y、33V / 39W / 44N / 121V、33V / 39W / 44N / 121Y、33V / 39W / 44N / 121Y / 175P / 210M、33V / 39W / 44N / 121Y / 230I、33V / 39W / 73N / 121Y / 197V、33V / 39W / 118L / 121W / 175P、33V / 39W / 118L / 121Y / 175P、33V / 39W / 118L / 121Y / 175P / 230I、33V / 39W / 121V、33V / 39W / 121W、33V / 39W / 121W / 210M / 230I, 33V / 39W / 121Y / 175P, 33V / 118L / 121W / 175P, 33V / 118L / 121W / 197V, 33V / 118L / 121Y, 33V / 121W, 33V / 121Y / 230 I. 37L / 91G, 39G / 68L / 72E / 239F, 39W / 44N / 118L / 121W / 175P / 210M, 39W / 118L / 121W / 210M, 39W / 118L / 121Y, 39W / 118L / 121Y / 210M, 39W / 121 172A, 172E, 172G, 172Q, 172R, 172V, 200G, 207N, 233R, 245D, 245G, 248G, and 248S. In some embodiments, the engineered nitroaldolase 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 sequence SEQ ID NO: 24 and 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: 24. NO:24 has one or more residue differences selected from the group consisting of: I7E, I9V / C13G, I9V / C13G / V39W, I9V / C13G / V39W / I68L / K72E / I239F, I9V / C13G / V39W / I68L / I239F, I9V / C13G / V39W / K72E / S122T, I9V / C13G / V39W / V97I / I239F, I9V / C13G / I68L / K72E / V97I / I239F, I9V / C 13G / I68L / V97I / S134D / I239F, I9V / C13G / I68L / V97I / I239F, I9V / C13G / K72E, I9V / C13G / S134D, S11N, I12A / G42W / F125R / D127G, I12A / H103S, I12A / H103S / D150P, C13F, C13G / V39W, C13G / V39W / I68L, C13G / V39W / I68L / K72E / V97I,C13G / V39W / K72E / V97I / S122T / S134D / K223P、C13G / V39W / V97I / I239F、C13G / V39W / I239F、C13G / I68L、C13G / I68L / K72E / V97I / S122T / I239F、A16M、A16V、L18C / A33V / V39W / H112R / Y118L / F121W / I197V、L18C / A33V / V39W / F121W / K175P / F210M、L18C / A33V / V39W / F121W / F210M、L18C / A33V / V39W / F121Y / K175P、L18C / A33V / Y118L / F121W、L18C / A33V / Y118L / F121Y、L18C / A33V / F121Y、L18C / A33V / F121Y / I197V、L18C / A33V / F121Y / F210M / V230I、L18C / V39W / D44N / F121Y / K175P / F210M、L18C / V39W / Y118L / F121W、L18C / V39W / F121W / K175P / F210M、L18C / V39W / F121W / I197V / F210M、L18C / Y118L / F121W、L18C / Y118L / F121Y、L18C / F121V、L18C / F121V / K175P、L18C / F121W、L18C / F121W / K175P、L18C / F121W / F210M、L18C / F121Y、L18C / F121Y / V230I、L18I / A33V / V39W / D44N / Y118L / F121W / K175P / I197V / F210M、L18I / A33V / V39W / D44N / F121T、L18I / A33V / V39W / F121T / K175P、L18I / A33V / V39W / F121V / K175P / I197V、L18I / A33V / V39W / F121W、L18I / A33V / Y118L / F121W、L18I / V39W / D44N / Y118L / F121W / V230I、L18I / V39W / D44N / F121V / K175P / F210M、L18I / V39W / Y118L / F121W / F210M、L18I / V39W / F121V / K175P、L18I / Y118L / F121T、L18I / Y118L / F121W、L18I / Y118L / F121W / K175P、L18I / Y118L / F121W / F210M、L18I / Y118L / F121Y、L18I / Y118L / F121Y / V230I, L18I / F121T / K175P, L18I / F121W, L18I / F121Y / F210M, Y20G, Y20K, Y20R, Y20T, K21V, A33V / V39G / D44N / F121W / T152A, A33 V / V39W / D44N / Y118L / F121W / F210M / V230I, A33V / V39W / D44N / Y118L / F121Y , A33V / V39W / D44N / F121V, A33V / V39W / D44N / F121Y, A33V / V39W / D44N / F121 Y / K175P / F210M, A33V / V39W / D44N / F121Y / V230I, A33V / V39W / K73N / F121Y / I197V、A33V / V39W / Y118L / F121W / K175P、A33V / V39W / Y118L / F121Y / K175P、 A33V / V39W / Y118L / F121Y / K175P / V230I, A33V / V39W / F121V, A33V / V39W / F1 21W, A33V / V39W / F121W / F210M / V230I, A33V / V39W / F121Y / K175P, A33V / Y11 8L / F121W / K175P, A33V / Y118L / F121W / I197V, A33V / Y118L / F121Y, A33V / F1 21W, A33V / F121Y / V230I, D37L / E91G, V39G / I68L / K72E / I239F, V39W / D44N / Y118L / F121W / K175P / F210M, V39W / Y118L / F121W / F210M, V39W / Y118L / F121 Y, V39W / Y118L / F121Y / F210M, V39W / F121W, V39W / F121W / F210M / V230I, I43 E, I43Q, I43S, I43W, D44V, L76P, E79F, V101L, Y118L / F121W, Y118L / F121Y, Y118L / F121Y / K175P, Y118L / F121Y / I197V, F121V, F121W, F121Y, F121Y / K1 75P / V230I, F121Y / F210M, L148F, L153W, S159Q, S161M, L172A, L172E, L172G, L172Q, L172R, L172V, V200G, D207N, A233R, L245D, L245G, I248G, and I248S.

[0155] In some embodiments, the engineered nitroaldolase 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:496 and one or more residue differences compared to SEQ ID NO:496 at residue positions selected from the group consisting of: 43 / 103, 43 / 103 / 172 / 238, 43 / 103 / 238 / 241, 43 / 238, 81 / 232, 103, 103 / 159 / 238, 103 / 194 / 238, 103 / 238, 122, 232, 238, and 238 / 241. In some embodiments, the engineered nitroaldolase 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: 496 and 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: 496. NO:496 differs by one or more residues selected from the group consisting of 43E / 103S / 238M / 241R, 43M / 103L, 43Q / 103S / 238M / 241R, 43Q / 238M, 43S / 103S / 172R / 238M, 43S / 238M, 81F / 232G, 103L, 103S, 103S / 159Q / 238M, 103S / 194L / 238M, 103S / 238M, 103V, 122C, 122I, 122K, 122L, 122M, 122Q, 122R, 122T, 122V, 232G, 238M, and 238M / 241R.In some embodiments, the engineered nitroaldolase 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 sequence SEQ ID NO: 496 and one or more residue differences compared to SEQ ID NO: 496 selected from the group consisting of I43E / H103S / Q238M / K241R, I43M / H103L, I43Q / H103S / Q238M / K241R, I43Q / Q238M, I43S / H103S / L172R / Q238M, I43S / Q238M, G81F / S232G, H103S / Q238M / K241R. 3L, H103S, H103S / S159Q / Q238M, H103S / Y194L / Q238M, H103S / Q238M, H103V, S122C, S 122I, S122K, S122L, S122M, S122Q, S122R, S122T, S122V, S232G, Q238M and Q238M / K241R.

[0156] In some embodiments, the engineered nitroaldolase 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: 496 and 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: 496. NO:496 differs from one or more residues at a residue position selected from the group consisting of: 12, 43, 43 / 47 / 103, 43 / 47 / 103 / 232, 43 / 103, 43 / 103 / 121 / 172 / 241, 43 / 103 / 172, 43 / 103 / 172 / 238, 43 / 103 / 238, 43 / 103 / 238 / 241, 43 / 121, 43 / 194 / 238, 43 / 238 , 44 / 103 / 121 / 238, 44 / 103 / 238, 44 / 103 / 238 / 241, 44 / 121 / 238 / 241, 47 / 103, 63, 103, 103 / 121, 103 / 121 / 172 / 238, 103 / 159 / 238, 103 / 172 / 238, 103 / 194 / 238, 103 / 238, 122, 131, 146, 178, 180 and 221.In some embodiments, the engineered nitroaldolase 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:496 and one or more residue differences compared to SEQ ID NO:496 selected from the group consisting of: 12V, 43E / 103S / 121W / 172R / 241R, 43E / 103S / 238M, 43E / 103S / 238M / 241R, 43M / 47G / 103L, 43M / 47G / 103V, 43M / 103L, 43M / 103S, 43M / 103V, 43Q / 4 7G / 103S / 232G, 43Q / 103L / 172R, 43Q / 103S, 43Q / 103S / 238M / 241R, 43Q / 121W, 43Q / 238 M, 43S, 43S / 103L, 43S / 103S, 43S / 103S / 172R, 43S / 103S / 172R / 238M, 43S / 103S / 238M / 2 41R, 43S / 194L / 238M, 43S / 238M, 44V / 103S / 121W / 238M, 44V / 103S / 238M, 44V / 103S / 23 8M / 241R, 44V / 121W / 238M / 241R, 47G / 103L, 63P, 103L, 103S, 103S / 121W, 103S / 121W / 17 103S / 159Q / 238M, 103S / 172R / 238M, 103S / 194L / 238M, 103S / 238M, 103V, 122H, 122I, 122K, 122L, 122M, 122N, 122R, 122T, 122V, 131Y, 146I, 178A, 180L, 180V, and 221G.In some embodiments, the engineered nitroaldolase 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 sequence SEQ ID NO: 496 and 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: 496. One or more residue differences compared to NO:496 selected from the group consisting of: I12V, I43E / H103S / Y121W / L172R / K241R, I43E / H103S / Q238M, I43E / H103S / Q238M / K241R, I43M / Q47G / H103L, I43M / Q47G / H103V, I43M / H103L, I43M / H103S, I43M / H103V, I43Q / Q47G / H103S / S232G, I43Q / H103L / L172R, I43Q / H103S, I43Q / H103S / Q238M / K241R, I43Q / Y121W, I43Q / Q238M, I43S, I43S / H103L, I43S / H103S, I43S / H103S / L172R, I43S / H103S / L172R / Q238M, I43S / H103S / Q238M / K2 41R, I43S / Y194L / Q238M, I43S / Q238M, N44V / H103S / Y121W / Q238M, N44V / H103S / Q238M, N44V / H103S / Q23 8M / K241R, N44V / Y121W / Q238M / K241R, Q47G / H103L, T63P, H103L, H103S, H103S / Y121W, H103S / Y121W / L1 8M, H103S / S159Q / Q238M, H103S / L172R / Q238M, H103S / Y194L / Q238M, H103S / Q238M, H103V, S122H, S122I, S122K, S122L, S122M, S122N, S122R, S122T, S122V, S131Y, V146I, F178A, E180L, E180V, and N221G.

[0157] In some embodiments, the engineered nitroaldolase 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: 860 and 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: 860. NO:860 differs from NO:860 at one or more residue positions selected from the group consisting of: 12, 12 / 43 / 63 / 103 / 146 / 180 / 232 / 241, 12 / 43 / 63 / 103 / 146 / 241, 12 / 43 / 81 / 103 / 241, 12 / 43 / 81 / 146 / 232 / 241, 12 / 43 / 81 / 180 / 241, 12 / 43 / 81 / 232 / 241, 12 / 43 / 81 / 241, 12 / 43 / 103, 12 / 43 / 103 / 146 / 180 / 232 / 241, 12 / 43 / 103 / 146 / 180 / 241, 12 / 43 / 103 / 180 / 241, 12 / 43 / 103 / 241, 12 / 43 / 146 / 180 / 232 / 241, 12 / 43 / 180 / 241, 12 / 63 / 81 / 103 / 241, 12 / 81, 12 / 81 / 180 / 241, 12 / 103 / 146 / 241, 12 / 103 / 180 / 241, 12 / 146, 12 / 146 / 180 / 241, 12 / 180 / 232 / 241, 12 / 241, 18, 43 / 81 / 232 / 241, 43 / 103 / 146 / 180, 43 / 103 / 180 / 241, 43 / 103 / 241, 43 / 180 / 232, 43 / 232 / 241, 63 / 103 / 180 / 232, 78, 81, 81 / 103 / 146, 81 / 14 6 / 180 / 241, 81 / 146 / 241, 81 / 180 / 241, 84, 103, 103 / 146, 103 / 146 / 180, 103 / 146 / 180 / 241, 103 / 180, 103 / 180 / 241, 103 / 261, 144, 145, 146 / 180 / 232, 146 / 180 / 241, 146 / 241, 150, 166, 172, 180 / 232, 232 / 241 and 241. In some embodiments, the engineered nitroaldolase 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: 860 and one or more residue differences compared to SEQ ID NO: 860 selected from the group consisting of: 12V,12V / 43M / 63P / 103L / 146I / 180L / 232G / 241R、12V / 43M / 81F / 103L / 241R、12V / 43M / 81F / 146I / 232G / 241R、12V / 43M / 81F / 180V / 241R、12V / 43M / 81F / 232G / 241R、12V / 43M / 81F / 241R、12V / 43M / 103L / 180L / 241R、12V / 43M / 103L / 241R、12V / 43M / 103V / 146I / 180V / 241R、12V / 43M / 103V / 241R、12V / 43M / 146I / 180L / 232G / 241R、12V / 43M / 180L / 241R、12V / 43M / 180V / 241R、12V / 43Q / 63P / 103L / 146I / 241R、12V / 43Q / 103L / 146I / 180V / 232G / 241R、12V / 43Q / 103L / 146I / 180V / 241R、12V / 43Q / 103L / 180V / 241R、12V / 43Q / 103V、12V / 63P / 81F / 103L / 241R、12V / 81F、12V / 81F / 180V / 241R、12V / 103L / 146I / 241R、12V / 103L / 180L / 241R、12V / 103V / 180V / 241R、12V / 146I、12V / 146I / 180L / 241R、12V / 180L / 232G / 241R、12V / 180V / 232G / 241R、12V / 241R、18V、43M / 81F / 232G / 241R、43M / 103L / 146I / 180V、43M / 103L / 180V / 241R、43M / 103L / 241R、43M / 103V / 146I / 180V、43M / 180L / 232G、43M / 232G / 241R、43Q / 103V / 146I / 180L、63P / 103L / 180L / 232G、78A、81F / 103L / 146I、81F / 146I / 180L / 241R、81F / 146I / 241R、81F / 180L / 241R、81Q、84Y、103A、103L、103L / 146I、103L / 146I / 180V、103L / 180L、103L / 180V / 241R、103N、103T、103T / 261V、103V / 146I / 180L / 241R、144V、145V、146I / 180L / 241R、146I / 180V / 232G、146I / 241R、150H、166L、172L、172V, 180L / 232G, 232G / 241R and 241R. In some embodiments, the engineered nitroaldolase 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 sequence SEQ ID NO: 860 and 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: 860. NO:860 has one or more residue differences selected from the group consisting of: I12V, I12V / S43M / T63P / S103L / V146I / E180L / S232G / K241R, I12V / S43M / G81F / S103L / K241R, I12V / S43M / G81F / V146I / S232G / K241R, I12V / S43M / G81F / E180V / K241R, I12V / S43M / G81F / S232G / K241R, I12V / S43M / G81F / K241R, I12V / S43M / S103L / E180L / K241R, I12V / S43M / S103L / K241R, I12V / S43M / S103V / V146I / E180V / K241R, I12V / S43M / S103V / K241 R、I12V / S43M / V146I / E180L / S232G / K241R、I12V / S43M / E180L / K241R、I12V / S43M / E180V / K241R、I12V / S43Q / T63P / S103L / V146 I / K241R, I12V / S43Q / S103L / V146I / E180V / S232G / K241R, I12V / S43Q / S103L / V146I / E180V / K241R, I12V / S43Q / S103L / E180V / I 12V / S103L / E180L / K241R, I12V / S103V / E180V / K241R, I12V / V146I, I12V / V146I / E180L / K241R, I12V / E180L / S232G / K241R, I1 2V / E180V / S232G / K241R, I12V / K241R, L18V, S43M / G81F / S232G / K241R, S43M / S103L / V146I / E180V, S43M / S103L / E180V / K241R,S43M / S103L / K241R, S43M / S103V / V146I / E180V, S43M / E180L / S232G, S43M / S232G / K241R, S43Q / S103V / V146I / E180L, T63P / S103L / E1 80L / S232G, G78A, G81F / S103L / V146I, G81F / V146I / E180L / K241R, G81F / V146I / K241R, G81F / E180L / K241R, G81Q, I84Y, S103A, S103L, S103L / V146I, S103L / V146I / E180V, S103L / E180L, S103L / E180V / K241R, S103N, S103T, S103T / A261V, S103V / V146I / E180L / K241R, T144V, A145V, V146I / E180L / K241R, V146I / E180V / S232G, V146I / K241R, D150H, V166L, R172L, R172V, E180L / S232G, S232G / K241R, and K241R.

[0158] In some embodiments, the engineered nitroaldolase 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: 860 and 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: 860. NO:860 differs from NO:860 at one or more residue positions selected from the group consisting of: 9, 12, 12 / 43 / 63 / 103 / 146 / 180 / 232 / 241, 12 / 43 / 63 / 103 / 146 / 241, 12 / 43 / 103 / 146 / 180 / 232 / 241, 12 / 43 / 103 / 146 / 180 / 241, 12 / 43 / 103 / 146 / 180 / 241, 12 / 43 / 103 / 146 / 232 / 241, 12 / 43 / 103 / 146 / 241, 12 / 43 / 103 / 180 / 241, 12 / 43 / 146 / 180 / 232 / 241, 12 / 43 / 146 / 241, 12 / 43 / 180 / 232, 12 / 43 / 180 / 241, 12 / 63 / 103 / 146 / 232 / 241, 12 / 103 / 146 / 241, 12 / 103 / 180 / 241, 12 / 146, 12 / 146 / 180 / 241, 12 / 146 / 232 / 241, 12 / 146 / 241, 12 / 180, 12 / 180 / 232 / 241, 12 / 180 / 241, 12 / 241, 18, 43 / 63 / 103 / 180 / 232 / 241, 43 / 103 / 146 / 180, 43 / 103 / 180, 43 / 103 / 180 / 232 / 241, 43 / 103 / 180 / 241, 43 / 180 / , 180 / 232, 180 / 232 / 241, 180 / 241, 208, 211 and 212. In some embodiments, the engineered nitroaldolase 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: 860 and one or more residue differences compared to SEQ ID NO: 860 selected from the group consisting of 9V, 12V, 12V / 43M / 63P / 103L / 146I / 180L / 232G / 241R, 12V / 43M / 103L / 146I / 241R,12V / 43M / 103L / 180L / 241R、12V / 43M / 103V / 146I / 180V / 241R、12V / 43M / 103V / 146I / 232G / 241R、12V / 43M / 146I / 180L / 232G / 241R、12V / 43M / 180L / 232G、12V / 43M / 180L / 241R、12V / 43Q / 63P / 103V / 146I / 241R、12V / 43Q / 103L / 146I / 180V / 232G / 241R、12V / 43Q / 103L / 146I / 180V / 241R、12V / 43Q / 103L / 146I / 241R、12V / 43Q / 103L / 180V / 241R、12V / 43Q / 146I / 241R、12V / 63P / 103L / 146I / 232G / 241R、12V / 103L / 180L / 241R、12V / 103L / 180V / 241R、12V / 103V / 146I / 241R、12V / 103V / 180L / 241R、12V / 103V / 180V / 241R、12V / 146I、12V / 146I / 180L / 241R、12V / 146I / 232G / 241R、12V / 146I / 241R、12V / 180L、12V / 180L / 232G / 241R、12V / 180L / 241R、12V / 180V、12V / 180V / 232G / 241R、12V / 180V / 241R、12V / 241R、18V、43M / 63P / 103L / 180V / 232G / 241R、43M / 103L / 146I / 180V、43M / 103L / 180V、43M / 103L / 180V / 241R、43M / 103V / 146I / 180V、43M / 180L / 232G、43Q / 103L / 180V / 232G / 241R、43Q / 103V / 146I / 180L、43Q / 103V / 180L / 241R、43Q / 103V / 180V / 232G / 241R、63P / 103L / 180L / 232G、103A、103L、103L / 146I / 180V、103L / 146I / 180V / 241R、103L / 180L、103L / 180V / 241R、103L / 241R、103N、103T、103V / 146I / 180L / 241R、103V / 180V / 241R、118L、118V、119H、122H、144H、144V、145V、146I / 180L / 241R、154R、160L、172C、172L、172V、180L / 232G, 180L / 241R, 180V / 232G, 180V / 232G / 241R, 208F, 211F and 212P. In some embodiments, the engineered nitroaldolase 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 sequence SEQ ID NO: 860 and 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: 860. One or more residue differences compared to NO:860 selected from the group consisting of: I9V, I12V, I12V / S43M / T63P / S103L / V146I / E180L / S232G / K241R, I12V / S43M / S103L / V146I / K241R, I12V / S43M / S103L / E180L / K241R, I12V / S43M / S103V / V146I / E180V / K241R, I12V / S43M / S103V / V146I / S232G / K241R , I12V / S43M / V146I / E180L / S232G / K241R, I12V / S43M / E180L / S232G, I12V / S43M / E180L / K241R, I12V / S43Q / T63P / S103V / V 146I / K241R, I12V / S43Q / S103L / V146I / E180V / S232G / K241R, I12V / S43Q / S103L / V146I / E180V / K241R, I12V / S43Q / S103L / V146I / K241R, I12V / S43Q / S103L / E180V / K241R, I12V / S43Q / V146I / K241R, I12V / T63P / S103L / V146I / S232G / K241R, I12V / S103L / E180L / K241R, I12V / S103L / E180V / K241R, I12V / S103V / V146I / K241R, I12V / S103V / E180L / K241R, I12V / S103V / E1 80V / K241R, I12V / V146I, I12V / V146I / E180L / K241R, I12V / V146I / S232G / K241R, I12V / V146I / K241R, I12V / E180L, I12V / E 180L / S232G / K241R, I12V / E180L / K241R, I12V / E180V, I12V / E180V / S232G / K241R, I12V / E180V / K241R, I12V / K241R, L18V,S43M / T63P / S103L / E180V / S232G / K241R, S43M / S103L / V146I / E180V, S43M / S103L / E180 V, S43M / S103L / E180V / K241R, S43M / S103V / V146I / E180V, S43M / E180L / S232G, S43Q / S1 03L / E180V / S232G / K241R, S43Q / S103V / V146I / E180L, S43Q / S103V / E180L / K241R, S43Q / S103V / E180V / S232G / K241R, T63P / S103L / E180L / S232G, S103A, S103L, S103L / V146I / E180V, S103L / V146I / E180V / K241R, S103L / E180L, S103L / E180V / K241R, S103L / K241R, S103N, S103T, S103V / V146I / E180L / K241R, S103V / E180V / K241R, Y118L, Y118V, K119H, S122H, T144H, T144V, A145V, V146I / E180L / K241R, A154R, N160L, R172C, R172L, R172V, E180L / S232G, E180L / K241R, E180V / S232G, E180V / S232G / K241R, Q208F, S211F, and R212P.

[0159] In some embodiments, the engineered nitroaldolase 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: 1120 and one or more residue differences compared to SEQ ID NO: 1120 at residue positions selected from the group consisting of: 3, 7, 7 / 158, 7 / 158 / 175, 7 / 158 / 197 / 202, 27 / 43 / 115 / 172, 27 / 43 / 172, 29, 32, 43 / 110 / 115 / 172, 44, 46, 48 / 172, 55, 59, 67, 71, 72, 73, 94 / 103 / 118 / 146, 1 03, 103 / 144 / 146 / 172 / 180, 103 / 146 / 154 / 172 / 180 / 232, 103 / 154, 118, 118 / 144 / 215, 118 / 172 / 180, 137, 142, 146, 146 / 154, 154, 158, 172 / 190, 184, 198, 205, 216, 223, 226, 229, 247, 261 and 263.In some embodiments, the engineered nitroaldolase 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: 1120 and 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: 1120. NO: 1120 has one or more residue differences selected from the group consisting of: 3D, 7V, 7V / 158Y, 7V / 158Y / 175P, 7V / 158Y / 197V / 202I, 27E / 43I / 115S / 172L, 27E / 43I / 172L, 29G, 29R, 32G, 32R, 43I / 110T / 115S / 172L, 44S, 46H, 46V, 48I / 172L, 55A, 55G, 55R, 55S, 59G, 59T, 67N, 71S, 72G, 73R, 94Q / 103L / 118V / 146I, 103L, 103L / 144V / 146I / 172L / 180V ,103L / 146I / 154R / 172L / 180V / 232G, 103L / 154R, 118V, 118V / 144V / 215H, 118V / 172L / 180V, 137M, 137R, 142V, 146I, 146I / 154R, 154R, 158Y, 172L / 19 0S, 184S, 198L, 205G, 205L, 216M, 216V, 223A, 223C, 223E, 223L, 223M, 223S, 226L, 226V, 229L, 229V, 229W, 247V, 261G, 261H, 263E, 263G, 263L and 263S.In some embodiments, the engineered nitroaldolase 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 sequence SEQ ID NO: 1120 and one or more residue differences compared to SEQ ID NO: 1120 selected from the group consisting of S3D, I7V, I7V / F158Y, I7V / F158Y / K175P, I7V / F158Y / I197V / V202I, Q27E / S43I / A115S / R172L, Q27E / S43I / R172L, A29G, A29R, N32G, N32R, S43I / I117V / V202I 0T / A115S / R172L, N44S, R46H, R46V, L48I / R172L, E55A, E55G, E55R, E55S, E59G, E59T, S67 N, G71S, K72G, K73R, P94Q / V103L / Y118V / V146I, V103L, V103L / T144V / V146I / R172L / L180 V, V103L / V146I / A154R / R172L / L180V / S232G, V103L / A154R, Y118V, Y118V / T144V / Q215H, Y118V / R172L / L180V, T137M, T137R, T142V, V146I, V146I / A154R, A154R, F158Y, R172L / T1 90S, D184S, R198L, E205G, E205L, L216M, L216V, K223A, K223C, K223E, K223L, K223M, K223S, K226L, K226V, C229L, C229V, C229W, Q247V, A261G, A261H, A263E, A263G, A263L, and A263S.

[0160] In some embodiments, the engineered nitroaldolase 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:1120 and one or more residue differences compared to SEQ ID NO:1120 at residue positions selected from the group consisting of: 73, 80, 103 / 146 / 209, 103 / 172 / 209, 118 / 119 / 172 / 209, 135, 144 / 209 / 232, 172 / 180 / 209, 181 and 223. In some embodiments, the engineered nitroaldolase 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: 1120 and one or more residue differences compared to SEQ ID NO: 1120 selected from the group consisting of 73R, 80A, 80G, 103L / 146I / 209C, 103L / 172C / 209C, 118V / 119H / 172C / 209C, 135V, 144V / 209C / 232G, 172C / 180V / 209C, 181S, and 223E. In some embodiments, the engineered nitroaldolase 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 sequence SEQ ID NO: 1120 and one or more residue differences compared to SEQ ID NO: 1120 selected from the group consisting of K73R, S80A, S80G, V103L / V146I / I209C, V103L / R172C / I209C, Y118V / K119H / R172C / I209C, N135V, T144V / I209C / S232G, R172C / L180V / I209C, Q181S, and K223E.

[0161] In some embodiments, the engineered nitroaldolase 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: 1348 and 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: 1348. NO:1348 differs from one or more residues at a residue position selected from the group consisting of: 2, 7 / 46, 39, 46, 46, 46 / 55, 46 / 55 / 72, 46 / 55 / 92, 46 / 55 / 118, 46 / 55 / 226 / 257 / 263, 46 / 55 / 263, 46 / 70, 46 / 70 / 92 / 118 / 261 / 263, 46 / 70 / 175 / 263, 46 / 72, 46 / 118, 55 / 257 / 261, 55 / 261 / 263, 55 / 263, 105, 118, 118 / 226 / 261 / 263, and 263. In some embodiments, the engineered nitroaldolase 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: 1348 and one or more residue differences compared to SEQ ID NO: 1348 selected from the group consisting of 2G, 7V / 46H, 39F, 39G, 39M, 39Q, 39S, 39T, 39V, 46H, 46H / 55R, 46H / 55R / 72C, 46H / 55R / 92A, 46H / 55R / 118V, 46H / 55R / 226V / 257Q / 263L, 46H / 55R / 264V / 265Q / 266V 3L, 46H / 70N, 46H / 70N / 92A / 118V / 261G / 263L, 46H / 70N / 175P / 263G, 46H / 72C, 46H / 118V , 55R / 257Q / 261G, 55R / 261G / 263I, 55R / 263G, 105G, 118V, 118V / 226V / 261G / 263I and 263G.In some embodiments, the engineered nitroaldolase 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 sequence SEQ ID NO: 1348 and an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1348. ID NO: 1348 has one or more residue differences selected from the group consisting of: V2G, I7V / R46H, W39F, W39G, W39M, W39Q, W39S, W39T, W39V, R46H, R46H / E55R, R46H / E55R / K72C, R46H / E55R / K92A, R46H / E55R / Y118V, R46H / E55R / K226V / S257Q / A263L, R46H / E55R / A2 63L, R46H / E70N, R46H / E70N / K92A / Y118V / A261G / A263L, R46H / E70N / K175P / A263G, R46H / K72C, R46H / Y118V, E55R / S257Q / A261G, E55R / A261G / A263I, E55R / A263G, A105G, Y118V, Y118V / K226V / A261G / A263I, and A263G.

[0162] In some embodiments, the engineered nitroaldolase 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: 1348 and 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: 1348. NO:1348 differs from one or more residues at a residue position selected from the group consisting of: 2, 7 / 46, 7 / 46 / 175, 22 / 46 / 55 / 118, 27 / 46 / 72, 27 / 46 / 146 / 158 / 202, 27 / 46 / 175, 27 / 118, 29 / 70 / 118, 39, 46, 46 / 55, 46 / 55 / 59 / 261 / 263, 46 / 55 / 72, 46 / 55 / 92, 46 / 55 / 118, 46 / 55 / 118 / 263, 46 / 55 / 146, 46 / 55 / 146 / 202, 46 / 55 / 226 / 257 / 263, 46 / 55 / 263. 6 / 70, 46 / 70 / 92 / 118 / 261 / 263, 46 / 70 / 175 / 263, 46 / 72, 46 / 92, 46 / 92 / 175, 46 / 118, 46 / 146, 46 / 146 / 257 / 263, 55 / 70 / 118, 55 / 257 / 261, 55 / 257 / 263, 55 / 261 / 263, 55 / 263, 70, 92 / 118, 105, 118, 118 / 226 / 261 / 263, 146 / 257 / 263, 146 / 261 / 263, 192 / 257 / 261, 202, 257 / 261 / 263, 261 / 263 and 263.In some embodiments, the engineered nitroaldolase 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: 1348 and one or more residue differences compared to SEQ ID NO: 1348 selected from the group consisting of 2G, 7V / 46H, 7V / 46H / 175P, 22V / 46H / 55R / 118V, 27G / 46H / 72C, 27G / 46H / 146I / 158Y / 202I, 27G / 46H / 175P, 27G / 118V, 29V / 70N / 118V, 39M, 39Q, 39S, 39T, 39V, 46H, 46H / 55R, 46H / 55R / 59T / 261G / 263L, 46H / 55R / 72C, 46H / 55R / 92A, 46H / 55R / 118V, 46H / 55 R / 118V / 263S, 46H / 55R / 146I, 46H / 55R / 146I / 202I, 46H / 55R / 226V / 257Q / 263L, 46H / 55R / 263 L, 46H / 70N, 46H / 70N / 92A / 118V / 261G / 263L, 46H / 70N / 175P / 263G, 46H / 72C, 46H / 92A, 46H / 92 A / 175P, 46H / 118V, 46H / 146I, 46H / 146I / 257Q / 263I, 55R / 70N / 118V, 55R / 257Q / 261G, 55R / 25 7Q / 263L, 55R / 261G / 263I, 55R / 263G, 70N, 92A / 118V, 105G, 118V, 118V / 226V / 261G / 263I, 146I / 257Q / 263S, 146I / 261G / 263L, 192V / 257Q / 261G, 202I, 257Q / 261G / 263G, 261G / 263I, and 263G.In some embodiments, the engineered nitroaldolase 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 sequence SEQ ID NO: 1348 and 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: 1348. NO: 1348 has one or more residue differences selected from the group consisting of: V2G, I7V / R46H, I7V / R46H / K175P, L22V / R46H / E55R / Y118V, Q27G / R46H / K72C, Q27G / R46H / V146I / F158Y / V202I, Q27G / R46H / K175P, Q27G / Y118V, A29V / E70N / Y118V, W39M, W39Q, W39S, W39T, W39V, R4 6H, R46H / E55R, R46H / E55R / E59T / A261G / A263L, R46H / E55R / K72C, R46H / E55R / K92A, R46H / E55R / Y118V, R46H / E5 5R / Y118V / A263S, R46H / E55R / V146I, R46H / E55R / V146I / V202I, R46H / E55R / K226V / S257Q / A263L, R46H / E55R / A26 3L, R46H / E70N, R46H / E70N / K92A / Y118V / A261G / A263L, R46H / E70N / K175P / A263G, R46H / K72C, R46H / K92A, R46H / K92A / K175P, R46H / Y118V, R46H / V146I, R46H / V146I / S257Q / A263I, E55R / E70N / Y118V, E55R / S257Q / A261G, E55R / : S257Q / A263L, E55R / A261G / A263I, E55R / A263G, E70N, K92A / Y118V, A105G, Y118V, Y118V / K226V / A261G / A263I, V146I / S257Q / A263S, V146I / A261G / A263L, E192V / S257Q / A261G, V202I, S257Q / A261G / A263G, A261G / A263I, and A263G.

[0163] As will be appreciated by the skilled artisan, in some embodiments, one of the above residue differences selected or a combination thereof can be kept constant (i.e. maintained) as a core feature in an engineered nitroaldolase, and additional residue differences at other residue positions can be incorporated into the sequence to produce additional engineered nitroaldolase polypeptides with improved properties. It should therefore be understood that for any engineered nitroaldolase comprising one or a subset of the above-described residue differences, the present invention contemplates other engineered nitroaldolases comprising one or a subset of the residue differences, and additionally comprising one or more residue differences at other residue positions disclosed herein.

[0164] As described above, the engineered nitroaldolase polypeptides are also capable of converting substrates (e.g., compounds (1) and (2)) into products (e.g., compounds (3)). In some embodiments, the engineered nitroaldolase polypeptides are capable of converting substrate compounds into product compounds with an activity that is at least 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 10 times, 20 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, 100 times, or more relative to the activity of the reference polypeptide SEQ ID NO: 2, 24, 496, 860, 1120, or 1348.

[0165] In some embodiments, the engineered nitroaldolase polypeptide capable of converting a substrate compound into a product compound with at least 2-fold greater activity relative to the activity of SEQ ID NO: 2, 24, 496, 860, 1120, or 1348 comprises an amino acid sequence selected from the even-numbered sequences of SEQ ID NO: 4-1484.

[0166] In some embodiments, the engineered nitroaldolase polypeptide is capable of converting a substrate compound to a product compound with stereoselectivity for the (S)-product compared to a reference polypeptide of SEQ ID NO: 2, 24, 496, 860, 1120, or 1348. In some embodiments, the engineered nitroaldolase polypeptide is capable of converting a substrate compound to a product compound with a stereoselectivity for the (S)-product (as measured by the amount of (S)-product to the total amount of product) of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater.

[0167] In some embodiments, the engineered nitroaldolase polypeptide capable of converting a substrate compound to a product compound with a stereoselectivity for the (S)-product of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or greater comprises an amino acid sequence selected from the even-numbered sequences of SEQ ID NOs: 4-1484.

[0168] In some embodiments, the engineered nitroaldolase has an amino acid sequence comprising one or more residue differences compared to SEQ ID NO: 2, 24, 496, 860, 1120 or 1348, increasing the expression of the engineered nitroaldolase activity in a bacterial host cell, particularly in E. coli.

[0169] In some embodiments, the engineered nitroaldolase polypeptide with improved properties has an amino acid sequence comprising a sequence selected from the even-numbered sequences within the range of SEQ ID NOs: 4-1484.

[0170] In some embodiments, the engineered nitroaldolase comprises an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to one of the even-numbered sequences within the range of SEQ ID NOs: 4-1484, and the amino acid residue differences that are present in any of the even-numbered sequences within the range of SEQ ID NOs: 4-1484 compared to SEQ ID NOs: 2, 24, 496, 860, 1120 or 1348, as provided in the Examples.

[0171] In addition to the residue positions specified above, any of the nitroaldolase polypeptides disclosed herein may also contain other residue differences at other residue positions (i.e., residue positions other than those contained herein) relative to SEQ ID NO: 2, 24, 496, 860, 1120 and / or 1348. 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 perform conversion of substrate to product. Thus, in some embodiments, in addition to the amino acid residue differences present in any of the engineered nitroaldolase polypeptides selected from the even-numbered sequences within the range of SEQ ID NOs: 4-1484, the sequence can also comprise 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12, 1-14, 1-15, 1-16, 1-18, 1-20, 1-22, 1-24, 1-26, 1-30, 1-35, 1-40, 1-45, or 1-50 residue differences at other amino acid residue positions compared to SEQ ID NOs: 2, 24, 496, 860, 1120, or 1348. 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, the residue differences can include conservative substitutions and non-conservative substitutions compared to the nitroaldolase polypeptide of SEQ ID NO: 2, 24, 496, 860, 1120 or 1348.

[0172] In some embodiments, the present invention also provides engineered polypeptides comprising fragments of engineered nitroaldolase polypeptides that retain the functional activity and / or improved properties of any engineered nitroaldolase enzyme described herein. Thus, in some embodiments, the present invention provides polypeptide fragments capable of converting a substrate into a product under suitable reaction conditions, wherein the fragment comprises at least about 90%, 95%, 96%, 97%, 98% or 99% of the full-length amino acid sequence of an engineered nitroaldolase of the present invention, such as an exemplary engineered nitroaldolase polypeptide selected from an even-numbered sequence within the range of SEQ ID NO: 4-1484. In some embodiments, the engineered nitroaldolase may have an amino acid sequence comprising a deletion in any one of the nitroaldolase polypeptide sequences described herein, such as an exemplary engineered polypeptide of an even-numbered sequence within the range of SEQ ID NO: 4-1484.

[0173] Thus, for various and each embodiments of the engineered nitroaldolase enzymes of the invention, the amino acid sequence can comprise a deletion 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 nitroaldolase 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 nitroaldolase enzymes described herein are maintained. In some embodiments, the deletion may comprise 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 can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residues. In some embodiments, the deletion can include the deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residues.

[0174] In some embodiments, the engineered nitroaldolase polypeptides described herein can have an amino acid sequence comprising an insertion compared to any of the engineered nitroaldolase polypeptides described herein, such as the exemplary engineered polypeptides of even-numbered sequences within the range of SEQ ID NO: 4-1484. Thus, for various and each embodiment of the nitroaldolase polypeptides of the present disclosure, the insertion can comprise one 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 nitroaldolase described herein are maintained. The insertion can be into the amino terminus or carboxyl terminus, or an internal portion of the nitroaldolase polypeptide.

[0175] In some embodiments, the engineered nitroaldolase herein can have an amino acid sequence comprising an even-numbered sequence selected from the range of SEQ ID NO: 4-1484 and optionally one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residues deleted, inserted, and / or substituted. 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 residues deleted, inserted, and / or substituted. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45 or 50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24 or 25 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the substitutions may be conservative substitutions or non-conservative substitutions.

[0176] In the above embodiments, suitable reaction conditions for engineering polypeptides are provided as described in the Examples herein.

[0177] In some embodiments, the polypeptides of the present invention are fusion polypeptides, wherein the engineered polypeptides are fused to other polypeptides, such as, for example but not limited to, antibody tags (e.g., myc epitopes), purification sequences (e.g., His tags for metal binding), and cell localization signals (e.g., secretion signals). Therefore, the engineered polypeptides described herein can be used fused to other polypeptides or not.

[0178] 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 contain naturally occurring and / or synthetic non-encoded amino acids in whole or in part. Some common non-encoded amino acids that the polypeptides described herein may contain include, but are not limited to: D-stereoisomers of genetically encoded amino acids; 2,3-diaminopropionic acid (Dpr); α-aminoisobutyric acid (Aib); ε-aminocaproic 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 Acid (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 Acid (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); naphth-1-ylalanine (1n Ala); naphthylalanine (2nAla); thiazolylalanine (taAla); benzothienylalanine (bAla); thienylalanine (tAla); furanylalanine (fAla); homophenylalanine (hPhe); homotyrosine (hTyr); homotryptophan (hTrp); pentafluorophenylalanine (5ff); styrylkalanine (sAla); anthrylalanine (aAla); 3,3-diphenylalanine (Dfa); 3-amino-5-phenylpentanoic acid (Afp); penicillamine (Pen); 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic);β-2-Thienylalanine (Thi); methionine sulfoxide (Mso); N(w)-nitroarginine (nArg); homolysine (hLys); phosphonomethylphenylalanine (pmPhe); phosphoserine (pSer); phosphothreonine (pThr); homoaspartic acid (hAsp); homoglutamic acid (hGlu); 1-aminocyclopentyl-(2 or 3)-ene-4-carboxylic acid; pipecolic acid (pipecolic acid acid, PA); azetidine-3-carboxylic acid (ACA); 1-aminocyclopentane-3-carboxylic acid; allylglycine (aGly); propargylglycine (pgGly); homoalanine (hAla); norvaline (nVal); homoleucine (hLeu), homovaline (hVal); homoisoleucine (hIle); homoarginine (hArg); N-acetyl lysine (AcLys); 2,4-diaminobutyric acid (Dbu); 2,3-diaminobutyric acid (Dab); N-methylvaline (MeVal); homocysteine ​​(hCys); homoserine (hSer); hydroxyproline (Hyp) and homoproline (hPro). Additional non-encoded amino acids that the polypeptides described herein may contain will be apparent to those skilled in the art (see, for example, the various amino acids provided in: Fasman,; CRC Practical Handbook ofBiochemistry and Molecular Biology , CRC Press, Boca Raton, FL, pp. 3-70

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

[0179] Those skilled in the art will recognize that amino acids or residues with side chain protecting groups may also constitute the polypeptides described herein. Non-limiting examples of such protected amino acids (which in this case belong to the aromatic category) include (protecting groups listed in parentheses), but are not limited to: Arg (tos), Cys (methylbenzyl), Cys (nitropyridylthio), Glu (δ-benzyl ester), Gln (xanthene), Asn (N-δ-xanthene), His (bom), His (benzyl), His (tos), Lys (fmoc), Lys (tos), Ser (O-benzyl), Thr (O-benzyl) and Tyr (O-benzyl).

[0180] The conformationally constrained non-coded amino acids that the polypeptides described herein may comprise include, but are not limited to, N-methyl amino acids (L-configuration); 1-aminocyclopentane-(2 or 3)-ene-4-carboxylic acid; pipecolic acid; azetidine-3-carboxylic acid; homoproline (hPro) and 1-aminocyclopentane-3-carboxylic acid.

[0181] In some embodiments, the engineered polypeptide can be in various forms, such as, for example, an isolated preparation, as a substantially purified enzyme, intact cells transformed with one or more genes encoding the enzyme, and / or as a cell extract and / or lysate of such cells. The enzyme can be lyophilized, spray dried, precipitated, or in the form of a crude paste, as discussed further below.

[0182] In some embodiments, the engineered polypeptide can be in the form of a biocatalytic composition. In some embodiments, the biocatalytic composition comprises (a) a means for converting ketone compounds and alkane compounds into β-nitro alcohol intermediates by contacting with a nitroaldolase polypeptide and (b) a suitable buffer. In some embodiments, the biocatalytic composition comprises a nitroaldolase that is active on ketone substrates. In some additional embodiments, the β-nitro alcohol intermediate is further reduced to β-hydroxy-α-amino alcohol. In some embodiments, the biocatalytic composition comprises a sodium citrate buffer.

[0183] In some embodiments, the engineered polypeptide can be provided on a solid support such as a membrane, a resin, a solid carrier or other solid phase material. The solid support can include organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy and polyacrylamide and their copolymers and grafts. The solid support can also be inorganic, such as glass, silica, controlled pore glass (CPG), reversed silica or metals such as gold or platinum. The configuration of the solid support can be in the form of beads, balls, particles, granules, gels, membranes or surfaces. The surface can be planar, generally planar or non-planar. The solid support can be porous or non-porous and can have swelling or non-swelling characteristics. The solid support can be configured in the form of a hole, a depression or other container, a vessel, a feature or a position.

[0184] In some embodiments, engineered nitroaldolase polypeptides of the present invention can be fixed on a solid support so that they retain their improved activity and / or other improved properties relative to reference polypeptide SEQ ID NO: 2, 24, 496, 860, 1120 or 1348. In such embodiments, the fixed polypeptide can promote the biocatalytic conversion of substrate compounds or other suitable substrates to products, and after the reaction is completed, it is easy to retain (e.g., by retaining the beads fixed with the polypeptide) and then reuse or recycle in subsequent reactions. Such immobilized enzyme methods allow further efficiency improvements and cost reductions. Therefore, it is also envisioned that any method using the nitroaldolase polypeptides of the present invention can be carried out using the nitroaldolase polypeptides bound to or fixed on a solid support.

[0185] Methods for enzyme immobilization are well known in the art. Engineered polypeptides can be non-covalently or covalently bound. Various methods for conjugating and fixing enzymes to solid supports (e.g., resins, membranes, beads, glass, etc.) are well known in the art (see, e.g., Yi et al., Proc. Biochem., 42(5):895-898

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

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

[2010] ; Truppo et al., Org. Proc. Res. Dev., online publication: dx.doi.org / 10.1021 / op200157c; Hermanson, BioconjugateTechniques , 2nd ed., 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 nitroaldolase of the present invention include, but are not limited to, beads or resins comprising: polymethacrylates with epoxide functional groups, polymethacrylates with aminoepoxide functional groups, styrene / DVB copolymers or polymethacrylates with octadecyl functional groups. Exemplary solid supports that can be used to immobilize the engineered nitroaldolase 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.

[0186] In some embodiments, the polypeptides described herein are provided in the form of a kit. The enzyme in the kit may be present alone or as more than one enzyme. The kit may also include reagents for performing enzymatic reactions, substrates for evaluating the activity of the enzyme, and reagents for detecting the product. The kit may also include a reagent dispenser and instructions for use of the kit.

[0187] In some embodiments, the kit of the present invention is included in an array comprising more than one different nitroaldolase polypeptides at different addressable positions, wherein different polypeptides are different variants of a reference sequence, and each of the variants has at least one different improved enzyme property. In some embodiments, more than one polypeptide fixed on a solid support is configured at a plurality of positions of an array, and the position is the automatic delivery of a reagent or addressable by a detection method and / or an instrument. The array can be used to test the conversion of various substrate compounds by polypeptides. Such arrays comprising a variety of engineered polypeptides and methods of use thereof are known in the art (see, for example, WO2009 / 008908A2).

[0188] Polynucleotides encoding engineered nitroaldolase, expression vectors and host cells

[0189] In another aspect, the invention provides polynucleotides encoding engineered nitroaldolase 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 a polypeptide. An expression construct containing a heterologous polynucleotide encoding an engineered nitroaldolase is introduced into an appropriate host cell to express the corresponding nitroaldolase polypeptide.

[0190] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of codons corresponding to a variety of amino acids provide an illustration of all polynucleotides capable of encoding subject polypeptides. The degeneracy of the genetic code in which the same amino acid is encoded by selectable or synonymous codons allows the generation of a very large number of nucleic acids, all of which encode improved nitroaldolases. Therefore, knowing a specific amino acid sequence, one skilled 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 present invention specifically contemplates each possible change of a polynucleotide encoding a polypeptide described herein that can be prepared by selecting a combination based on possible codon selections, and for any polypeptide described herein, all such changes are considered to be specifically disclosed, including amino acid sequences disclosed as even-numbered sequences within the range of SEQ ID NO: 4-1484 presented in Table 2-1, Table 2-2, Table 3-1, Table 3-2, Table 4-1, Table 4-2, Table 5-1, Table 5-2, Table 6-1, Table 6-2, Table 7-1, and Table 7-2 and incorporated by reference into the sequence table herein.

[0191] In various embodiments, codons are preferably selected to adapt to the host cell in which the protein is produced. For example, the preferred codons used in bacteria are used to express genes 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, not all codons need to be replaced to optimize the codon usage of nitroaldolase, because the native sequence will include preferred codons and may not be required for all amino acid residues due to the preferred codon usage. Therefore, the codon-optimized polynucleotide encoding nitroaldolase can contain preferred codons at about 40%, 50%, 60%, 70%, 80% or more than 90% of the codon positions in the full-length coding region.

[0192] In some embodiments, the polynucleotide comprises a codon-optimized nucleotide sequence encoding a nitroaldolase polypeptide amino acid sequence as represented by SEQ ID NO: 2, 24, 496, 860, 1120 or 1348. 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 more identity to a codon-optimized nucleic acid sequence encoding an even-numbered sequence within the range of SEQ ID NO: 4-1484. 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 more identity to a codon-optimized nucleic acid sequence encoding an odd-numbered sequence within the range of SEQ ID NO: 3-1483. In some embodiments, codon-optimized sequences of odd-numbered sequences within the range of SEQ ID NOs: 3-1483 enhance expression of the encoded nitroaldolase enzyme, providing a preparation of the enzyme capable of converting a substrate to a product.

[0193] 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 NOs: 3-1483, or the complement thereof, and encodes a nitroaldolase.

[0194] In some embodiments, as described above, the polynucleotide encodes an engineered nitroaldolase polypeptide having improved properties compared to SEQ ID NO: 2, 24, 496, 860, 1120 or 1348, 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: 2, 24, 496, 860, 1120 or 1348, and one or more residue differences compared to SEQ ID NO: 2, 24, 496, 860, 1120 or 1348, wherein the sequence is selected from the even numbered sequences within the range of SEQ ID NO: 4-1484. In some embodiments, the reference amino acid sequence is selected from the even numbered sequences within the range of SEQ ID NO: 4-1484. In some embodiments, the reference amino acid sequence is SEQ ID NO: 2, while in some other embodiments, the reference sequence is SEQ ID NO: 24, while in some other embodiments, the reference sequence is SEQ ID NO: 496. In some embodiments, the reference amino acid sequence is SEQ ID NO: 860, while in some other embodiments, the reference sequence is SEQ ID NO: 1120, while in some other embodiments, the reference sequence is SEQ ID NO: 1348.

[0195] In some embodiments, the polynucleotide encodes a nitroaldolase polypeptide capable of converting one or more substrates into products with improved properties compared to SEQ ID NO: 2, 24, 496, 860, 1120 or 1348, 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 sequence SEQ ID NO: 2, 24, 496, 860, 1120 or 1348.

[0196] In some embodiments, the polynucleotide encoding the engineered nitroaldolase comprises a polynucleotide sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1, 23, 495, 859, 1119 or 1347. In some embodiments, the polynucleotide encoding the engineered nitroaldolase comprises a polynucleotide sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence selected from the odd numbered sequences within the range of SEQ ID NO: 3-1483. In some embodiments, the polynucleotide encoding the engineered nitroaldolase comprises a polynucleotide sequence selected from the odd-numbered sequences within the range of SEQ ID NOs: 3-1483.

[0197] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from an odd-numbered sequence within the range of SEQ ID NO: 3-1483, or its complement, and encodes a nitroaldolase polypeptide having one or more improved properties described herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a nitroaldolase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 2,The nitroaldolase has an amino acid sequence comprising one or more residue differences compared to SEQ ID NO:2 at residue positions selected from the group consisting of: 7 / 9, 7 / 9 / 33 / 68 / 72 / 158, 7 / 9 / 33 / 72 / 175 / 197 / 230, 7 / 9 / 33 / 72 / 230, 7 / 9 / 68 / 72 / 197, 7 / 9 / 158 / 175, 7 / 9 / 175, 7 / 9 / 197, 7 / 33, 7 / 68 / 72 / 175, 7 / 72, 7 / 72 / 97 / 175 / 230, 9 / 11 / 33 / 72 / 197 / 230, 9 / 33 / 68 / 158 / 197, 9 / 33 / 72 / 230, 9 / 9 / 33 / 72 / 197. 7 / 230、9 / 68 / 72、9 / 97 / 175 / 202 / 218、9 / 175、9 / 202、11、13、14 / 33 / 68 / 72 / 97、18、18 / 33 / 68 / 97 / 158 / 175 / 239、18 / 33 / 72 / 97 / 158 / 175、18 / 33 / 72 / 97 / 158 / 239、18 / 33 / 72 / 97 / 175 / 202、18 / 33 / 72 / 175 / 202 / 237 / 239、18 / 33 / 72 / 197 / 237、18 / 68 / 72、18 / 68 / 72 / 158 / 175 / 237 / 239、1 8 / 68 / 72 / 175 / 202 / 239、18 / 68 / 72 / 239、18 / 68 / 175 / 223 / 237 / 239、18 / 68 / 239、18 / 72 / 97 / 175 / 237 / 239、18 / 72 / 175 / 197、18 / 72 / 237、18 / 72 / 239、18 / 97 / 175 / 237 / 239、18 / 97 / 223 / 237 / 239、18 / 175、18 / 197 / 239、18 / 239、20 / 124、33 / 68 / 72、33 / 68 / 72 / 175 / 230、33 / 68 / 223 / 239 , 33 / 72, 33 / 72 / 97 / 197, 33 / 72 / 175, 33 / 175, 39, 44 / 97, 52, 68 / 72, 68 / 72 / 97, 68 / 72 / 97 / 197, 68 / 72 / 197, 68 / 72 / 197 / 239, 68 / 97 / 175, 68 / 97 / 175 / 197 / 223, 72 / 97 / 158 / 223 / 237, 72 / 175 / 197, 97 / 158 / 175 / 197, 97 / 175, 103, 121, 122, 124, 125, 128, 133, 148, 175, 197 and 197 / 202.

[0198] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from an odd-numbered sequence within the range of SEQ ID NO: 3-1483, or its complement, and encodes a nitroaldolase polypeptide having one or more improved properties described herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a nitroaldolase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 2, the nitroaldolase having an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 3. NO:2 compared to an amino acid sequence that differs at one or more residues at a residue position selected from the group consisting of: 7 / 11 / 33 / 72 / 97, 7 / 11 / 33 / 97, 7 / 11 / 72, 7 / 11 / 158, 7 / 11 / 237, 7 / 33 / 72 / 97 / 158 / 175 / 197 / 237 / 239, 9 / 11 / 33, 9 / 11 / 33 / 72, 9 / 11 / 72, 9 / 33 / 68 / 97 / 237 / 239, 9 / 72 / 97 / 158 / 237 / 239, 9 / 218 / 239, 10, 11, 12, 13, 14, 18, 33 / 68 / 72 / 97 / 237 / 239, 33 / 72 / 158 / 23 9, 39, 40, 41, 48, 49, 51, 54, 54 / 126, 57, 68 / 175 / 239, 82, 84, 85, 86, 103, 104, 105, 107, 115, 117, 119, 119 / 120, 120, 122, 123, 124, 127, 130, 131, 133, 134, 137 / 145, 144, 145, 146, 147, 148, 149, 150, 152, 157, 158, 159, 169, 173, 174, 175, 180, 181, 183, 203, 204, 208, 209, 210, 215, 218, 236 and 237.

[0199] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from an odd-numbered sequence within the range of SEQ ID NO: 3-1483, or its complement, and encodes a nitroaldolase polypeptide having one or more improved properties described herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a nitroaldolase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24, the nitroaldolase having an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24. NO:24 compared to an amino acid sequence that differs at one or more residues at a residue position selected from the group consisting of: 9 / 39 / 122 / 157, 18, 18 / 33 / 39 / 230, 18 / 33 / 175, 18 / 39 / 44 / 118, 18 / 175, 20, 33 / 39, 33 / 39 / 44 / 118 / 121, 33 / 39 / 44 / 121, 33 / 39 / 44 / 152, 33 / 3 9 / 44 / 175、33 / 39 / 44 / 197、33 / 39 / 44 / 210 / 230、33 / 39 / 118 / 121 / 175、33 / 39 / 121 / 175、33 / 39 / 197 / 210、33 / 39 / 210、33 / 39 / 210 / 230、33 / 118 / 121、33 / 118 / 121 / 175、39、39 / 44 / 103、39 / 44 / 118、39 / 44 / 152 / 230、39 / 44 / 175 / 197 / 230、39 / 103、39 / 103 / 125、39 / 103 / 125 / 127 / 146、39 / 103 / 125 / 127 / 146 / 150、39 / 103 / 125 / 146、39 / 103 / 127 / 150、39 / 103 / 150、39 / 118 / 121、 39 / 197, 39 / 197 / 210, 43, 44 / 103, 81, 103, 103 / 125 / 127, 103 / 125 / 146, 103 / 127, 103 / 150, 118 / 121, 118 / 121 / 175, 118 / 121 / 197, 121, 152, 152 / 197, 154, 159, 163, 175, 210, 232 and 238.

[0200] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from an odd-numbered sequence within the range of SEQ ID NO: 3-1483, or its complement, and encodes a nitroaldolase polypeptide having one or more improved properties described herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a nitroaldolase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24, the nitroaldolase having an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24. NO:24 an amino acid sequence that differs at one or more residues compared to a residue position selected from the group consisting of: 7, 9 / 13, 9 / 13 / 39, 9 / 13 / 39 / 68 / 72 / 239, 9 / 13 / 39 / 68 / 239, 9 / 13 / 39 / 72 / 122, 9 / 13 / 39 / 97 / 239, 9 / 13 / 68 / 72 / 97 / 239, 9 / 13 / 68 / 97 / 134 / 239, 9 / 13 / 68 / 97 / 239, 9 / 13 / 72, 9 / 13 / 134, 11, 12 / 42 / 125 / 127, 12 / 103, 12 / 103 / 150, 13, 13 / 39, 13 / 39 / 68, 13 / 39 / 68 / 72 / 97, 13 / 39 / 72 / 97 / 122 / 134 / 223, 13 / 39 / 97 / 239, 13 / 39 / 239, 13 / 68, 13 / 68 / 72 / 97 / 122 / 239, 16, 18 / 33 / 39 / 44 / 118 / 121 / 175 / 197 / 210, 18 / 33 / 39 / 44 / 121, 18 / 33 / 39 / 112 / 118 / 121 / 197、18 / 33 / 39 / 121、18 / 33 / 39 / 121 / 175、18 / 33 / 39 / 121 / 175 / 197、18 / 33 / 39 / 121 / 175 / 210、18 / 33 / 39 / 121 / 210、18 / 33 / 118 / 121、18 / 33 / 121、18 / 33 / 121 / 197、18 / 33 / 121 / 210 / 230、18 / 39 / 44 / 118 / 121 / 230 、18 / 39 / 44 / 121 / 175 / 210、18 / 39 / 118 / 121、18 / 39 / 118 / 121 / 210、18 / 39 / 121 / 175、18 / 39 / 121 / 175 / 210、18 / 39 / 121 / 197 / 210、18 / 118 / 121、18 / 118 / 121 / 175、18 / 118 / 121 / 210、18 / 118 / 121 / 230、18 / 121、18 / 121 / 175、18 / 121 / 210、18 / 121 / 230, 20, 21, 33 / 39 / 44 / 118 / 121, 33 / 39 / 44 / 118 / 121 / 210 / 230, 33 / 39 / 44 / 121, 33 / 39 / 44 / 121 / 152, 33 / 39 / 44 / 121 / 175 / 210, 33 / 39 / 44 / 121 / 230, 33 / 39 / 73 / 121 / 197, 33 / 39 / 118 / 121 / 175, 33 / 39 / 118 / 121 / 175 / 230, 33 / 39 / 121, 33 / 39 / 121 / 175, 33 / 39 / 121 / 210 / 230, 33 / 118 / 121, 33 / 118 , 33 / 118 / 121 / 197, 33 / 121, 33 / 121 / 230, 37 / 91, 39 / 44 / 118 / 121 / 175 / 210, 39 / 68 / 72 / 239, 39 / 118 / 121, 39 / 118 / 121 / 210, 39 / 121, 39 / 121 / 210 / 230, 43, 44, 76, 79, 101, 118 / 121, 118 / 121 / 175, 118 / 121 / 197, 121, 121 / 175 / 230, 121 / 210, 148, 153, 159, 161, 172, 200, 207, 233, 245 and 248.

[0201] In some embodiments, the polynucleotide is capable of hybridizing under high stringency conditions to a reference polynucleotide sequence selected from an odd-numbered sequence within the range of SEQ ID NO: 3-1483, or its complement, and encodes a nitroaldolase polypeptide having one or more improved properties described herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a nitroaldolase comprising an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:496, the nitroaldolase having an amino acid sequence that comprises one or more residue differences compared to SEQ ID NO:496 at residue positions selected from the group consisting of: 43 / 103, 43 / 103 / 172 / 238, 43 / 103 / 238 / 241, 43 / 238, 81 / 232, 103, 103 / 159 / 238, 103 / 194 / 238, 103 / 238, 122, 232, 238, and 238 / 241.

[0202] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from an odd-numbered sequence within the range of SEQ ID NO: 3-1483, or its complement, and encodes a nitroaldolase polypeptide having one or more improved properties described herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a nitroaldolase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 496, the nitroaldolase having an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 496 NO:496 compared to an amino acid sequence that differs at one or more residues at a residue position selected from the group consisting of: 12, 43, 43 / 47 / 103, 43 / 47 / 103 / 232, 43 / 103, 43 / 103 / 121 / 172 / 241, 43 / 103 / 172, 43 / 103 / 172 / 238, 43 / 103 / 238, 43 / 103 / 238 / 241, 43 / 121, 43 / 194 / 238, 43 / 194 / 238 238, 44 / 103 / 121 / 238, 44 / 103 / 238, 44 / 103 / 238 / 241, 44 / 121 / 238 / 241, 47 / 103, 63, 103, 103 / 121, 103 / 121 / 172 / 238, 103 / 159 / 238, 103 / 172 / 238, 103 / 194 / 238, 103 / 238, 122, 131, 146, 178, 180 and 221.

[0203] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from an odd-numbered sequence within the range of SEQ ID NO: 3-1483, or its complement, and encodes a nitroaldolase polypeptide having one or more improved properties described herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a nitroaldolase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 860, the nitroaldolase having an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 860 NO:860 compared to an amino acid sequence that differs at one or more residues at a residue position selected from the group consisting of: 12, 12 / 43 / 63 / 103 / 146 / 180 / 232 / 241, 12 / 43 / 63 / 103 / 146 / 241, 12 / 43 / 81 / 103 / 241, 12 / 43 / 81 / 146 / 232 / 241, 12 / 43 / 81 / 180 / 241, 12 / 43 / 81 / 232 / 241, 12 / 43 / 81 / 241, 1 2 / 43 / 103、12 / 43 / 103 / 146 / 180 / 232 / 241、12 / 43 / 103 / 146 / 180 / 241、12 / 43 / 103 / 180 / 241、12 / 43 / 103 / 241、12 / 43 / 146 / 180 / 232 / 241、12 / 43 / 180 / 241、12 / 63 / 81 / 103 / 241、12 / 81、12 / 81 / 180 / 241、12 / 103 / 146 / 241 、12 / 103 / 180 / 241、12 / 146、12 / 146 / 180 / 241、12 / 180 / 232 / 241、12 / 241、18、43 / 81 / 232 / 241、43 / 103 / 146 / 180、43 / 103 / 180 / 241、43 / 103 / 241、43 / 180 / 232、43 / 232 / 241、63 / 103 / 180 / 232、78、81、81 / 103 / 146、81 / 1 , 46 / 180 / 241, 81 / 146 / 241, 81 / 180 / 241, 84, 103, 103 / 146, 103 / 146 / 180, 103 / 146 / 180 / 241, 103 / 180, 103 / 180 / 241, 103 / 261, 144, 145, 146 / 180 / 232, 146 / 180 / 241, 146 / 241, 150, 166, 172, 180 / 232, 232 / 241 and 241.

[0204] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from an odd-numbered sequence within the range of SEQ ID NO: 3-1483, or its complement, and encodes a nitroaldolase polypeptide having one or more improved properties described herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a nitroaldolase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 860, the nitroaldolase having an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 860 NO:860 compared to an amino acid sequence that differs at one or more residues at a residue position selected from the group consisting of: 9, 12, 12 / 43 / 63 / 103 / 146 / 180 / 232 / 241, 12 / 43 / 63 / 103 / 146 / 241, 12 / 43 / 103 / 146 / 180 / 232 / 241, 12 / 43 / 103 / 146 / 180 / 241, 12 / 43 / 103 / 146 / 232 / 241, / 43 / 103 / 146 / 241、12 / 43 / 103 / 180 / 241、12 / 43 / 146 / 180 / 232 / 241、12 / 43 / 146 / 241、12 / 43 / 180 / 232、12 / 43 / 180 / 241、12 / 63 / 103 / 146 / 232 / 241、12 / 103 / 146 / 241、12 / 103 / 180 / 241、12 / 146、12 / 146 / 180 / 241, 12 / 146 / 232 / 241, 12 / 146 / 241, 12 / 180, 12 / 180 / 232 / 241, 12 / 180 / 241, 12 / 241, 18, 43 / 63 / 103 / 180 / 232 / 241, 43 / 103 / 146 / 180, 43 / 103 / 180, 43 / 103 / 180 / 232 / 241, 43 / 103 / 180 / 241, 43 / 18 0 / 232, 63 / 103 / 180 / 232, 103, 103 / 146 / 180, 103 / 146 / 180 / 241, 103 / 180, 103 / 180 / 241, 103 / 241, 118, 119, 122, 144, 145, 146 / 180 / 241, 154, 160, 172, 180 / 232, 180 / 232 / 241, 180 / 241, 208, 211, and 212.

[0205] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from an odd-numbered sequence within the range of SEQ ID NO: 3-1483, or its complement, and encodes a nitroaldolase polypeptide having one or more improved properties described herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a nitroaldolase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1120, the nitroaldolase having an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1120 An amino acid sequence that differs from NO:1120 at one or more residues at a residue position selected from the group consisting of: 3, 7, 7 / 158, 7 / 158 / 175, 7 / 158 / 197 / 202, 27 / 43 / 115 / 172, 27 / 43 / 172, 29, 32, 43 / 110 / 115 / 172, 44, 46, 48 / 172, 55, 59, 67, 71, 72, 73, 94 / 103 / 118 / 14 6, 103, 103 / 144 / 146 / 172 / 180, 103 / 146 / 154 / 172 / 180 / 232, 103 / 154, 118, 118 / 144 / 215, 118 / 172 / 180, 137, 142, 146, 146 / 154, 154, 158, 172 / 190, 184, 198, 205, 216, 223, 226, 229, 247, 261 and 263.

[0206] In some embodiments, the polynucleotide is capable of hybridizing under high stringency conditions to a reference polynucleotide sequence selected from an odd-numbered sequence within the range of SEQ ID NO: 3-1483, or its complement, and encodes a nitroaldolase polypeptide having one or more improved properties described herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a nitroaldolase comprising an amino acid sequence that is at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:1120, the nitroaldolase having an amino acid sequence that comprises one or more residue differences compared to SEQ ID NO:1120 at residue positions selected from the group consisting of: 73, 80, 103 / 146 / 209, 103 / 172 / 209, 118 / 119 / 172 / 209, 135, 144 / 209 / 232, 172 / 180 / 209, 181 and 223.

[0207] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from an odd-numbered sequence within the range of SEQ ID NO: 3-1483, or its complement, and encodes a nitroaldolase polypeptide having one or more improved properties described herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a nitroaldolase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1348, the nitroaldolase having an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1348. NO:1348 has an amino acid sequence that differs at one or more residues compared to residue positions selected from the group consisting of: 2, 7 / 46, 39, 46, 46 / 55, 46 / 55 / 72, 46 / 55 / 92, 46 / 55 / 118, 46 / 55 / 226 / 257 / 263, 46 / 55 / 263, 46 / 70, 46 / 70 / 92 / 118 / 261 / 263, 46 / 70 / 175 / 263, 46 / 72, 46 / 118, 55 / 257 / 261, 55 / 261 / 263, 55 / 263, 105, 118, 118 / 226 / 261 / 263 and 263.

[0208] In some embodiments, the polynucleotide is capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from an odd-numbered sequence within the range of SEQ ID NO: 3-1483, or its complement, and encodes a nitroaldolase polypeptide having one or more improved properties described herein. In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes a nitroaldolase comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1348, the nitroaldolase having an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1348. NO:1348 compared to an amino acid sequence that differs at one or more residues at a residue position selected from the group consisting of: 2, 7 / 46, 7 / 46 / 175, 22 / 46 / 55 / 118, 27 / 46 / 72, 27 / 46 / 146 / 158 / 202, 27 / 46 / 175, 27 / 118, 29 / 70 / 118, 39, 46, 46 / 55, 46 / 55 / 59 / 261 / 263, 46 / 55 / 72, 46 / 55 / 92, 46 / 55 / 118, 46 / 55 / 118 / 263, 46 / 55 / 146, 46 / 55 / 146 / 202, 46 / 55 / 226 / 257 / 263. 3. 46 / 70, 46 / 70 / 92 / 118 / 261 / 263, 46 / 70 / 175 / 263, 46 / 72, 46 / 92, 46 / 92 / 175, 46 / 118, 46 / 146, 46 / 146 / 257 / 263, 55 / 70 / 118, 55 / 257 / 261, 55 / 257 / 263, 55 / 261 / 263, 55 / 263, 70, 92 / 118, 105, 118, 118 / 226 / 261 / 263, 146 / 257 / 263, 146 / 261 / 263, 192 / 257 / 261, 202, 257 / 261 / 263, 261 / 263 and 263.

[0209] In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes an engineered nitroaldolase polypeptide with improved properties comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 2, 24, 496, 860, 1120 or 1348. 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%, 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered nitroaldolase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NOs: 3-1483.

[0210] In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes an engineered nitroaldolase polypeptide with improved properties, the engineered nitroaldolase 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 more identity to SEQ ID NO: 2. 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% sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered nitroaldolase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NO: 3-461.

[0211] In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes an engineered nitroaldolase polypeptide with improved properties comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 24. 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%, 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered nitroaldolase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NO: 459-841.

[0212] In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes an engineered nitroaldolase polypeptide with improved properties comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 496. 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%, 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered nitroaldolase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NO: 843-979.

[0213] In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes an engineered nitroaldolase polypeptide with improved properties comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 860. 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%, 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered nitroaldolase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NO: 981-1211.

[0214] In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes an engineered nitroaldolase polypeptide with improved properties comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1120. 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%, 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered nitroaldolase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NO: 1213-1365.

[0215] In some embodiments, the polynucleotide capable of hybridizing under highly stringent conditions encodes an engineered nitroaldolase polypeptide with improved properties comprising an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO: 1348. 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%, 99% or more sequence identity at the nucleotide level to a reference polynucleotide encoding an engineered nitroaldolase. In some embodiments, the reference polynucleotide sequence is selected from SEQ ID NO: 1367-1483.

[0216] In some embodiments, the polynucleotide encoding the separation of any engineered nitroaldolase polypeptide provided herein is manipulated in various ways to provide expression of the polypeptide. In some embodiments, the polynucleotide encoding the polypeptide is provided with an expression vector, and there are one or more control sequences in the expression vector to regulate the expression of the polynucleotide and / or polypeptide. Depending on the expression vector, the manipulation of the polynucleotide to the separation can be desirable or necessary before the polynucleotide to be separated is inserted into the carrier. The technology utilizing recombinant DNA methods to modify polynucleotides and nucleotide sequences is well known in the art.

[0217] 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 known in the art, a suitable promoter 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 Escherichia coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis α-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens α-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic β-lactamase genes (see, e.g., Villa-Kamaroff et al., Proc. Natl. Am. Am. Res., 1996). Acad. Sci. USA 75:3727-3731

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

[1983] Exemplary promoters for filamentous fungal host cells include promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline proteinase, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 99 / 11665). 96 / 00787), and the NA2-tpi promoter (a hybrid of the promoters from the Aspergillus niger neutral α-amylase gene and the Aspergillus oryzae triose phosphate isomerase gene), and mutants, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be derived from the genes for 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] ). In some embodiments, the control sequence is a suitable transcription terminator sequence (a sequence recognized by the host cell to terminate transcription). The terminator sequence is operably connected to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in the host cell of selection 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, for example, Romanos et al., above). In some embodiments, the control sequence is a suitable leader sequence (non-translated region of mRNA important for translation by the host cell). The leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the selected host cell can be used. Exemplary leaders for filamentous fungal host cells are obtained from the following genes: Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells include, but are not limited to, those obtained from the following genes: Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-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 which is recognized by the host cell as a signal for adding polyadenosine residues to the transcribed mRNA when transcribed. Any polyadenylation sequence that is functional 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 obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger α-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art (see, e.g., Guo and Sherman, Mol. Cell. Bio., 15:5983-5990

[1995] ). In some embodiments, the control sequence is a signal peptide coding region, which encodes an amino acid sequence connected to the amino terminus of the polypeptide and directs the encoded polypeptide to the secretory pathway of the cell. The 5' end of the coding sequence of the nucleic acid sequence may inherently contain a signal peptide coding region, which is naturally connected to the segment of the coding region of the polypeptide that encodes secretion in accordance with the translation reading frame. Alternatively, 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 to the secretory pathway of the selected host cell can be used for the expression of the engineered nitroaldolase polypeptide provided herein. The effective signal peptide coding region for bacterial host cells includes, but is not limited to, the signal peptide coding region obtained from the following genes: Bacillus NClB 11837 maltogenic amylase, Bacillus stearothermophilus α-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM) and Bacillus subtilis prsA. Additional signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 57: 109-137

[1993] ). Signal peptide coding regions effective for filamentous fungal host cells include, but are not limited to, those obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to, those from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. In some embodiments, the control sequence is a propeptide coding region encoding an amino acid sequence positioned at the amino terminus of a polypeptide. The polypeptide produced is referred to as a "proenzyme", "propolypeptide" or in some cases a "zymogen". A propolypeptide can be converted into a mature active polypeptide by catalyzing or autocatalyzing the cleavage of a propeptide from a propolypeptide. The propeptide coding region includes, but is not limited to, those from the following genes: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae α-factor, Rhizomucor miehei aspartic protease, and Myceliophthora thermophila lactase (see, e.g., WO95 / 33836). When both the signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned adjacent to the amino terminus of the polypeptide and the signal peptide region is positioned adjacent to the amino terminus of the propeptide region. In some embodiments, regulatory sequences are also utilized. These sequences promote the regulation of polypeptide expression relative to host cell growth. Examples of regulatory systems are those that cause the expression of genes to be turned on or off in response to chemical or physical stimuli (including the presence of regulatory compounds). In prokaryotic host cells, suitable regulatory sequences include but are not limited to lac, tac and trp operator systems. In yeast host cells, suitable regulatory systems include but are not limited to ADH2 systems or GAL1 systems. In filamentous fungi, suitable regulatory sequences include but are not limited to TAKA α-amylase promoter, Aspergillus niger glucoamylase promoter and Aspergillus oryzae glucoamylase promoter. The present invention also provides recombinant expression vectors, which comprise polynucleotides encoding engineered nitroaldolase polypeptides and one or more expression regulatory regions, such as promoters and terminators, replication origins, etc., depending on the type of the host into which they are introduced. In some embodiments, various nucleic acids described above and control sequences are combined to produce recombinant expression vectors, which comprise one or more convenient restriction sites to allow insertion or replacement of the nucleic acid sequence encoding variant nitroaldolase polypeptides at such sites. Alternatively, one or more polynucleotide sequences of the present invention are expressed by inserting the polynucleotide sequence or the nucleic acid construct comprising the polynucleotide sequence into a suitable vector for expression. When creating an expression vector, the encoding sequence is positioned in the vector in such a way that the encoding sequence is operably connected to a suitable control sequence for expression. The recombinant expression vector can be any vector (e.g., plasmid or virus) that can be easily subjected to recombinant DNA procedures and can result in the expression of the variant nitroaldolase polynucleotide sequence. The selection of the 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. In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome). The vector may include any means for ensuring self-replication. In some optional embodiments, the vector may be a vector that, when introduced into a host cell, is integrated into the genome and replicates with one or more chromosomes into which it is integrated. In addition, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the host cell genome, or a transposon may be used. In some embodiments, the expression vector preferably comprises one or more selectable markers that allow easy selection of transformed cells. A "selectable marker" is a gene whose product provides biocide or virus resistance, resistance to heavy metals, prototrophy to auxotrophs, etc. Examples of bacterial selectable markers include, but are not limited to, dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and equivalents thereof. In another aspect, the invention provides a host cell comprising a polynucleotide encoding at least one engineered nitroaldolase polypeptide of the invention, the polynucleotide being operably linked to one or more control sequences for expressing one or more engineered nitroaldolases in the host cell. Host cells for use in expressing polypeptides encoded by expression vectors of the present 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 Accession No. 201178]); insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary host cells are Escherichia coli strains (such as W3110 (ΔfhuA) and BL21). Therefore, on the other hand, the invention provides a method for producing an engineered nitroaldolase polypeptide, wherein the method comprises cultivating a host cell capable of expressing a polynucleotide encoding an engineered nitroaldolase polypeptide under conditions suitable for expressing the polypeptide. In some embodiments, the method further comprises the step of separating and / or purifying the nitroaldolase polypeptide as described herein. Suitable culture media and growth conditions for the host cells described above are well known in the art. Polynucleotides for expressing the nitroaldolase polypeptide can be introduced into cells by various methods known in the art. Techniques include, among others, electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion. Engineered nitroaldolases with characteristics disclosed herein can be obtained by subjecting polynucleotides encoding naturally occurring or engineered nitroaldolase 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, e.g., 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 No. 6,537,746). Other directed evolution procedures that can be used include, among others, the staggered extension process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 16:258-261

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

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

[1996] ). For example, methods of mutagenesis and directed evolution can be readily applied to polynucleotides to generate libraries of variants that can be expressed, screened, and assayed. Mutagenesis and directed evolution methods are well known in the art (see, e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201, 6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,0 No. 53, No. 6,303,344, No. 6,309,883, No. 6,319,713, No. 6,319,714, No. 6,323,030, No. 6,326,204, No. 6,335,160, No. 6,335,198, No. 6,344,356, No. 6,352,859, No. 6,355,484, No. 6,358,740, No. 6,358,742, No. 6,365,377, No. 6,365,408, No. 6,368,861, No. 6,372,497, No. 6,337,186, No. 6,376,246, No. 6,379 ,964, No. 6,387,702, No. 6,391,552, No. 6,391,640, No. 6,395,547, No. 6,406,855, No. 6,406,910, No. 6,413,745, No. 6,413,774, No. 6,420,175, No. 6,423,542, No. 6,426,224, No. 6,436,675, No. 6,444,468, No. 6,455,253, No. 6,479,652, No. 6,482,647, No. 6,483,011, No. 6,484,105, No. 6,489,146, No. 6,5 00,617, No. 6,500,639, No. 6,506,602, No. 6,506,603, No. 6,518,065, No. 6,519,065, No. 6,521,453, No. 6,528,311, No. 6,537,746, No. 6,573,098, No. 6,576,467, No. 6,579,678, No. 6,586,182, No. 6,602,986, No. 6,605,430, No. 6,613,514, No. 6,653,072, No. 6,686,515, No. 6,703,240, No. 6,716,631, No. 6,No. 825,001, 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. 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. 8,17 0,806, 8,224,580, 8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326 and all related U.S. and PCT and non-U.S. counterparts; Ling et al., Anal. Biochem., 254(2):157-78

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

[1996] ; Smith, Ann. Rev. Genet., 19:423-462 [ 1985]; Botstein et al., Science, 229:1193-1201

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

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

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

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

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

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

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

[1997] ; Zhang et al., Proc. Nat. Acad. Sci. USA, 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, all of which are incorporated herein by reference). In some embodiments, the enzyme clones obtained after mutagenesis are screened by subjecting the enzyme to a specified temperature (or other assay conditions, such as testing the activity of the enzyme to a wide range of substrates), and measuring the amount of enzyme activity remaining after the heat treatment or other assay conditions. The clones containing the polynucleotide encoding the nitroaldolase polypeptide are then sequenced to identify changes in the nucleotide sequence (if any), and used to express the enzyme in a host cell. Measuring the enzyme activity from the expression library can be performed using any suitable method known in the art (e.g., standard biochemical techniques, such as HPLC analysis). In some embodiments, clones obtained after mutagenesis can be screened for engineered nitroaldolases with one or more desired improved enzyme properties (e.g., improved regioselectivity). Enzyme activity from expression libraries can be measured using standard biochemical techniques such as GC analysis, HPLC analysis, and / or product derivatization (before or after separation), such as using dansyl chloride or OPA (see, e.g., Yaegaki et al., J Chromatogr. 356(1):163-70

[1986] ). When the sequence of the engineered polypeptide is known, the polynucleotide encoding the enzyme can be prepared by a standard solid phase method according to a known synthetic method. In some embodiments, fragments up to about 100 bases can be synthesized individually, and then connected (for example, by enzymatic or chemical connection method or polymerase-mediated method) to form any desired continuous sequence. For example, the polynucleotides and oligonucleotides of the part encoding nitroaldolase can be prepared by chemical synthesis known in the art (for example, Beaucage et al., Tet. Lett., 22: 1859-69

[1981] classical phosphoramidite method or Matthes et al., EMBO J., 3: 801-05

[1984] described method), as usually practiced in automated synthesis methods. According to the phosphoramidite method, oligonucleotides are synthesized (for example, in an automatic DNA synthesizer), purified, annealed, connected and cloned into a suitable vector. In addition, any nucleic acid can be obtained from any of various commercial sources. In some embodiments, additional changes can be made by synthesizing oligonucleotides containing deletions, insertions, and / or substitutions and combining the oligonucleotides in various permutations to generate engineered nitroaldolase enzymes with improved properties. Thus, in some embodiments, a method for making an engineered nitroaldolase polypeptide comprises: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity to an amino acid sequence selected from an even-numbered sequence of SEQ ID NOs: 4-1484, and having one or more residue differences compared to SEQ ID NOs: 2, 24, 496, 860, 1120, or 1348; and (b) expressing the nitroaldolase polypeptide encoded by the polynucleotide. In some embodiments of the method, the polynucleotide encoding optionally has one or several (e.g., up to 3, 4, 5 or up to 10) amino acid residue deletions, insertions and / or substitutions of the engineered nitroaldolase. In some embodiments, the amino acid sequence optionally has 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45 or 1-50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45 or 50 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24 or 25 amino acid residue deletions, insertions and / or substitutions. In some embodiments, the substitutions may be conservative substitutions or non-conservative substitutions. In some embodiments, any engineered nitroaldolase expressed in a host cell can be recovered from the cells and / or culture medium using any one or more of the well-known techniques for protein purification, including, among others, lysozyme treatment, ultrasound, filtration, salting out, ultracentrifugation, and chromatography. Suitable solutions for lysis and efficient protein extraction from bacteria such as E. coli are commercially available (e.g., CelLytic B TM , Sigma-Aldrich, St. Louis MO). Chromatographic techniques used to separate nitroaldolase polypeptides include, but are not limited to, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. The conditions used to purify a particular enzyme will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and will be apparent to those skilled in the art. In some embodiments, affinity techniques can be used to separate improved nitroaldolase. For affinity chromatography purification, any antibody that specifically binds to the nitroaldolase polypeptide can be used. For the production of antibodies, a variety of host animals including but not limited to rabbits, mice, rats, etc. can be immunized by injection with nitroaldolase polypeptides or fragments thereof. Nitroaldolase polypeptides or fragments can be attached to a suitable carrier such as BSA by means of side chain functional groups or joints attached to side chain functional groups. In some embodiments, affinity purification can use a specific ligand or dye affinity column to which nitroaldolase binds (see, e.g., EP0641862; Stellwagen, "Dye Affinity Chromatography," in CurrentProtocolsinProteinScience ,Unit 9.2-9.2.16

[2001] ). Methods of using engineered nitroaldolase In some embodiments, the nitroaldolase enzymes described herein can be used in methods of converting one or more suitable substrates into a product. In another aspect, the engineered nitroaldolase polypeptides disclosed herein can be used in a method for converting a substrate compound (1) or a structural analog thereof and a substrate compound (2) or a structural analog thereof into a product of compound (3) or a corresponding structural analog thereof. Structural analogs of compound (1) include other ketones with halogen modifications and / or ketones with various modifications on the α carbon. Structural analogs of compound (2) include other alkanes with nitro groups or other nitro-containing compounds with available α-hydrogen. Structural analogs of compound (3) include various β-nitro alcohols. In some embodiments, the present disclosure provides a method for preparing compound (3); The method comprises reacting a substrate of compound (1) under suitable reaction conditions. Substrate with compound (2) The step of contacting with an engineered nitroaldolase polypeptide as disclosed herein, thereby producing a product of compound (3). In the embodiments provided herein and illustrated in the Examples, various suitable reaction conditions that can be used in the methods include, but are not limited to, substrate loading, co-substrate loading, pH, temperature, buffer, solvent system, polypeptide loading, and reaction time. Additional suitable reaction conditions for biocatalytic conversion of substrate compounds to product compounds using the engineered nitroaldolase described herein can be easily optimized by routine experimentation according to the guidance provided herein, including but not limited to contacting the engineered nitroaldolase polypeptide and one or more substrate compounds under experimental reaction conditions of concentration, pH, temperature, and solvent conditions, and detecting product compounds. In some embodiments, suitable reaction conditions include for each of one or more substrates, at least about 0.5g / L to about 60g / L, 1g / L to about 40g / L, 20g / L to about 35g / L, about 10g / L to about 25g / L or 25g / L to about 35g / L substrate compound loading. In some embodiments, suitable reaction conditions include for each of one or more substrates, at least about 0.5g / L, at least about 1g / L, at least about 5g / L, at least about 10g / L, at least about 15g / L, at least about 20g / L, at least about 30g / L, at least about 35g / L or at least about 40g / L or even larger substrate compound loading. In the method for carrying out nitroaldolase mediation described herein, engineered polypeptides can be added to the reaction mixture in the form of purified enzyme, partially purified enzyme, whole cells transformed with one or more genes encoding the enzyme, cell extracts and / or lysates of such cells, and / or as enzymes fixed on a solid support. Whole cells transformed with one or more genes encoding engineered nitroaldolase, or its cell extracts, lysates, and separated enzymes can be used in various forms, including solids (e.g., freeze-dried, spray-dried, etc.) or semisolids (e.g., crude pastes). Cell extracts or cell lysates can be partially purified by precipitation (ammonium sulfate, polyethyleneimine, heat treatment, etc.), followed by desalting procedures (e.g., ultrafiltration, dialysis, etc.), then freeze-dried. Any enzyme preparation (including whole cell preparations) can be stabilized by cross-linking or fixing to a solid phase (e.g., Eupergit C, etc.) using a known cross-linking agent (such as, for example, glutaraldehyde). One or more genes encoding engineered nitroaldolase polypeptides can be transformed into host cells respectively or together into the same host cell. For example, in some embodiments, one group of host cells can be transformed with one or more genes encoding a kind of engineered nitroaldolase polypeptide, and another group of host cells can be transformed with one or more genes encoding another kind of engineered nitroaldolase polypeptide. Both groups of transformed host cells can be used in the form of whole cells, or in the form of lysates or extracts derived therefrom for reaction mixtures. In other embodiments, host cells can be transformed with one or more genes encoding multiple engineered nitroaldolase polypeptides. In some embodiments, engineered polypeptides can be expressed in the form of secreted polypeptides and the culture medium containing the secreted polypeptides can be used for nitroaldolase reactions. In some embodiments, the improved activity and / or stereoselectivity of the engineered nitroaldolase polypeptide disclosed herein provides a method in which a higher conversion percentage can be achieved with a lower concentration of the engineered polypeptide. In some embodiments of the method, suitable reaction conditions include 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), 150% (w / w), 175% (w / w), 200% (w / w) or more of the engineered polypeptide amount of the substrate compound load. In some embodiments, the engineered polypeptide is present at about 0.01 g / L to about 130 g / L; about 0.05 g / L to about 100 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 nitroaldolase 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, 50 g / L, 80 g / L, or 100 g / L. During the reaction process, the pH of the reaction mixture can change. The pH of the reaction mixture can be maintained at a desired pH or within a desired pH range. This can be achieved by adding acid or alkali before the reaction process and / or during the reaction process. Alternatively, pH can be controlled by using a buffer. Accordingly, in some embodiments, reaction conditions include a buffer. Suitable buffers for maintaining a desired pH range are known in the art, and include, for example but not limited to, sodium citrate, borate, phosphate, 2-(N-morpholinyl) ethanesulfonic acid (MES), 3-(N-morpholinyl) propanesulfonic acid (MOPS), acetate, triethanolamine and 2-amino-2-hydroxymethyl-propane-1,3-diol (Tris) etc. In some embodiments, reaction conditions include water as a suitable solvent, without the presence of a buffer. In the embodiment of the method, reaction conditions include suitable pH. The desired pH or desired pH range can be maintained by using an acid or base, a suitable buffer or a combination of buffering and adding an acid or base. The pH of the reaction mixture can be controlled before the reaction process and / or during the reaction process. In some embodiments, suitable reaction conditions include a solution pH of about 4 to about 10, a pH of about 4 to about 6, a pH of about 5 to about 6, a pH of about 6 to about 9, a pH of about 5 to about 7. In some embodiments, reaction conditions include a solution pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10. In the embodiment of the method herein, considering, for example, the increase of the reaction rate at a higher temperature and the activity of the enzyme during the reaction time period, suitable temperature is used for reaction conditions. Therefore, in some embodiments, suitable reaction conditions include a temperature of about 10 ℃ to about 60 ℃, about 10 ℃ to about 55 ℃, about 15 ℃ to about 60 ℃, about 20 ℃ to about 60 ℃, about 20 ℃ to about 55 ℃, about 25 ℃ to about 55 ℃ or about 30 ℃ to about 50 ℃. In some embodiments, suitable reaction conditions include a temperature of about 10 ℃, 15 ℃, 20 ℃, 22 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ or 60 ℃. In some embodiments, the temperature during the enzymatic reaction can be maintained at a specific temperature during the entire reaction process. In some embodiments, the temperature during the enzymatic reaction can be adjusted during the reaction process along with the temperature curve. In some embodiments, the method of the present invention is carried out in a solvent. Suitable solvents include water, aqueous buffer solutions, organic solvents, polymer solvents and / or co-solvent systems, which generally include aqueous solvents, organic solvents and / or polymer solvents. Aqueous solvents (water or aqueous co-solvent systems) can be pH buffered or non-buffered. In some embodiments, the method using engineered nitroaldolase polypeptides can be carried out in an aqueous co-solvent system comprising: organic solvents (e.g., ethanol, isopropanol (IPA), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethyl acetate, butyl acetate, 1-octanol, heptane, octane, methyl tert-butyl ether (MTBE), toluene, etc.), ions or polar solvents (e.g., 1-ethyl-4-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium tetrafluoroborate, 1-butyl-3-methylimidazolium hexafluorophosphate, glycerol, polyethylene glycol, etc.). In some embodiments, cosolvent can be a polar solvent, such as polyol, dimethyl sulfoxide (DMSO) or lower alcohol. The non-aqueous cosolvent component in the aqueous cosolvent system can be miscible with the aqueous component, providing a single liquid phase, or can be miscible or immiscible with the aqueous component part, providing a two-liquid phase. Exemplary aqueous cosolvent systems can comprise water and one or more cosolvents selected from organic solvents, polar solvents and polyol solvents. Typically, the cosolvent component of the aqueous cosolvent system is selected so that the nitroaldolase is not deactivated unfavorably under reaction conditions. By utilizing enzyme activity assays, such as those described herein, measuring the enzyme activity of the engineered nitroaldolase specified in the candidate solvent system to the substrate of interest to limit, suitable cosolvent systems can be easily identified. In some embodiments of the method, suitable reaction conditions include aqueous co-solvents, wherein the co-solvent includes about 1% to about 50% (v / v), about 1% to about 40% (v / v), about 2% to about 40% (v / v), about 5% to about 30% (v / v), about 10% to about 30% (v / v) or about 10% to about 20% (v / v) of DMSO. In some embodiments of the method, suitable reaction conditions may include an aqueous co-solvent comprising 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) of ethanol. In some embodiments, the reaction conditions include a surfactant for stabilizing or enhancing the reaction. The surfactant may include a nonionic surfactant, a cationic surfactant, an anionic surfactant, and / or an amphiphilic surfactant. Exemplary surfactants include, for example, but are not limited to, nonylphenoxypolyethoxyethanol (NP40), TRITON TMX-100 polyethylene glycol tert-octylphenyl ether, polyoxyethylene-stearamide, hexadecyl trimethyl ammonium bromide, oleyl amido sodium sulfate, polyoxyethylene sorbitan monostearate, hexadecyl 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 0.1 mg / ml to 50 mg / ml, particularly 1 mg / ml to 20 mg / ml. In some embodiments, the reaction conditions include a defoamer, which helps reduce or prevent the formation of foam in the reaction solution, such as when the reaction solution is mixed or sparged. Defoamers include non-polar oils (e.g., mineral oils, silicones, etc.), polar oils (e.g., fatty acids, alkylamines, alkylamides, alkyl sulfates, etc.), and hydrophobes (e.g., treated silica, polypropylene, etc.), some of which also act as surfactants. Exemplary defoamers include (Dow Corning), polyglycol copolymers, oxy / ethoxylated alcohols, and polydimethylsiloxanes. In some embodiments, the defoamer can be present at about 0.001% (v / v) to about 5% (v / v), about 0.01% (v / v) to about 5% (v / v), about 0.1% (v / v) to about 5% (v / v), or about 0.1% (v / v) to about 2% (v / v). In some embodiments, the defoamer can be present at about 0.001% (v / v), about 0.01% (v / v), about 0.1% (v / v), about 0.5% (v / v), about 1% (v / v), about 2% (v / v), about 3% (v / v), about 4% (v / v), or about 5% (v / v) or more, as desired to promote the reaction. The amount of the reactant used in the nitroaldolase reaction will generally vary according to the amount of desired product and the amount of the concomitant nitroaldolase substrate used. One of ordinary skill in the art will readily understand how to change these amounts to tailor them to the level of desired productivity and production scale. In some embodiments, the order in which the reactants are added is not critical. The reactants can be added together simultaneously to a solvent (e.g., a single-phase solvent, a two-phase aqueous co-solvent system, etc.), or alternatively, some reactants can be added separately, and some can be added together at different time points. For example, cofactors, cosubstrates, and substrates can be added to a solvent first. Solid reactants (e.g., enzymes, salts, etc.) can be provided to the reaction in a variety of different forms including powders (e.g., freeze-dried, spray-dried, etc.), solutions, emulsions, suspensions, etc. The reactants can be readily freeze-dried or spray-dried using methods and equipment known to those of ordinary skill in the art. For example, a protein solution can be frozen at -80°C in small aliquots and subsequently added to a pre-cooled freeze drying chamber before applying a vacuum. When an aqueous co-solvent system is used, the nitroaldolase and co-substrate may be added and mixed into the aqueous phase first to improve mixing efficiency. The nitroaldolase substrate may be added and mixed in, followed by the organic phase, or the substrate may be dissolved in the organic phase and mixed in. Alternatively, the nitroaldolase substrate may be premixed in the organic phase and subsequently added to the aqueous phase. The method of the present invention is generally allowed to proceed until further conversion of substrate to product does not vary significantly with reaction time (e.g., less than 10% of substrate is converted or less than 5% of substrate is converted). In some embodiments, the reaction is allowed to continue until there is complete or near-complete conversion of substrate to product. Conversion of substrate to product can be monitored using known methods by detecting substrate and / or product (with or without derivatization). Suitable analytical methods include gas chromatography, HPLC, MS, etc. 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, 30 g / L, 40 g / L, or more for each of the one or more substrates, and wherein the method produces at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more 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. When used in a method under appropriate reaction conditions, the engineered nitroaldolase polypeptides of the invention produce an excess of the desired product over one or more undesired products of at least 30%, 40%, 50%, 60% or greater enantiomeric excess. In some other embodiments of the method for converting one or more substrate compounds into product compounds using engineered nitroaldolase polypeptides, suitable reaction conditions can include each of one or more substrates to the initial substrate loading of the reaction solution, and then contact with the polypeptide. The reaction solution is then further supplemented with other substrate compounds added continuously or in batches, for each of one or more substrates, at least about 1g / L / h, at least about 2g / L / h, at least about 4g / L / h, at least about 6g / L / h or more speed over time. Therefore, according to these suitable reaction conditions, for each of one or more substrates, the polypeptide is added to the solution with an initial substrate loading of at least about 1g / L, 5g / L or 10g / L. After this addition of the polypeptide, additional substrate is then continuously added to the solution at a rate of about 2 g / L / h, 4 g / L / h or 6 g / L / h for each of the one or more substrates until a much higher final substrate loading of at least about 30 g / L or more is reached for each of the 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, followed by 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 more is reached for each of the one or more substrate compounds. The substrate supplementation reaction conditions allow for more substrate loadings to be achieved while maintaining a high conversion rate of at least about 5%, 25%, 50%, 75%, 90% or more of substrate conversion to product for each or both of the one or more substrates. Any method disclosed herein for preparing compound (3) using an engineered polypeptide can be carried out under a suitable range of reaction conditions (including but not limited to a ketone substrate range, a nitroaldolase substrate range, a temperature, a pH, a solvent system, a substrate loading, a polypeptide loading, and a reaction time). In one example, in some embodiments, the preparation of compound (3) can be carried out under suitable reaction conditions including: (a) a ketone substrate loading of the substrate compound of about 0.01M to 1M; (b) an amino acid substrate loading of the substrate compound of about 0.01M to 1M; (c) an engineered polypeptide of about 0.5g / L to 100g / L; (d) a 0.01M-1M sodium citrate buffer; (e) at a pH of 5-8; and (f) a temperature of about 15°C to 60°C. In some embodiments, suitable reaction conditions include: (a) about 0.5 M of compound (1) substrate compound; (b) about 0.3 M of compound (2) substrate compound; (c) about 50 g / L of each engineered polypeptide; (d) 0.1 M sodium citrate buffer; (e) at a static pH of 6, and (g) about 22°C. In some embodiments, additional reaction components or additional techniques are performed to supplement the reaction conditions. These may include taking measures to stabilize the enzyme or prevent enzyme inactivation, reduce product inhibition, and shift the reaction equilibrium toward the formation of the desired product. In further embodiments, any of the above described methods for converting one or more substrate compounds into product compounds may also include one or more steps selected from the following: 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 and / or can be obtained by routine experimentation. In addition, illustrative methods are provided in the Examples below. Various features and embodiments of the invention are illustrated in the following representative examples, which are intended to be illustrative rather than limiting. experiment The following examples, including the experiments and results obtained, are provided for illustrative purposes only and should not be construed as limiting the present invention. In the examples hereinafter, the following abbreviations apply: ppm (parts per million); M (mole / liter); mM (millimol / liter), uM and μM (micromol / liter); nM (nanomoles / 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). number); 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 Bactotryptone, 24 g / L yeast extract, 4 mL / L glycerol, 65 mM potassium phosphate, pH 7.0, 1 mM MgSO4); PLP (pyridoxal 5'-phosphate), TEoA (triethanolamine buffer), 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 common laboratory strain of Escherichia coli, available from the Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput); HPLC (high pressure liquid chromatography); GC (gas chromatography); FIOPC (fold increase over positive control); Microfluidics (Microfluidics, Corp., Westwood, MA); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Difco (Difco Laboratories, BDDiagnostic Systems, Detroit, MI); Agilent (Agilent Technologies, Inc., Santa Clara, CA); Clara, CA); Corning (Corning, Inc., Palo Alto, CA); Dow Corning (Dow Corning, Corp., Midland, MI); and Gene Oracle (Gene Oracle, Inc., Mountain View, CA). Example 1 Production of engineered polypeptides in pCK110900 A polynucleotide (SEQ ID NO: 1) encoding a polypeptide having nitroaldolase activity (SEQ ID NO: 2) was cloned into the pCK110900 vector system (see, e.g., U.S. Pat. No. 9,714,437, which is hereby incorporated by reference in its entirety), and then expressed in E. coli W3110fhuA under the control of the lac promoter. The polynucleotide and related polypeptides are derived from nitrilase present in Saccharomyces cerevisiae. Single colonies were picked in a 96-well format and grown in 190 μL LB medium containing 1% glucose and 30 μg / mL CAM at 30°C, 200 rpm and 85% humidity. After overnight growth, 20 μL of the grown culture was transferred to a deep well plate containing 380 μL TB medium and 30 μg / mL CAM. The culture was grown at 30°C, 250 rpm and 85% humidity for about 2.5 hours. When the optical density (OD 600 ) reached 0.4-0.6, the expression of the nitroaldolase gene was induced by adding IPTG at a final concentration of 1 mM. After induction, growth was continued at 30°C, 250 rpm and 85% humidity for 18-20 hours. The cells were harvested by centrifugation at 4,000 rpm and 4°C for 10 minutes; the supernatant was then discarded. The cell pellet was stored at -80°C until ready for use. Before measuring, the cell pellet was thawed and resuspended in 200 μL of lysis buffer containing 1 g / L lysozyme, 0.5 g / L PMBS and 0.1 M sodium citrate buffer (at pH 6) of 0.025 μL / mL commercial DNA enzyme (New England BioLabs, M0303L). The plate was stirred at room temperature on a microtiter plate oscillator with medium speed shaking for 2 hours. The plate was then centrifuged at 4000 rpm for 15 minutes at 4 ° C, and the clarified supernatant was used for the HTP assay reaction described in the following examples. The shake flask procedure can be used to produce engineered nitroaldolase polypeptide shake flask powder (SFP), which can be used for secondary screening assays and / or for biocatalytic processes described herein. Compared with the cell lysate used in the HTP assay, the shake flask powder preparation of the enzyme provides a more purified preparation of the engineered enzyme (e.g., up to 30% of the total protein), and also allows the use of a more concentrated enzyme solution. In order to start cultivation, a single colony of the Escherichia coli containing a plasmid encoding an interested engineered polypeptide is inoculated into 5mL of an LB cell culture medium containing 30 μg / mL CAM and 1% glucose. The culture is grown overnight (at least 16 hours) at 37°C, 250rpm in an incubator. The growth culture is then added to the TB culture medium containing 30 μg / mL CAM of 250mL in a 1L shake flask. The 250mL culture is grown at 30°C and 250rpm for 3.5 hours until OD 600 Reach 0.6-0.8. Expression of the nitroaldolase gene was induced by adding IPTG at a final concentration of 1 mM, and growth was continued for another 18-20 hours. The cells were harvested by transferring the culture to a pre-weighed centrifuge bottle, which was then centrifuged at 4000 rpm for 20 minutes at 4°C. The supernatant was discarded and the remaining cell pellet was weighed. In some embodiments, the cell pellet is stored at -80°C until ready for use. For lysis, the cell pellet is resuspended in 6 mL / g wet cell weight of 25 mM triethanolamine-HCl buffer, pH 7.5 or 25 mM sodium citrate buffer, pH 6, and 110 L The cells were lysed using a processor system (Microfluidics). Cell debris was removed by centrifugation at 10,000 rpm for 60 minutes at 4°C. The clarified lysate was collected, frozen at -80°C, and then lyophilized using standard methods known in the art. Lyophilized frozen clarified lysate provided a dry shake flask powder containing a crude engineered polypeptide. Example 2 Evolution and screening of engineered polypeptides derived from SEQ ID NO: 2 to improve the production of compound (3) The engineered polynucleotides (SEQ ID NO: 1) encoding the polypeptide SEQ ID NO: 2 having nitroaldolase activity were used to generate the engineered polypeptides of Table 2-1. Compared to the starting polypeptide, these polypeptides exhibit improved nitroaldolase activity under the desired conditions, for example, improved formation of β-nitro alcohol (compound (3)) from the substrates trifluoroacetone and nitromethane (compounds (1) and (2) respectively). Similarly, the engineered polypeptides of Table 2-2 exhibit improved nitroaldolase selectivity for the desired (S)-alcohol. As described below, engineered polypeptides having amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 2. Directed evolution was initiated from the polynucleotide set forth in SEQ ID NO: 1. Libraries of engineered polypeptides were generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assays and analytical methods that measure the ability of the polypeptides to produce compound (3). Enzyme assays were performed in 96-well deep well (1.1 mL total volume) plates with a total reaction volume of 100 μl / well. Reactions contained undiluted 67 v / v% nitroaldolase lysate prepared as described in Example 1, 40 mM trifluoroacetone (1), and 500 mM nitromethane (2) dissolved in 100 mM sodium citrate pH 6. The reaction plates were heat sealed and shaken at 250 rpm at 22° C. for ~22 hours. After overnight incubation (~22 hours), 300 μL / well of methyl tert-butyl ether (MTBE) was added to the reaction plate and mixed thoroughly. The plate was sealed and centrifuged at 4,000 rpm for 10 min. An aliquot of the upper organic phase was removed and added to a shallow 96-well plate for analysis by gas chromatography (GC) as described in Example 8. The hit variants were grown in 250 mL shake flasks as described in Example 1 to produce lyophilized enzyme powders. The activity of the enzyme powders was evaluated by measuring the enzyme dose response of 0-25 g / L nitroaldolase shake flask powders under the assay conditions as described in this Example. These hit variants are listed in Tables 2-1 and 2-2. * express. Example 3 Evolution and screening of engineered polypeptides derived from SEQ ID NO: 24 to improve the production of compound (3) The engineered polypeptides of Table 3-1 were generated using an engineered polynucleotide (SEQ ID NO:23) encoding the polypeptide SEQ ID NO:24 having nitroaldolase activity. Compared to the starting polypeptide, these polypeptides exhibit improved nitroaldolase activity under desired conditions, for example, improved formation of β-nitro alcohol (compound (3)) from substrates trifluoroacetone and nitromethane (compounds (1) and (2) respectively). Similarly, the engineered polypeptides of Table 3-2 exhibit improved nitroaldolase selectivity for the desired (S)-alcohol. As described below, engineered polypeptides having amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO:24. Directed evolution was initiated from the polynucleotide set forth in SEQ ID NO: 23. Libraries of engineered polypeptides were generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assays and analytical methods that measure the ability of the polypeptides to produce compound (3). Enzyme assays and analyses were performed using methods similar to those described in Example 2. High throughput cell pellets were lysed in 400 μL / well lysis buffer and reacted with 93 v / v% clarified lysate, 0.3 M trifluoroacetone (1) and 0.5 M nitromethane (2). The hit variants were grown in 250 mL shake flasks and enzyme powders were generated as described in Example 1. The activity of the enzyme powder was evaluated using an assay similar to that described in Example 2 at 0-100 g / L nitroaldolase SFP, 0.3 M trifluoroacetone (1), 0.5 M nitromethane (2), 0.1 M sodium citrate (pH 6) at 22° C. for 22 hours. The hit variants are listed in Tables 3-1 and 3-2. * express. Example 4 Evolution and screening of engineered polypeptides derived from SEQ ID NO: 496 to improve the production of compound (3) The engineered polypeptides of Table 4-1 were generated using an engineered polynucleotide (SEQ ID NO:495) encoding the polypeptide SEQ ID NO:496 having nitroaldolase activity. Compared to the starting polypeptide, these polypeptides exhibit improved nitroaldolase activity under desired conditions, for example, an improvement in the formation of β-nitro alcohol (compound (3)) from substrates trifluoroacetone and nitromethane (compounds (1) and (2) respectively). Similarly, the engineered polypeptides of Table 4-2 exhibit improved nitroaldolase selectivity for the desired (S)-alcohol. As described below, engineered polypeptides having amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO:496. Directed evolution was initiated from the polynucleotide set forth in SEQ ID NO: 495. Libraries of engineered polypeptides were generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assays and analytical methods that measure the ability of the polypeptides to produce compound (3). Enzyme assays and analyses were performed using the same methods as described in Example 3. The hit variants were grown in 250 mL shake flasks and enzyme powders were generated as described in Example 1. The activity of the enzyme powders was evaluated as described in Example 3. These hit variants are listed in Tables 4-1 and 4-2. * express. Example 5 Evolution and screening of engineered polypeptides derived from SEQ ID NO: 860 to improve the production of compound (3) The engineered polynucleotides (SEQ ID NO: 859) encoding the polypeptide SEQ ID NO: 860 having nitroaldolase activity were used to generate the engineered polypeptides of Table 5-1. Compared to the starting polypeptides, these polypeptides exhibit improved nitroaldolase activity under the desired conditions, for example, an improvement in the formation of β-nitro alcohol (compound (3)) from the substrates trifluoroacetone and nitromethane (compounds (1) and (2) respectively). Similarly, the engineered polypeptides of Table 5-2 exhibit improved nitroaldolase selectivity for the desired (S)-alcohol. As described below, engineered polypeptides having amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 860. Directed evolution was initiated from the polynucleotide set forth in SEQ ID NO: 859. Libraries of engineered polypeptides were generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assays and analytical methods that measure the ability of the polypeptides to produce compound (3). Enzyme assays and analyses were performed using the same methods as described in Example 3. The hit variants were grown in 250 mL shake flasks and enzyme powders were generated as described in Example 1. The activity of the enzyme powders was evaluated as described in Example 3. These hit variants are listed in Tables 5-1 and 5-2. * express. Example 6 Evolution and screening of engineered polypeptides derived from SEQ ID NO: 1120 to improve the production of compound (3) The engineered polynucleotides (SEQ ID NO: 1119) encoding the polypeptide SEQ ID NO: 1120 having nitroaldolase activity were used to generate the engineered polypeptides of Table 6-1. Compared to the starting polypeptides, these polypeptides exhibit improved nitroaldolase activity under the desired conditions, for example, an improvement in the formation of β-nitro alcohol (compound (3)) from the substrates trifluoroacetone and nitromethane (compounds (1) and (2) respectively). Similarly, the engineered polypeptides of Table 6-2 exhibit improved nitroaldolase selectivity for the desired (S)-alcohol. As described below, engineered polypeptides having amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 1120. Directed evolution was initiated from the polynucleotide set forth in SEQ ID NO: 1119. Libraries of engineered polypeptides were generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assays and analytical methods that measure the ability of the polypeptides to produce compound (3). Enzyme assays and analyses were performed using the methods described in Example 3, except that the nitromethane (2) concentration was increased to 0.75 M and the pH was lowered to pH 5.5. The hit variants were grown in 250 mL shake flasks and enzyme powders were generated as described in Example 1. The activity of the enzyme powders was evaluated as described in Example 3, except that the concentration of trifluoroacetone (1) was increased to 0.525 M, the concentration of nitromethane was increased to 0.75 M, and the pH was decreased to pH 5.5; the hit variants were indicated in Tables 6-1 and 6-2 using * express. Example 7 Evolution and screening of engineered polypeptides derived from SEQ ID NO: 1348 to improve the production of compound (3) The engineered polynucleotides (SEQ ID NO: 1347) encoding the polypeptide SEQ ID NO: 1348 having nitroaldolase activity were used to generate the engineered polypeptides of Table 7-1. Compared to the starting polypeptides, these polypeptides exhibit improved nitroaldolase activity under the desired conditions, for example, an improvement in the formation of β-nitro alcohol (compound (3)) from the substrates trifluoroacetone and nitromethane (compounds (1) and (2) respectively). Similarly, the engineered polypeptides of Table 7-2 exhibit improved nitroaldolase selectivity for the desired (S)-alcohol. As described below, engineered polypeptides having amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 1348. Directed evolution was initiated from the polynucleotide set forth in SEQ ID NO: 1347. Libraries of engineered polypeptides were generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using HTP assays and analytical methods that measure the ability of the polypeptides to produce compound (3). Enzyme assays and analyses were performed using the methods described in Example 6, except that the trifluoroacetone (1) concentration was increased to 0.525 M and the reaction time was decreased to 6 hours. The hit variants were grown in 250 mL shake flasks and enzyme powders were generated as described in Example 1. The activity of the enzyme powders was evaluated as described in Example 6, except that the reaction time was reduced to 6 hours; the hit variants were indicated in Tables 7-1 and 7-2 using * express. Example 8 Analysis and detection of the conversion of compounds (1) and (2) into compound (3) The data described in Example 2 to Example 4 were collected using the analysis methods provided in Table 8-1 and Table 8-2. The methods provided herein can be used to analyze variants generated using the present invention. However, it is not intended that the present invention be limited to the methods described herein, as there are other suitable known methods in the art that can be applied to analyze variants provided herein and / or variants generated using the methods provided herein. The data described in Example 5 were collected using the analysis methods provided in Table 8-3 and Table 8-4. The data described in Example 6 and Example 7 were collected using the analysis methods provided in Table 8-4. The methods provided herein can be used to analyze variants generated using the present invention. However, it is not intended that the present invention be limited to the methods described herein, as there are other suitable known methods in the art that can be applied to analyze variants provided herein and / or variants generated using the methods provided herein. All publications, patents, patent applications, and other documents cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes. While various specific embodiments have been illustrated and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention.

Claims

1. An engineered nitroaldolase polypeptide, comprising a polypeptide sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2, 24, 496, 860, 1120 and / or 1348, or a functional fragment thereof, wherein the engineered nitroaldolase polypeptide comprises at least one substitution or set of substitutions in the polypeptide sequence, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 2, 24, 496, 860, 1120 and / or 1348.

2. The engineered nitroaldolase polypeptide of claim 1, wherein the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2,wherein the engineered nitroaldolase polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 11, 7 / 9, 7 / 9 / 33 / 68 / 72 / 158, 7 / 9 / 33 / 72 / 175 / 197 / 230, 7 / 9 / 33 / 72 / 230, 7 / 9 / 68 / 72 / 197, 7 / 9 / 158 / 175, 7 / 9 / 175, 7 / 9 / 197, 7 / 33, 7 / 68 / 72 / 175, 7 / 72, 7 / 72 / 97 / 175 / 230, 9, 9 / 11 / 33 / 72 / 197 / 230, 9 / 33 / 68 / 158 / 197, 9 / 33 / 72 / 230 , 9 / 33 / 97 / 230, 9 / 68 / 72, 9 / 97 / 175 / 202 / 218, 9 / 175, 9 / 202, 11, 13, 14 / 33 / 68 / 72 / 97, 18, 18 / 33 / 68 / 97 / 158 / 175 / 239, 18 / 33 / 72 / 97 / 158 / 175, 18 / 33 / 72 / 97 / 158 / 239, 18 / 33 / 72 / 97 / 175 / 202, 18 / 33 / 72 / 175 / 202 / 237 / 239, 18 / 33 / 72 / 197 / 237, 18 / 68 / 72, 18 / 68 / 72 / 158 / 175 / 237 / 239, 18 / 68 / 72 / 175 / 202 / 239、18 / 68 / 72 / 239、18 / 68 / 175 / 223 / 237 / 239、18 / 68 / 239、18 / 72 / 97 / 175 / 237 / 239、18 / 72 / 175 / 197、18 / 72 / 237、18 / 72 / 239、18 / 97 / 175 / 237 / 239、18 / 97 / 223 / 237 / 239、18 / 175、18 / 197 / 239、18 / 239、20 / 124、33 / 68 / 72、33 / 68 / 72 / 175 / 230、33 / 68 / 223 / 239、33 / 72、33 / 72 / 97 / 197, 33 / 72 / 175, 33 / 175, 39, 44 / 97, 52, 68 / 72, 68 / 72 / 97, 68 / 72 / 97 / 197, 68 / 72 / 197, 68 / 72 / 197 / 239, 68 / 97 / 175, 68 / 97 / 175 / 197 / 223, 72 / 97 / 158 / 223 / 237, 72 / 175 / 197, 97 / 158 / 175 / 197, 97 / 175, 103, 121, 122, 124, 125, 128, 133, 148, 175, 197, and 197 / 202, and wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO: 2., 3. The engineered nitroaldolase polypeptide of claim 1, wherein the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 2, wherein the engineered nitroaldolase polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 7 / 11 / 33 / 72 / 97, 7 / 11 / 33 / 9 7, 7 / 11 / 72, 7 / 11 / 158, 7 / 11 / 237, 7 / 33 / 72 / 97 / 158 / 175 / 197 / 237 / 239, 9 / 11 / 33, 9 / 11 / 33 / 72, 9 / 11 / 72, 9 / 33 / 68 / 97 / 237 / 239, 9 / 72 / 97 / 158 / 237 / 239, 9 / 218 / 239, 10, 11, 12, 13, 14, 18, 33 / 68 / 72 / 97 / 237 / 239, 33 / 72 / 158 / 239, 39, 40, 41, 48, 49, 51, 54, 54 / 126, 57, 68 / 175 / 239, 82, 84, 85, 86, 103, 104, 105, 107, 115, 117, 119, 119 / 120, 120, 122, 123, 124, 127, 130 , 131, 133, 134, 137 / 145, 144, 145, 146, 147, 148, 149, 150, 152, 157, 158, 159, 169, 173, 174, 175, 180, 181, 183, 203, 204, 208, 209, 210, 215, 218, 236 and 237, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

2.

4. The engineered nitroaldolase polypeptide of claim 1, wherein the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 24, wherein the engineered nitroaldolase polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 9 / 39 / 122 / 157, 18, 18 / 33 / 39 / 230, 18 / 33 / 175, 18 / 39 / 44 / 118 、18 / 175、20、33 / 39、33 / 39 / 44 / 118 / 121、33 / 39 / 44 / 121、33 / 39 / 44 / 152、33 / 39 / 44 / 175、33 / 39 / 44 / 197、33 / 39 / 44 / 210 / 230、33 / 39 / 118 / 121 / 175、33 / 39 / 121 / 175、33 / 39 / 197 / 210、33 / 39 / 210、33 / 39 / 210 / 230、33 / 118 / 121, 33 / 118 / 121 / 175, 39, 39 / 44 / 103, 39 / 44 / 118, 39 / 44 / 152 / 230, 39 / 44 / 175 / 197 / 230, 39 / 103, 39 / 103 / 125, 39 / 103 / 125 / 127 / 146, 39 / 103 / 125 / 127 / 146 / 150, 39 / 103 / 125 / 146, 39 / 103 / 127 / 150, 39 / 103 / 150, 39 / 118 / 121, 39 / 197, 39 / 197 / 210, 43, 44 / 103, 81, 103, 103 / 125 / 127, 103 / 125 / 146, 103 / 127, 103 / 150, 118 / 121, 118 / 121 / 175, 118 / 121 / 197, 121, 152, 152 / 197, 154, 159, 163, 175, 210, 232 and 238, and wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:

24.

5. The engineered nitroaldolase polypeptide of claim 1, wherein the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 24, wherein the engineered nitroaldolase polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 9 / 13, 9 / 13 / 39, 9 / 13 / 39 / 68 / 72 / 239, 9 / 13 / 39 / 68 / 239, 9 / 13 / 39 / 72 / 122, 9 / 13 / 39 / 97 / 239, 9 / 13 / 68 / 72 / 97 / 239, 9 / 13 / 68 / 97 / 239. 39, 9 / 13 / 68 / 97 / 239, 9 / 13 / 72, 9 / 13 / 134, 11, 12 / 42 / 125 / 127, 12 / 103, 12 / 103 / 150, 13, 13 / 39, 13 / 39 / 68, 13 / 39 / 68 / 72 / 97, 13 / 39 / 72 / 97 / 122 / 134 / 223, 13 / 39 / 97 / 239, 13 / 39 / 239, 13 / 68, 13 / 68 / 72 / 97 / 122 / 239, 16, 18 / 33 / 39 / 44 / 118 / 121 / 175 / 197 / 210, 18 / 33 / 39 / 44 / 121, 18 / 33 / 39 / 112 / 118 / 121 / 197、18 / 33 / 39 / 121、18 / 33 / 39 / 121 / 175、18 / 33 / 39 / 121 / 175 / 197、18 / 33 / 39 / 121 / 175 / 210、18 / 33 / 39 / 121 / 210、18 / 33 / 118 / 121、18 / 33 / 121、197、18 / 33 / 121 / 210 / 230、18 / 39 / 44 / 118 / 121 / 230、18 / 39 / 44 / 121 / 175 / 210、18 / 39 / 118 / 121、18 / 39 / 118 / 121 / 210、18 / 39 / 121 / 175、18 / 39 / 121 / 175 / 210、18 / 39 / 121 / 197 / 210、18 / 118 / 121、18 / 118 / 121 / 175、18 / 118 / 121 / 210、18 / 118 / 121 / 230、18 / 121、18 / 121 / 175、18 / 121 / 210、18 / 121 / 230、20、21、33 / 39 / 44 / 118 / 121、33 / 39 / 44 / 118 / 121 / 210 / 230、33 / 39 / 44 / 121、33 / 39 / 44 / 121 / 152、33 / 39 / 44 / 121 / 175 / 210、33 / 39 / 44 / 121 / 230、33 / 39 / 73 / 121 / 197、33 / 39 / 118 / 121 / 175、33 / 39 / 118 / 121 / 175 / 230、33 / 39 / 121、33 / 39 / 121 / 175、33 / 39 / 121 / 210 / 230、33 / 118 / 121、33 / 118 / 121 / 175、33 / 118 / 121 / 197、33 / 121、33 / 121 / 230、37 / 91、39 / 44 / 118 / 121 / 175 / 210, 39 / 68 / 72 / 239, 39 / 118 / 121, 39 / 118 / 121 / 210, 39 / 121, 39 / 121 / 210 / 230, 43, 44, 76, 79, 101, 118 / 121, 118 / 121 / 175, 118 / 121 / 197, 121, 121 / 175 / 230, 121 / 210, 148, 153, 159, 161, 172, 200, 207, 233, 245 and 248, and wherein the amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO:

24.

6. The engineered nitroaldolase polypeptide of claim 1, wherein the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 496, wherein the engineered nitroaldolase polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 43 / 103, 43 / 103 / 172 / 238, 43 / 103 / 238 / 241, 43 / 238, 81 / 232, 103, 103 / 159 / 238, 103 / 194 / 238, 103 / 238, 122, 232, 238 and 238 / 241, and wherein the amino acid positions of the polypeptide sequence are referenced to SEQ ID NO: NO:

496.

7. The engineered nitroaldolase polypeptide of claim 1, wherein the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 496, wherein the engineered nitroaldolase polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 12, 43, 43 / 47 / 103, 43 / 47 / 103 / 232, 43 / 103, 43 / 103 / 121 / 172 / 241, 43 / 103 / 172, 43 / 103 / 172 / 238, 43 / 103 / 238, : 43 / 103 / 238 / 241, 43 / 121, 43 / 194 / 238, 43 / 238, 44 / 103 / 121 / 238, 44 / 103 / 238, 44 / 103 / 238 / 241, 44 / 121 / 238 / 241, 47 / 103, 63, 103, 103 / 121, 103 / 121 / 172 / 238, 103 / 159 / 238, 103 / 172 / 238, 103 / 194 / 238, 103 / 238, 122, 131, 146, 178, 180 and 221, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

496.

8. The engineered nitroaldolase polypeptide of claim 1, wherein the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 860, wherein the engineered nitroaldolase polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 12, 12 / 43 / 63 / 103 / 146 / 180 / 232 / 241, 12 / 43 / 63 / 103 / 146 / 241, 12 / 43 / 81 / 103 / 241, 12 / 43 / 63 / 103 / 146 / 241. / 81 / 146 / 232 / 241、12 / 43 / 81 / 180 / 241、12 / 43 / 81 / 232 / 241、12 / 43 / 81 / 241、12 / 43 / 103、12 / 43 / 103 / 146 / 180 / 232 / 241、12 / 43 / 103 / 146 / 180 / 241、12 / 43 / 103 / 180 / 241、12 / 43 / 103 / 241、12 / 43 / 146 / 180 / 232 / 241、12 / 43 / 180 / 241、12 / 63 / 81 / 103 / 24 1, 12 / 81, 12 / 81 / 180 / 241, 12 / 103 / 146 / 241, 12 / 103 / 180 / 241, 12 / 146, 12 / 146 / 180 / 241, 12 / 180 / 232 / 241, 12 / 241, 18, 43 / 81 / 232 / 241, 43 / 103 / 146 / 180, 43 / 103 / 180 / 241, 43 / 103 / 241, 43 / 180 / 232, 43 / 232 / 241, 63 / 103 / 180 / 232, 78, 81, 81 / 103 / 146, 81 / 146 / 180 / 241, 81 / 146 / 241, 81 / 180 / 241, 84, 103, 103 / 146, 103 / 146 / 180, 103 / 146 / 180 / 241, 103 / 180, 103 / 180 / 241, 103 / 261, 144, 145, 146 / 180 / 232, 146 / 180 / 241, 146 / 241, 150, 166, 172, 180 / 232, 232 / 241 and 241, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

860.

9. The engineered nitroaldolase polypeptide of claim 1, wherein the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 860, wherein the engineered nitroaldolase polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 9, 12, 12 / 43 / 63 / 103 / 146 / 180 / 232 / 241, 12 / 43 / 63 / 103 / 146 / 241, 12 / 43 / 10 3 / 146 / 180 / 232 / 241、12 / 43 / 103 / 146 / 180 / 241、12 / 43 / 103 / 146 / 232 / 241、12 / 43 / 103 / 146 / 241、12 / 43 / 103 / 180 / 241、12 / 43 / 146 / 180 / 232 / 241、12 / 43 / 146 / 241、12 / 43 / 180 / 232、12 / 43 / 180 / 241、12 / 63 / 103 / 146 / 232 / 241、12 / 103 / 14 6 / 241、12 / 103 / 180 / 241、12 / 146、12 / 146 / 180 / 241、12 / 146 / 232 / 241、12 / 146 / 241、12 / 180、12 / 180 / 232 / 241、12 / 180 / 241、12 / 241、18、43 / 63 / 103 / 180 / 232 / 241、43 / 103 / 146 / 180、43 / 103 / 180、43 / 103 / 180 / 232 / 241、43 / 103 / 180 / 24 1, 43 / 180 / 232, 63 / 103 / 180 / 232, 103, 103 / 146 / 180, 103 / 146 / 180 / 241, 103 / 180, 103 / 180 / 241, 103 / 241, 118, 119, 122, 144, 145, 146 / 180 / 241, 154, 160, 172, 180 / 232, 180 / 232 / 241, 180 / 241, 208, 211 and 212, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:

860.

10. The engineered nitroaldolase polypeptide of claim 1, wherein the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1120, wherein the engineered nitroaldolase polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 3, 7, 7 / 158, 7 / 158 / 175, 7 / 158 / 197 / 202, 27 / 43 / 115 / 172, 27 / 43 / 172, 29, 32, 43 / 110 / 115 / 172, 44, 46, 48 / 110 / 115 / 172, 172, 55, 59, 67, 71, 72, 73, 94 / 103 / 118 / 146, 103, 103 / 144 / 146 / 172 / 180, 103 / 146 / 154 / 172 / 180 / 232, 103 / 154, 118, 118 / 144 / 215, 118 / 172 / 180, 137, 142, 146, 146 / 154, 154, 158, 172 / 190, 184, 198, 205, 216, 223, 226, 229, 247, 261 and 263, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1120.

11. The engineered nitroaldolase polypeptide of claim 1, wherein the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1120, wherein the engineered nitroaldolase polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 73, 80, 103 / 146 / 209, 103 / 172 / 209, 118 / 119 / 172 / 209, 135, 144 / 209 / 232, 172 / 180 / 209, 181 and 223, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 1120.

12. The engineered nitroaldolase polypeptide of claim 1, wherein the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1348, wherein the engineered nitroaldolase polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 2, 7 / 46, 39, 46, 46 / 55, 46 / 55 / 72, 46 / 55 / 92, 4 6 / 55 / 118, 46 / 55 / 226 / 257 / 263, 46 / 55 / 263, 46 / 70, 46 / 70 / 92 / 118 / 261 / 263, 46 / 70 / 175 / 263, 46 / 72, 46 / 118, 55 / 257 / 261, 55 / 261 / 263, 55 / 263, 105, 118, 118 / 226 / 261 / 263 and 263, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1348.

13. The engineered nitroaldolase polypeptide of claim 1, wherein the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1348, wherein the engineered nitroaldolase polypeptide comprises at least one substitution or set of substitutions at one or more positions in the polypeptide sequence selected from the group consisting of: 2, 7 / 46, 7 / 46 / 175, 22 / 46 / 55 / 118、27 / 46 / 72、27 / 46 / 146 / 158 / 202、27 / 46 / 175、27 / 118、29 / 70 / 118、39、46、46 / 55、46 / 55 / 59 / 261 / 263、46 / 55 / 72、46 / 55 / 92、46 / 55 / 118、46 / 55 / 118 / 263、46 / 55 / 146、46 / 5 5 / 146 / 202、46 / 55 / 226 / 257 / 263、46 / 55 / 263、46 / 70、46 / 70 / 92 / 118 / 261 / 263、46 / 70 / 175 / 263、46 / 72、46 / 92、46 / 92 / 175、46 / 118、46 / 146、46 / 146 / 257 / 263、55 / 70 / 118、55 / 257 / 26 1, 55 / 257 / 263, 55 / 261 / 263, 55 / 263, 70, 92 / 118, 105, 118, 118 / 226 / 261 / 263, 146 / 257 / 263, 146 / 261 / 263, 192 / 257 / 261, 202, 257 / 261 / 263, 261 / 263 and 263, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO:1348.

14. The engineered nitroaldolase polypeptide of any one of claims 1-13, wherein the engineered nitroaldolase polypeptide comprises an amino acid sequence having at least 80% sequence identity to any even-numbered sequence set forth in SEQ ID NOs: 4-1484.

15. The engineered nitroaldolase polypeptide of any one of claims 1-14, wherein the engineered nitroaldolase polypeptide comprises a polypeptide sequence set forth in the even-numbered sequence of SEQ ID NOs: 4-1484.

16. The engineered nitroaldolase polypeptide of any one of claims 1-15, wherein the engineered nitroaldolase polypeptide comprises a polypeptide sequence that exhibits at least one improved property compared to the engineered nitroaldolase polypeptide of SEQ ID NO:

2.

17. The engineered nitroaldolase polypeptide of claim 16, wherein the improved properties include improved production of compound (3) 18. The engineered nitroaldolase polypeptide of claim 16, wherein the improved properties comprise an improved stereoselectivity for the (S)-product of at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more.

19. The engineered nitroaldolase polypeptide of claim 16, wherein the improved properties include improved activity towards substrates of trifluoroacetone, compound (1), and nitromethane, compound (2).

20. The engineered nitroaldolase polypeptide of any one of claims 1-19, wherein the engineered nitroaldolase polypeptide is purified.

21. A composition comprising at least one engineered nitroaldolase polypeptide provided by any one of claims 1-20.

22. An engineered polynucleotide encoding at least one engineered nitroaldolase polypeptide according to any one of claims 1-20.

23. An engineered polynucleotide sequence encoding at least one engineered polypeptide, wherein the polypeptide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:2, 24, 496, 860, 1120 and / or 1348, wherein the sequence of the polypeptide comprises at least one substitution at one or more positions.

24. An engineered polynucleotide sequence according to claim 22 or 23, wherein the polynucleotide sequence comprises at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, 23, 495, 860, 1119 and / or 1347.

25. The engineered polynucleotide sequence of any one of claims 22-24, wherein the polynucleotide sequence comprises SEQ ID NO: 3, 23, 495, 859, 1119 or 1347.

26. An engineered polynucleotide comprising an odd-numbered sequence listed in SEQ ID NO: 3-1483.

27. A vector comprising the engineered polynucleotide according to any one of claims 22 to 26.

28. The vector of claim 27, further comprising at least one control sequence.

29. A host cell comprising the vector according to claim 27 and / or 28.

30. A host cell, wherein the host cell produces at least one engineered nitroaldolase polypeptide according to any one of claims 1-20.

31. A method for producing an engineered nitroaldolase polypeptide in a host cell, the method comprising culturing the host cell of claim 29 and / or 30 in a culture medium under suitable conditions such that at least one engineered nitroaldolase polypeptide is produced.

32. The method of claim 31 , further comprising the step of recovering the engineered nitroaldolase polypeptide.

33. The method of claim 31 and / or 32, further comprising the step of purifying the at least one engineered nitroaldolase polypeptide.

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