SHC enzymes and enzyme variants

Abstract

Squalenehopene cyclase (SHC) enzymes and variants thereof, and uses thereof for producing (-)-ambrox from homofarnesol and ambroxide from bishomofarnesol.

Description

[Technical Field]

[0001] Technical Field The present invention generally relates to SHC / HAC enzymes and variants thereof. The present invention further relates to various uses of SHC / HAC enzymes and variants thereof, such as, for example, the enzymatic conversion of (3E,7E)-homofarnesol (EEH) to (-)-ambrox or the enzymatic conversion of E,E-bishomofarnesol (bisEEH) to ambra oxide. The present invention also relates to the products of the enzymatic reactions, such as (-)-ambrox or ambra oxide, produced using SHC / HAC enzymes and variants thereof, and various uses of the products. [Background technology]

[0002] background Squalene Hopene Cyclase (SHC) is a membrane-bound enzyme that acts as a biocatalyst for the cyclization of the linear triterpenoid squalene to hopene and hopanol.

[0003] Numerous wild-type and variant SHC enzymes from various bacteria have been demonstrated to be useful for converting (3E,7E)-homofarnesol to (-)-ambrox (e.g., WO 2016 / 170099; WO 2018 / 157021; Neumann & Simon 1986, Biol Chem Hoppe-Seyler 367, 723-729; JP2009060799; Seckler & Poralla 1986, Biochem Biophys Act 356-363; Ochs et al. 1990, J Bacteriol 174, 298-302; WO 2010 / 139719; US 8759043; WO 2012 / 066059; Seitz et al. 2012, J Molecular Catalysis B:Enzymatic 84, 72-77; and Seitz 2012 PhD thesis ( http: / / elib.uni-stuttgart.de / handle / 1 1682 / 1400), the contents of which are incorporated herein by reference. It would be desirable to provide new and improved methods for making (-)-ambrox, e.g., using new SHC enzymes or enzyme variants. It would also be desirable to provide new and improved methods for cyclizing other substrates to form compounds useful, e.g., in or as fragrances. Summary of the Invention

[0004] overview In a first aspect, a process for preparing (-)-ambrox or a mixture comprising (-)-ambrox is provided, the process comprising enzymatically converting (3E,7E)-homofarnesol (EEH) or a mixture of homofarnesol isomers comprising EEH to (-)-ambrox or a mixture comprising (-)-ambrox using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 1 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 , 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 1 47, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 8, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209 , 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363 36, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and wherein the amino acid sequence of the SHC / HAC enzyme variant is , 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 9 6, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 8, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159 , 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190,191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232 2, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 5, 376, 377, 378, 379, 380, 381, 382, ​​or 383 at positions corresponding to W169, A306, and / or G600 of SEQ ID NO: 1, 2, or 3, or at positions in the amino acid sequence of the wild-type SHC / HAC enzyme corresponding to W169, A306, and / or G600 of SEQ ID NO: 1, 2, or 3.

[0005] In embodiments, a process for preparing (-)-ambrox or a mixture containing (-)-ambrox is provided, the process comprising enzymatically converting (3E,7E)-homofarnesol (EEH) or a mixture of homofarnesol isomers containing EEH to (-)-ambrox or a mixture containing (-)-ambrox using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant has an amino acid sequence having at least 30.0% identity or similarity to SEQ ID NOs: 1, 13, 15, 23, 32, and wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications relative to SEQ ID NO: 1 at positions corresponding to positions W169, A306, and / or G600 of SEQ ID NO: 1, or at positions in the amino acid sequence of a wild-type SHC / HAC enzyme corresponding to W169, A306, and / or G600 of SEQ ID NO: 1.

[0006] In an embodiment, a process for preparing ambulaoxide or a mixture containing ambulaoxide is provided, the process comprising enzymatically converting (2,E)-bishomofarnesol (bisEEH) or a mixture of bishomofarnesol isomers containing bisEEH to ambulaoxide or a mixture containing ambulaoxide using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 1 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82 , 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 1 18, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 1 49, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 18 0, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 36, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and wherein the amino acid sequence of the SHC / HAC enzyme variant is SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and 7, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 0, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 , 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, at positions corresponding to positions W169, A306, and / or G600 of SEQ ID NO: 1, 2, or 3, or at positions in the amino acid sequence of the wild-type SHC / HAC enzyme corresponding to W169, A306, and / or G600 of SEQ ID NO: 1, 2, or 3.

[0007] In one embodiment, a process for preparing amblyoxide or a mixture containing amblyoxide is provided, the process comprising enzymatically converting (2,E)-bishomofarnesol (bisEEH) or a mixture of bishomofarnesol isomers containing bisEEH to amblyoxide or a mixture containing amblyoxide using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant has an amino acid sequence having at least 30.0% identity or similarity to SEQ ID NO:1, 13, 15, 23, 32, and wherein the amino acid sequence of the SHC / HAC enzyme variant has amino acid modifications relative to SEQ ID NO:1 at positions corresponding to positions W169, A306, and / or G600 of SEQ ID NO:1, or at positions in the amino acid sequence of a wild-type SHC / HAC enzyme corresponding to W169, A306, and / or G600 of SEQ ID NO:1.

[0008] In an aspect, a process is provided, wherein: - the W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is replaced by G, A or V or functional equivalents thereof, preferably wherein W at position 169 is replaced by G; - A at position 306 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is replaced by V or a functional equivalent thereof; and / or - the G at position 600 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is replaced by A, V, L, I or M or a functional equivalent thereof.

[0009] In an aspect, a process is provided, wherein: - W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is replaced by G.

[0010] In an aspect, a process is provided, wherein: - W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by G; and - an A at position 306 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is replaced by a V.

[0011] In an aspect, a process is provided, wherein: - W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by G; and - G at position 600 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is replaced by M.

[0012] In an aspect, a process is provided, wherein: - W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is replaced by G; - A at position 306 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by V; and - G at position 600 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is replaced by M.

[0013] In an aspect, a process is provided, wherein: SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 1 42, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 1 73, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204 4, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235 , 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 is S; or 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 1 33, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164 4, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195 , 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268 , 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, the amino acid at position 168 of the amino acid sequence of wild-type SHC is S.

[0014] In an embodiment, a process is provided as defined above, wherein the amino acid at position 168 of SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is S (or the amino acid at the position in the amino acid sequence of wild-type SHC corresponding to 168 in SEQ ID NO:1 is S).

[0015] In an embodiment, there is provided a process as defined above, wherein: - the SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 1, 4, 5, 6, or 350 and has at least one of the following mutations: W169G, A306V, and G600M; or - the SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 7, 8, 9, or 386 and has the following mutations: M132R, A224V, I432T, A557T, R613S, and has at least one of the following mutations: W169G, A306V, and G600M; or - the SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 10, 11, 12, or 385, and has the following mutations: M132R, A224V, I432T, and has at least one of the following mutations: W169G, A306V, and G600M.

[0016] In embodiments, processes are provided in which W at position 169 is replaced by G. In an aspect, a process is provided, wherein: The W at position 169 is replaced by G and the A at position 169 is replaced by V. In an aspect, a process is provided, wherein: The W at position 169 is replaced by a G and the G at position 600 is replaced by an M.

[0017] In an aspect, a process is provided, wherein: The W at position 169 is replaced by G, the A at position 306 is replaced by V, and the G at position 600 is replaced by M.

[0018] In an embodiment, there is provided a process as defined above, wherein: - the SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 23, 304, 359, or 360 and has at least one of the following mutations: W172G, A311V, and G609M (Tel SHC / HAC variant); or - the SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 32, 311, 357, or 358 and has the following mutations: W196G, A335V, and G629M (Sco SHC / HAC variant); or - the SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 13, 312, 353, or 354 and has at least one of the following mutations: W222G, A368V, and G667M (Zmo SHC1 variant); or - The SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 15, 313, 355, or 356 and has at least one of the following mutations: W177G, A321V, and G619M (Zmo SHC2 variant).

[0019] In an aspect, a process is provided, wherein: The SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 23, 304, 359, or 360, and has a W172G mutation. In an aspect, a process is provided, wherein: The SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 23, 304, 359, or 360, and has the W172G and A311V mutations.

[0020] In an aspect, a process is provided, wherein: The SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 23, 304, 359, or 360, and has the W172G and G609M mutations. In an aspect, a process is provided, wherein: The SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 23, 304, 359, or 360, and has the following mutations: W172G, A311V, and G609M.

[0021] In an aspect, a process is provided, wherein: The SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 32, 311, 357, or 358, and has a W196G mutation. In an aspect, a process is provided, wherein: wherein the SHC / HAC enzyme variant has an amino acid sequence having at least 70.0% identity to SEQ ID NO: 32, 311, 357, or 358, and has the W196G and A335V mutations.

[0022] In an aspect, a process is provided, wherein: The SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 32, 311, 357, or 358, and has the W196G and G629M mutations. In an aspect, a process is provided, wherein: The SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 32, 311, 357, or 358, and has the following mutations: W196G, A335V, and G629M.

[0023] In an aspect, a process is provided, wherein: The SHC / HAC enzyme variant has an amino acid sequence having at least 70.0% identity to SEQ ID NO: 13, 312, 353, or 354, and has a W222G mutation. In an aspect, a process is provided, wherein: The SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 13, 312, 353, or 354, and has the W222G and A368V mutations.

[0024] In an aspect, a process is provided, wherein: The SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 13, 312, 353, or 354, and has the W222G and G667M mutations. In an aspect, a process is provided, wherein: The SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 13, 312, 353, or 354, and has the following mutations: W222G, A368V, and G667M.

[0025] In an aspect, a process is provided, wherein: The SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 15, 313, 355, or 356, and has a W177G mutation. In an aspect, a process is provided, wherein: The SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 15, 313, 355, or 356, and has the W177G and A321V mutations.

[0026] In an aspect, a process is provided, wherein: The SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 15, 313, 355, or 356, and has the W177G and G619M mutations. In an aspect, a process is provided, wherein: The SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 15, 313, 355, or 356, and has the following mutations: W177G, A321V, and G619M.

[0027] In an embodiment, there is provided a process as defined above, wherein the SHC / AHC enzyme variant is selected from the group consisting of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 305, 306, 304, 302, 311, 312, 313, 353, 354, 355, 356, 357, 358, 359, 360, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 350, 351, or 352.

[0028] In embodiments, there is provided a process as defined above, wherein the SHC / HAC enzyme variant disclosed herein exhibits: Reference SHC enzymes (e.g., wild-type SHCs, e.g., SEQ ID NOs: 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 4, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135 , 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 1 98, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229 9, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260 , 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291,292, 293, 294, 295, 296, 307, 308, 309, 310, 215G2 AacSHC or SHC#65, or the parent SHC enzyme from which the variant is derived, e.g., represented by SEQ ID NOs: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383), - increased substrate specificity for EEH (or for bisEEH) when a homofarnesol or bis-homofarnesol substrate is used; - increased product selectivity when homofarnesol or bis-homofarnesol substrates are used relative to (-)-ambrox (or ambroxide); - increased specificity for specific isomers of the substrate when substrates other than homofarnesol or bis-homofarnesol are used (e.g., ethyl-homofarnesol, hydroxyfarnesylacetone, etc. - see e.g., WO2021 / 110858 and WO2021 / 209482, respectively); - increased productivity, and / or - an increased degree of conversion of EEH (or bisEEH) over the first 3 to 6 hours (or over the first 4, 5, or 6 hours) of the reaction, as well as an increased conversion rate of EEH (or bisEEH).

[0029] In an embodiment, a process as defined above is provided, wherein the enzymatic conversion occurs at a temperature in the range of about 30°C to about 50°C, such as from about 40°C to about 50°C, and / or at a pH in the range of about 5 to about 6.

[0030] In an embodiment there is provided a process as defined above, wherein the process comprises culturing a recombinant host cell that produces the SHC / HAC enzyme variant. In an embodiment, a process as defined above is provided, wherein the recombinant host cell comprises a nucleic acid sequence selected from SEQ ID NOs: 38, 39, 40, 41, 42, 43, 44, 45, or 46.

[0031] In an embodiment, there is provided a process as defined above, wherein the mixture of homofarnesol isomers comprising EEH is an EE:EZ isomer mixture, preferably the EE:EZ isomer mixture is in a weight ratio of 80:20.

[0032] In embodiments, a process is provided as defined above, wherein the weight ratio of the recombinant host cell producing the SHC / HAC enzyme variant to EEH, or to a mixture of homofarnesol isomers including EEH in an 80:20 weight ratio EE:EZ isomer mixture, is about 2:1, 1:1, or about 0.5:1, or about 0.1:1.

[0033] In an aspect, a process is provided, wherein: The weight ratio of recombinant host cells producing the SHC / HAC enzyme variant to EEH or to the mixture of homofarnesol isomers containing EEH in the EE:EZ isomer mixture is 0.4, and the concentration of EEH is 450 g / l.

[0034] In an aspect, a process is provided, wherein: The weight ratio of recombinant host cells producing the SHC / HAC enzyme variant to EEH or to the mixture of homofarnesol isomers containing EEH in the EE:EZ isomer mixture is 1, and the concentration of EEH is 250 g / l.

[0035] In an aspect, a process is provided, wherein: The weight ratio of recombinant host cells producing the SHC / HAC enzyme variant to EEH or to the mixture of homofarnesol isomers containing EEH in the EE:EZ isomer mixture is 1, and the concentration of EEH is 300 g / l.

[0036] In a further aspect, there is provided a SHC / HAC enzyme variant as defined herein. In a further aspect, there is provided (-)-ambrox in solid form, in amorphous or crystalline form, obtained by or obtainable by a process as defined herein.

[0037] In a further aspect, there is provided the use of (-)-ambrox as provided herein as part of a fragrance product or a cosmetic product or a consumer product, such as a fabric care, toiletry, beauty product, cleaning product, detergent product, and / or soap product.

[0038] In a further aspect, there is provided a fragrance or cosmetic or consumer product comprising (-)-ambrox as provided herein. In a further aspect, there is provided a nucleic acid sequence encoding a SHC / HAC enzyme variant as defined herein.

[0039] In a further aspect, there is provided a construct comprising a nucleic acid sequence as defined herein. In a further aspect, there is provided a recombinant host cell comprising a nucleic acid sequence or construct as defined herein. [Brief explanation of the drawings]

[0040] Brief description of the figure [Figure 1] Figure 1: EEH conversion between AacSHC and 215G2SHC P1, P2, and P3 variants. Reactions were performed at 8 g / L EEH (AacSHC wt 4 g / L EEH only), with cells at OD650nm of 10, and under individually optimized reaction conditions (T, pH, SDS).

[0041] [Figure 2]FIG. 2 shows the reaction products of the SHC-catalyzed cyclization of homofarnesol using a mixture of E,E- and E,Z-homofarnesol isomers.

[0042] [Figure 3] Figure 3: Substrate specificity and product selectivity for P1, P2, and P3 SHC variants. Reactions were performed in 8 g / L EEH with an OD of 10 cells, using individually optimized reaction conditions (T, pH, SDS).

[0043] [Figure 4] Figure 4: Homofarnesol cyclization with SHC variants. Reactions were performed at 125 g / L EEH and 250 g / L cells at T, pH, and SDS (SDS:cell ratio) conditions defined as optimal for each variant.

[0044] [Figure 5] Figure 5: Biotransformation of homofarnesol with SHC variants. Reactions were performed at 125 g / L EEH and 250 g / L cells at T, pH, and SDS (SDS:cell ratio) conditions defined as optimal for each variant.

[0045] [Figure 6] Figure 6: Substrate specificity and product selectivity for the biotransformation of homofarnesol by SHC enzyme variants. Reactions were performed at 125 g / L E,Z-homofarnesol and 250 g / L cells under the optimal T, pH, and SDS (SDS:cell ratio) conditions for each of the SHC enzymes.

[0046] [Figure 7] Figure 7: Biotransformation of homofarnesol with SHC variants. Reactions were performed at 250 g / L EEH and 250 g / L cells under conditions of T, pH, and SDS (SDS:cell ratio) defined as optimal for each variant.

[0047] [Figure 8]Figure 8: Biotransformation of homofarnesol with SHC variants. Reactions were performed with A: 250 g / L EEH and SHC#65 P2, and B: 300 g / L EEH and SHC#65 at various [EEH]:[cell] ratios and T, pH, and SDS (SDS:cell ratio) conditions defined as optimal for each variant.

[0048] [Figure 9-1] Figure 9: Alignment of squalene hopene cyclase amino acid sequences. The figure shows an amino acid sequence alignment of squalene hopene cyclase enzymes listed in Table 3 and / or Table 16 prepared with CLUSTAL O (1.2.4). The amino acids at positions 169, 306, and 600 of the Alicyclobacillus acidocaldarius (AacSHC) sequence are highlighted in white on a black background. The W at position 169 is almost strictly conserved throughout the aligned amino acid sequences (15 of 16 sequences), as is the G at position 600, a single conservative substitution. The A at position 306 is less conserved (9 of 16 sequences). [Figure 9-2] Figure 9: Alignment of squalene hopene cyclase amino acid sequences. The figure shows an amino acid sequence alignment of squalene hopene cyclase enzymes listed in Table 3 and / or Table 16 prepared with CLUSTAL O (1.2.4). The amino acids at positions 169, 306, and 600 of the Alicyclobacillus acidocaldarius (AacSHC) sequence are highlighted in white on a black background. The W at position 169 is almost strictly conserved throughout the aligned amino acid sequences (15 of 16 sequences), as is the G at position 600, a single conservative substitution. The A at position 306 is less conserved (9 of 16 sequences). [Figure 9-3]Figure 9: Alignment of squalene hopene cyclase amino acid sequences. The figure shows an amino acid sequence alignment of squalene hopene cyclase enzymes listed in Table 3 and / or Table 16 prepared with CLUSTAL O (1.2.4). The amino acids at positions 169, 306, and 600 of the Alicyclobacillus acidocaldarius (AacSHC) sequence are highlighted in white on a black background. The W at position 169 is almost strictly conserved throughout the aligned amino acid sequences (15 of 16 sequences), as is the G at position 600, a single conservative substitution. The A at position 306 is less conserved (9 of 16 sequences). [Figure 9-4] Figure 9: Alignment of squalene hopene cyclase amino acid sequences. The figure shows an amino acid sequence alignment of squalene hopene cyclase enzymes listed in Table 3 and / or Table 16 prepared with CLUSTAL O (1.2.4). The amino acids at positions 169, 306, and 600 of the Alicyclobacillus acidocaldarius (AacSHC) sequence are highlighted in white on a black background. The W at position 169 is almost strictly conserved throughout the aligned amino acid sequences (15 of 16 sequences), as is the G at position 600, a single conservative substitution. The A at position 306 is less conserved (9 of 16 sequences).

[0049] [Figure 10] Figure 10: Cyclization of bis-homofarnesol in SHC variants. Reactions were performed with 4 g / L E,E-bis-homofarnesol and cells with an OD of 10, applying conditions individually defined as optimal for SHC#65 and SHC#65 P2 with respect to T, pH, and SDS concentration.

[0050] [Figure 11] Figure 11: Cyclization of homofarnesol with wild-type and variant SHC enzymes. Reactions were carried out in deionized water at 30°C with 2.36 g / L homofarnesol and cells with an OD650nm of 6.

[0051] [Figure 12] Figure 12: Biotransformation of homofarnesol in SHC#65 P2. Reactions were performed with 400 g / L EEH and cells producing SHC#65 P2 at [cell]:[EEH] ratios of 0.6, 0.5, 0.4, and 0.3. Reactions were performed at 35°C, pH 5.8, and a constant [SDS]:[cell] ratio of 0.025.

[0052] [Figure 13] Figure 13: Biotransformation of homofarnesol in SHC#65 P2. Reactions were carried out with 450 g / L EEH and 180 g / L cells ([cells]:[EEH] ratio of 0.4) or 250 g / L EEH and 250 g / L cells ([cells]:[EEH] ratio of 1.0) at 35°C, pH 5.8, and a constant [SDS]:[cells] ratio of 0.030. DETAILED DESCRIPTION OF THE INVENTION

[0053] Array Overview SEQ ID NO: 1 is the wild-type Alicyclobacillus acidocaldarius (Aac) SHC amino acid sequence. SEQ ID NO:2 corresponds to SEQ ID NO:1 with the substitutions M132R, A224V, I432T, A557T, and R613S, and is sometimes referred to herein as SHC / HAC enzyme variant #65 or SHC#65 variant.

[0054] SEQ ID NO: 3, sometimes referred to as 215G2 SHC (or 215G2 SHC variant), corresponds to the wild-type AacSHC amino acid sequence with the mutations M132R, A224V, and I432T. SEQ ID NO: 4 corresponds to SEQ ID NO: 1 with the substitution W169G (also called Aac SHC P1).

[0055] SEQ ID NO: 5 corresponds to SEQ ID NO: 1 with the substitutions W169G and G600M (also called Aac SHC P2). SEQ ID NO: 6 corresponds to SEQ ID NO: 1 with the substitutions W169G, A306V, and G600M (also called Aac SHC P3).

[0056] SEQ ID NO: 7 corresponds to SEQ ID NO: 2 with the substitution W169G (also referred to as the SHC#65 P1 variant). SEQ ID NO: 8 corresponds to SEQ ID NO: 2 with the substitutions W169G and G600M (also referred to as the SHC#65 P2 variant).

[0057] SEQ ID NO: 9 corresponds to SEQ ID NO: 2 with the substitutions W169G, A306V, and G600M (also referred to as the SHC#65 P3 variant). SEQ ID NO: 10 corresponds to SEQ ID NO: 3 with the substitution W169G (also referred to as the 215G2 SHC P1 variant).

[0058] SEQ ID NO: 11 corresponds to SEQ ID NO: 3 with the substitutions W169G and G600M (also referred to as the 215G2 SHC P2 variant). SEQ ID NO: 12 corresponds to SEQ ID NO: 3 with the substitutions W169G, A306V, and G600M (also referred to as the 215G2 SHC P3 variant).

[0059] SEQ ID NOs: 13 to 34, SEQ ID NO: 384 are wild-type SHC enzymes that may be used as SEQ ID NO: 1 in the present invention (see also Tables 3 and 16). SEQ ID NO: 13 is identical to SEQ ID NO: 384. SEQ ID NO: 35 is the nucleotide sequence encoding wild-type AacSHC.

[0060] SEQ ID NO: 36 is a nucleotide sequence encoding the polypeptide of SEQ ID NO: 2 (SHC#65 variant). SEQ ID NO: 37 is the nucleotide sequence encoding the 215G2 variant (SEQ ID NO: 3). SEQ ID NO:38 is the nucleotide sequence encoding AacSHC P1 represented by SEQ ID NO:4.

[0061] SEQ ID NO:39 is the nucleotide sequence encoding AacSHC P2 represented by SEQ ID NO:5. SEQ ID NO:40 is the nucleotide sequence encoding AacSHC P3 represented by SEQ ID NO:6. SEQ ID NO:41 is the nucleotide sequence encoding the SHC#65 P1 variant represented by SEQ ID NO:7.

[0062] SEQ ID NO:42 is the nucleotide sequence encoding the SHC#65 P2 variant represented by SEQ ID NO:8. SEQ ID NO:43 is the nucleotide sequence encoding the SHC#65 P3 variant represented by SEQ ID NO:9.

[0063] SEQ ID NO:44 is the nucleotide sequence encoding the 215G2 P1 variant represented by SEQ ID NO:10. SEQ ID NO:45 is the nucleotide sequence encoding the 215G2 P2 variant represented by SEQ ID NO:11.

[0064] SEQ ID NO:46 is the nucleotide sequence encoding the 215G2 P3 variant represented by SEQ ID NO:12. SEQ ID NO:47 corresponds to SEQ ID NO:1 with the substitutions M132R, A224V, I432T, Y81H, A557T, and R613S, and is sometimes referred to herein as SHC / HAC enzyme variant #66.

[0065] SEQ ID NO:48 corresponds to SEQ ID NO:1 with the substitutions M132R, A224V, I432T, Y81H, H431L, and A557T, and is sometimes referred to herein as SHC / HAC enzyme variant #110B8. SEQ ID NO:49 corresponds to SEQ ID NO:1 with the substitutions M132R, A224V, I432T, T90A, and R613S, and is sometimes referred to herein as SHC / HAC enzyme variant #90C7.

[0066] SEQ ID NO:50 corresponds to SEQ ID NO:1 with the substitutions M132R, A224V, I432T, A172T, and M277K, and is sometimes referred to herein as SHC / HAC enzyme variant #115A7. SEQ ID NO: 51 is the amino acid sequence of the SHC / HAC variant 215G2 SHC (represented by SEQ ID NO: 3) with the additional mutation L37Q.

[0067] SEQ ID NO: 52 is the amino acid sequence of the SHC / HAC variant 215G2 SHC (represented by SEQ ID NO: 3) with the additional mutation V174I. SEQ ID NO: 53 is the amino acid sequence of the SHC / HAC variant 215G2 SHC (represented by SEQ ID NO: 3) with the additional mutations V174I and F601Y.

[0068] SEQ ID NO: 54 is the amino acid sequence of SHC / HAC variant 215G2 SHC (represented by SEQ ID NO: 3) with additional mutations L37Q, V174I, and F601Y. SEQ ID NOs: 55-296, 307-310 are wild-type amino acid sequences of SHC / HAC as identified in Table 2 or 16.

[0069] SEQ ID NOs: 297 to 300 are nucleotide sequences encoding SEQ ID NOs: 47 to 50. SEQ ID NO: 301 is the amino acid sequence of a GmoSHC variant having V45L, Q54E, M184I, T326S, and F624Y. SEQ ID NO: 302 is the amino acid sequence of TelSHC with the following mutations: W172G, P311V, F425Y, G609A.

[0070] SEQ ID NO: 303 is the amino acid sequence of ZmoSHC1 with the following mutations: Q221S, W222G, A368V, F486Y, and G667A. SEQ ID NO: 304 is the amino acid sequence of TelSHC with the W172G mutation (P1).

[0071] SEQ ID NO: 305 is the amino acid sequence of AacSHC with the following mutations: W169G, G600M, M132R, A224V, and I432T. SEQ ID NO: 306 is the amino acid sequence of AacSHC with the following mutations: W169G, G600M, M132R, and I432T.

[0072] SEQ ID NOs: 307-310 (together with SEQ ID NOs: 55-296) are the wild-type amino acid sequences of SHC / HAC as identified in Tables 2 or 16. SEQ ID NO: 311 is the amino acid sequence of ScoSHC1 with the W196G mutation (P1).

[0073] SEQ ID NO: 312 is the amino acid sequence of ZmoSHC1 with the W222G mutation (P1). SEQ ID NO: 313 is the amino acid sequence of ZmoSHC2 with the W177G mutation (P1). SEQ ID NO: 314 is the motif DXDDTA found in SHC / HAC.

[0074] SEQ ID NOs: 315 to 360 are amino acid sequences of SHC variants, and in particular SEQ ID NOs: 315 to 347, 348, and 350 to 352 (excluding 349) are SHC variants derived from AaC SHC (SEQ ID NO: 1).

[0075] SEQ ID NO: 315 corresponds to SEQ ID NO: 1 with the substitution W169G. SEQ ID NO: 316 corresponds to SEQ ID NO: 1 with the substitution W169A. SEQ ID NO: 317 corresponds to SEQ ID NO: 1 with the substitution W169V. SEQ ID NO: 318 corresponds to SEQ ID NO: 1 with the substitutions W169G and G600A. SEQ ID NO: 319 corresponds to SEQ ID NO: 1 with the substitutions W169G and G600V.

[0076] SEQ ID NO: 320 corresponds to SEQ ID NO: 1 with the substitutions W169G and G600L. SEQ ID NO: 321 corresponds to SEQ ID NO: 1 with the substitutions W169G and G600I. SEQ ID NO: 322 corresponds to SEQ ID NO: 1 with the substitutions W169G and G600M. SEQ ID NO: 323 corresponds to SEQ ID NO: 1 with the substitutions W169A and G600A. SEQ ID NO: 324 corresponds to SEQ ID NO: 1 with the substitutions W169A and G600V.

[0077] SEQ ID NO: 325 corresponds to SEQ ID NO: 1 with the substitutions W169A and G600L. SEQ ID NO: 326 corresponds to SEQ ID NO: 1 with the substitutions W169A and G600I. SEQ ID NO: 327 corresponds to SEQ ID NO: 1 with the substitutions W169A and G600M. SEQ ID NO: 328 corresponds to SEQ ID NO: 1 with the substitutions W169V and G600A. SEQ ID NO: 329 corresponds to SEQ ID NO: 1 with the substitutions W169V and G600V.

[0078] SEQ ID NO: 330 corresponds to SEQ ID NO: 1 with the substitutions W169V and G600L. SEQ ID NO: 331 corresponds to SEQ ID NO: 1 with the substitutions W169V and G600I. SEQ ID NO: 332 corresponds to SEQ ID NO: 1 with the substitutions W169V and G600M. SEQ ID NO:333 corresponds to SEQ ID NO:1 with the substitutions W169G, G600A, and A306V. SEQ ID NO:334 corresponds to SEQ ID NO:1 with the substitutions W169G, G600V, and A306V.

[0079] SEQ ID NO:335 corresponds to SEQ ID NO:1 with the substitutions W169G, G600L, and A306V. SEQ ID NO:336 corresponds to SEQ ID NO:1 with the substitutions W169G, G600I, and A306V. SEQ ID NO:337 corresponds to SEQ ID NO:1 with the substitutions W169G, G600M, and A306V. SEQ ID NO:338 corresponds to SEQ ID NO:1 with the substitutions W169A, G600A, and A306V. SEQ ID NO:339 corresponds to SEQ ID NO:1 with the substitutions W169A, G600V, and A306V.

[0080] SEQ ID NO:340 corresponds to SEQ ID NO:1 with the substitutions W169A, G600L, and A306V. SEQ ID NO:341 corresponds to SEQ ID NO:1 with the substitutions W169A, G600I, and A306V. SEQ ID NO:342 corresponds to SEQ ID NO:1 with the substitutions W169A, G600M, and A306V. SEQ ID NO:343 corresponds to SEQ ID NO:1 with the substitutions W169V, G600A, and A306V. SEQ ID NO:344 corresponds to SEQ ID NO:1 with the substitutions W169V, G600V, and A306V.

[0081] SEQ ID NO:345 corresponds to SEQ ID NO:1 with the substitutions W169V, G600L, and A306V. SEQ ID NO:346 corresponds to SEQ ID NO:1 with the substitutions W169V, G600I, and A306V. SEQ ID NO:347 corresponds to SEQ ID NO:1 with the substitutions W169V, G600I, and A306V. SEQ ID NO:348 corresponds to SEQ ID NO:1 with the substitutions M132R, A224V, and I432T. SEQ ID NO: 349 is a nucleotide sequence encoding an SHC variant derived from AacSHC.

[0082] SEQ ID NO: 350 corresponds to SEQ ID NO: 1 with the substitutions W169G and A306V. SEQ ID NO: 351 corresponds to SEQ ID NO: 1 with the substitutions W169A and A306V. SEQ ID NO: 352 corresponds to SEQ ID NO: 1 with the substitutions W169V and A306V. SEQ ID NO: 353 corresponds to SEQ ID NO: 13 with substitutions W222G and G667M (also referred to as the ZmoSHC1 P2 variant).

[0083] SEQ ID NO: 354 corresponds to SEQ ID NO: 13 with the substitutions W222G, A368V, and G667M (also referred to as the ZmoSHC1 P3 variant). SEQ ID NO: 355 corresponds to SEQ ID NO: 15 with substitutions W177G and G619M (also referred to as the ZmoSHC2 P2 variant).

[0084] SEQ ID NO:356 corresponds to SEQ ID NO:15 with the substitutions W177G, A321V, and G619M (also referred to as the ZmoSHC2 P3 variant). SEQ ID NO: 357 corresponds to SEQ ID NO: 32 with substitutions W196G and G629M (also referred to as the ScoSHC P2 variant).

[0085] SEQ ID NO: 358 corresponds to SEQ ID NO: 32 with the substitutions W196G, A335V, and G629M (also referred to as the SScoSHC P3 variant). SEQ ID NO: 359 corresponds to SEQ ID NO: 23 with substitutions W172G and G609M (also referred to as the TelSHC P2 variant).

[0086] SEQ ID NO: 360 corresponds to SEQ ID NO: 23 with the substitutions W172G, A311V, and G609M (also referred to as the TelSHC P3 variant). SEQ ID NOs: 361-383 are the amino acid sequences of SHC variants derived from AacSHC (SEQ ID NO: 1) as identified in Table 18.

[0087] SEQ ID NO: 361 corresponds to SEQ ID NO: 1 with the substitution T77A. SEQ ID NO: 362 corresponds to SEQ ID NO: 1 with the substitution I92V. SEQ ID NO: 363 corresponds to SEQ ID NO: 1 with the substitution F129L. SEQ ID NO: 364 corresponds to SEQ ID NO: 1 with the substitution M132R. SEQ ID NO: 365 corresponds to SEQ ID NO: 1 with the substitution A224V.

[0088] SEQ ID NO: 366 corresponds to SEQ ID NO: 1 with the substitution I432T. SEQ ID NO: 367 corresponds to SEQ ID NO: 1 with the substitution Q579H. SEQ ID NO: 368 corresponds to SEQ ID NO: 1 with the substitution F601Y. SEQ ID NO: 369 corresponds to SEQ ID NO: 1 with the substitutions M132R and I432T. SEQ ID NO: 370 corresponds to SEQ ID NO: 1 with the substitution F601Y.

[0089] SEQ ID NO:371 corresponds to SEQ ID NO:1 with the substitutions T77A, I92V, and F129L. SEQ ID NO: 372 corresponds to SEQ ID NO: 1 with substitutions Q579H and F601Y. SEQ ID NO: 373 corresponds to SEQ ID NO: 1 with the substitution F129L. SEQ ID NO: 374 corresponds to SEQ ID NO: 1 with substitutions F129L and F601Y. SEQ ID NO:375 corresponds to SEQ ID NO:1 with the substitutions F129L, M132R, and I432T.

[0090] SEQ ID NO:376 corresponds to SEQ ID NO:1 with the substitutions M132R, I432T, and F601Y. SEQ ID NO:377 corresponds to SEQ ID NO:1 with the substitutions F129L, M132R, I432T, and F601Y. SEQ ID NO: 378 corresponds to SEQ ID NO: 1 with the substitution F605W. SEQ ID NO:379 corresponds to SEQ ID NO:1 with the substitutions M132R, A224V, I432T, A557T, and H431L, and is sometimes referred to herein as SHC / HAC enzyme variant #49 or SHC#49 variant.

[0091] SEQ ID NO:380 corresponds to SEQ ID NO:1 with the substitutions M132R, A224V, I432T, Y81H, A557T, and R613S, and is sometimes referred to herein as SHC / HAC enzyme variant #66 or SHC#66 variant. SEQ ID NO:381 corresponds to SEQ ID NO:1 with the substitutions M132R, A224V, I432T, Y81H, H431L, and A557T, and is sometimes referred to herein as SHC / HAC enzyme variant #110B8 or SHC #110B8 variant.

[0092] SEQ ID NO:382 corresponds to SEQ ID NO:1 with the substitutions M132R, A224V, I432T, T90A, and R613S, and is sometimes referred to herein as SHC / HAC enzyme variant #90C7 or SHC#90C7 variant. SEQ ID NO:383 corresponds to SEQ ID NO:1 with the substitutions M132R, A224V, I432T, A172T, and M277K, and is sometimes referred to herein as SHC / HAC enzyme variant #115A7 or SHC#115A7 variant.

[0093] SEQ ID NO: 385 corresponds to SEQ ID NO: 3 with the substitutions W169G and A306V. SEQ ID NO: 386 corresponds to SEQ ID NO: 2 with the substitutions W169G and A306V.

[0094] Detailed Description SHC / HAC enzymes and their variants As used herein, the term "SHC enzyme" refers to a wild-type (WT) squalene hopene cyclase enzyme that occurs naturally in thermophilic bacteria, such as, for example, Alicyclobacillus acidocaldarius (Aac). SHCs that act in the cyclization of homofarnesol to ambrox are also sometimes referred to as mofarnesol ambrox cyclase (HAC) enzymes. Thus, the term "SHC / HAC enzyme" is sometimes used herein.

[0095] As used herein, the term "variant" should be understood as a polypeptide that differs from a polypeptide from which it is derived by one or more changes or modifications in the amino acid sequence. The polypeptide from which a variant is derived is also known as the parent or reference polypeptide. Typically, a variant is artificially constructed, preferably by genetic engineering means. Typically, the polypeptide from which a variant is derived is a wild-type protein or a domain of a wild-type protein. However, variants usable in the present disclosure may also be derived from homologs, orthologs, or paralogs of the parent polypeptide or from artificially constructed variants, provided that the variant exhibits at least one biological activity of the parent polypeptide. The change in the amino acid sequence may be an amino acid exchange (substitution), insertion, deletion, N-terminal truncation, C-terminal truncation, or any combination of these changes, and may occur at one or several sites.

[0096] As used herein, the term "SHC / HAC enzyme variant" refers to an enzyme that is derived from a wild-type SHC enzyme (such as the Aac SHC enzyme represented by SEQ ID NO: 1), but has one or more amino acid modifications compared to the wild-type SHC enzyme, and thus does not naturally occur in prokaryotes. Table 2 provides a list of wild-type SHCs that may be used to generate SHC / HAC enzyme variants in the present invention. Tables 3 and 16 provide lists of preferred wild-type SHCs to be used to generate SHC / HAC enzyme variants in the present invention. Table 3 lists the following SHC / AHC enzymes: AaC SHC represented by SEQ ID NO: 1, ZmoSHC1 represented by SEQ ID NO: 13 or 384, ZmoSCH2 represented by SEQ ID NO: 15, BjpSHC represented by SEQ ID NO: 16, Burkhoderia ambifaria SHC represented by SEQ ID NO: 17 or 18, Bacillus anthracis SHC represented by SEQ ID NO: 19, Frankia alni SHC represented by SEQ ID NO: 20, Rhodopseudomonas palustris SHC represented by SEQ ID NO: 21, GmoSCO SHC represented by SEQ ID NO: 22, Tel SHC represented by SEQ ID NO: 23 SHC, ApaSHC1 represented by SEQ ID NO: 24, BmeSHC represented by SEQ ID NO: 25, SalSHC represented by SEQ ID NO: 26, ApaSHCA represented by SEQ ID NO: 27, BamSHC1 represented by SEQ ID NO: 28, BamSHC2 represented by SEQ ID NO: 29, PcaSHC2 (or Syntrophotalea carbinolica DSM 2380) represented by SEQ ID NO: 30, RpaSHC1 represented by SEQ ID NO: 31, ScoSHC represented by SEQ ID NO: 32, SfuSHC represented by SEQ ID NO: 33, TtuSHC represented by SEQ ID NO: 34.More preferred wild-type SHCs to be used to generate SHC / HAC enzyme variants in the present invention are: AaC SHC represented by SEQ ID NO: 1, ZmoSHC1 represented by SEQ ID NO: 13, ZmoSCH2 represented by SEQ ID NO: 15, Tel SHC represented by SEQ ID NO: 23, and ScoSHC represented by SEQ ID NO: 32.

[0097] The one or more amino acid modifications may, for example, modify (e.g., increase) the enzymatic activity toward a substrate (e.g., EEH). In the context of the present invention, an SHC / HAC enzyme variant may be derived from an SHC / HAC variant. Examples of SHC / HAC variants include SEQ ID NOs: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383.

[0098] Assays for determining and quantifying the activity of SHC / HAC enzymes and / or SHC / HAC enzyme variants are described herein and are known in the art. For example, the activity of an SHC / HAC enzyme and / or SHC / HAC enzyme variant can be determined by incubating a purified SHC / HAC enzyme or enzyme variant, or an extract from a host cell or a complete recombinant host organism producing the SHC / HAC enzyme or enzyme variant, with an appropriate substrate under appropriate conditions and analyzing the reaction products (e.g., by gas chromatography (GC) or HPLC analysis). Further details on assaying the activity of an SHC / HAC enzyme and / or SHC / HAC enzyme variant and analyzing the reaction products are provided in the Examples. These assays involve producing an SHC / HAC enzyme variant in a recombinant host cell (e.g., E. coli).

[0099] As used herein, the term "activity" refers to the ability of an enzyme to react with a substrate to provide a desired product. Activity can be determined using what is known as an activity test to monitor the formation of the desired product. Derivatives of the SHC / HAC enzymes of the present disclosure can be characterized by their ability to cyclize homofarnesol (e.g., EEH) to (-)-ambrox and demonstrate biological activity, such as HAC activity. Derivatives of the SHC / HAC enzymes of the present disclosure can be characterized by their ability to cyclize bishomofarnesol (e.g., E,E-bishomofarnesol) to ambroxide.

[0100] In the context of this application, the activity or biological activity of an SHC / HAC variant is compared under the same conditions to the corresponding activity or biological activity of the wild-type, parental, reference SHC / HAC from which it is derived. Examples of wild-type SHC / HAC enzymes are identified in Tables 2, 3, or 16. In embodiments, the wild-type SHC / HAC enzyme is represented by SEQ ID NO: 1 or is set forth in SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 1 8, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 25 03, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257,258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310. Exemplary parent or reference SHC / HAC enzymes used to create the SHC / HAC variants of the present invention may be represented by any of SEQ ID NOs: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383.

[0101] "Biological activity," as used herein, refers to any activity that a polypeptide may exhibit, including, but not limited to, enzymatic activity; substrate specificity, selectivity (such as substrate and / or product selectivity), conversion rate (such as EEH:EZH conversion ratio), yield, binding activity to another compound (e.g., binding to another polypeptide, especially binding to a receptor, or binding to a nucleic acid); inhibitory activity (e.g., enzyme inhibitory activity); activating activity (e.g., enzyme activating activity); or toxic effects. It is not required that a variant exhibit such activity to the same extent as a parent or wild-type or reference polypeptide. A variant is considered a variant within the context of the present application if it exhibits a relevant activity that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or at least 200% of the activity of the parent polypeptide. Likewise, a variant will be considered a variant within the context of the present application if it exhibits the relevant biological activity to the extent of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, or at least 200% of the activity of the parent polypeptide (as the terms derivative and variant are used interchangeably throughout this disclosure).

[0102] In embodiments, the SHC / HAC enzyme variants described herein exhibit better yields (i.e., increased yields) compared to the yields obtained using a reference SHC enzyme (e.g., a wild-type SHC / HAC enzyme such as an AacSHC / AHC enzyme, or a known SHC / HAC enzyme variant or the parent SHC from which the variant is derived). The term "yield" refers to grams of recoverable product (i.e., (-)-ambrox or ambroxide) per gram of feedstock (which may be calculated as a percent molar conversion). In this context, "increased" may mean an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 200%, or at least 400% over the yield obtained using the reference SHC enzyme.

[0103] In embodiments, "selectivity" refers to "product selectivity," describing the ability to produce a specific compound (e.g., (-)-ambrox or ambroxide) in a substantially enriched / predominant form ("product selectivity") from a mixture of several substrate isomers in an enzymatically catalyzed method as described above. In embodiments, the mixture of homofarnesol substrate isomers is selected from one or more of the following mixtures: [(3Z,7Z), (3E,7Z), (3Z,7E), and (3E,7E)], [(3Z,7E) and (3E,7E)], [(3Z,7E), (3E,7Z)], and / or [(3E,7E) and (3E,7Z)]. In embodiments, the mixture is [(3E,7E), (3Z,7E)], also designated [EE:EZ] or EEH:EZH. In embodiments, the ratio of EEH:EZH in this mixture may range from about 50:50 to about 99:01, or from about 60:40 to about 99:1, or from about 70:30 to about 95:5, or from about 80:20 to about 95:5.

[0104] In a preferred embodiment, "selectivity" refers to "product selectivity," which describes the ability to produce a specific compound (e.g., (-)-ambrox, i.e., Compound (I)) in a substantially enriched / predominant form ("product selectivity") in an enzymatically catalyzed method as described above from an 80:20 or 90:10 EEH:EZH mixture.

[0105] In this context, "substantially enriched / predominant form" may mean that the total product formed as a result of the cyclization reaction of the enzymatic variant of WT SHC consists essentially of or consists of, for example: a compound of formula (I) as described herein ((-)-ambrox) when using any of the homofarnesol substrate isomer mixtures identified above (preferably EEH:EZH, more preferably in a ratio of 80:20 or 90:10); or - the compound of formula (I) ((-)-ambrox) and the compound of formula (III) described herein when using any of the homofarnesol substrate isomer mixtures identified above (preferably EEH:EZH, more preferably in a ratio of 80:20 or 90:10).

[0106] This means that the compounds of formula (II) and (IV) may not be detectable at the end of the process or during the first 3, 4, 5, or 6 hours, or 12 or 18 hours of the process. Detection of the compounds of formula (I), (II), (III), and (IV) may be achieved using techniques known to those skilled in the art, preferably the techniques used in the experimental part. This also means that in the absence of any downstream processing steps, only the compound of formula (I) or only the compounds of formula (I) and (III) are produced. This means that downstream processing (DSP) may be simplified because both the degree of substrate conversion and the conversion rate of the substrate are increased. In other words, DSP is simplified because the amount of unreacted homofarnesol present at the end of the reaction may be reduced. It is known that unreacted homofarnesol dissolves at least a portion of the solid (-)-ambrox formed, and that the solid (-)-ambrox formed may also dissolve in other liquid by-products, the absence of which (e.g., compounds II and / or IV) may simplify the DSP step.

[0107] In this context, "downstream processing steps" refers to the separation of solid (-)-ambrox from unreacted homofarnesol substrate and the separation of (-)-ambrox from other by-products (e.g., compounds II and / or III and / or IV). DSP steps may include, but are not limited to, one or more of centrifugation, filtration, steam or organic solvent extraction or distillation, or selective crystallization steps. In this context, an example of simplified DSP is when a distillation step may be more efficient at separating (-)-ambrox from unreacted homofarnesol than at separating (-)-ambrox from the by-products produced. Each of these downstream processing steps is defined later herein. Exemplary downstream processing steps are provided in WO2022 / 023464, the contents of which are incorporated herein by reference.

[0108] In embodiments, the SHC / HAC enzyme variants described herein are similar to or are not similar to a reference SHC enzyme (e.g., a wild-type SHC / HAC enzyme such as the AacSHC / AHC enzyme represented by SEQ ID NO: 1, or SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, , 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 12 0, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 14, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245 5, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, or any other wild-type SHC / HAC enzyme represented by SEQ ID NOs: 2, 3, 47, 48, 49, 50, 51, 52, The product selectivity of the SHC / HACs (SHC / HACs) represented by formula (I) above is 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383) shows increased product selectivity (i.e., increased selectivity for (-)-ambrox or amblyoxide) compared to the product selectivity obtained using the SHC / HACs (SHC / HACs represented by formula (I) above) above. The term "product selectivity" also refers to grams of recoverable product (i.e., (-)-ambrox or amblyoxide) per gram of total product formed, which may be calculated as a percent molar conversion. In this context, "increase" may mean an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 200%, or at least 400% over the yield obtained using a reference SHC enzyme.

[0109] In embodiments, "selectivity" refers to the ability of an enzyme-catalyzed method as described herein to preferentially convert a specific substrate isomer (e.g., an EEH isomer) to a specific product from a plurality / mixture of several substrate isomers ("substrate selectivity"). More specifically, this means that the specific product (-)-ambrox is enriched for enzymatic conversion of a specific substrate (EEH) isomer from a mixture of several substrate isomers.

[0110] Thus, the "substrate selectivity" of an enzyme or enzyme variant refers to the ability of the enzyme or enzyme variant to react with one particular substrate isomer compared to another substrate isomer while the enzyme is in contact with a mixture containing at least two distinct substrate isomers. For example, a WT enzyme that is selective for EEH over other isomers of homofarnesol, or selective for bisEEH over other isomers of bishomofarnesol, SHC enzymes (represented by SEQ ID NO: 1 or any of the SHC / HACs disclosed in Tables 2, 3, or 16, or SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63 6, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 , 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 199, 198, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232 83, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270 0, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310), or WT An enzyme variant of SHC (such as those represented by any of SEQ ID NOs: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383) means that the WT SHC enzyme or an enzyme variant of WT SHC is more likely to convert EEH than other isomers of homofarnesol, or more likely to convert bisEEH than other isomers of bishomofarnesol. In embodiments, the mixture of homofarnesol substrate isomers, comprising at least two distinct substrate isomers of homofarnesol, comprises an EEH isomer and is selected from one or more of the following mixtures: [(3Z,7Z), (3E,7Z), (3Z,7E), and (3E,7E)], [(3Z,7E) and (3E,7E)], [(3Z,7E), (3E,7Z)], and / or [(3E,7E) and (3E,7Z)]. In embodiments, the mixture is [(3E,7E), (3Z,7E)], also designated [EE:EZ] or EEH:EZH. In embodiments, the ratio of EEH:EZH in this mixture may range from about 55:45, from about 50:50 to about 99:01, or from about 60:40 to about 99:1, or from about 70:30 to about 95:5, or from about 80:20 to about 95:5.

[0111] For example, the wt% of total products formed as a result of the reaction of a WT or reference or parent SHC enzyme variant with a mixture containing at least two distinct substrate isomers of homofarnesol (the mixture containing EEH) may be at least about 1 percentage point greater than the wt% of total products formed as a result of the reaction of the corresponding WT or parent or reference SHC enzyme with the same mixture containing EEH. For example, the wt% of total products formed as a result of the reaction of the enzyme variant may be at least about 2, or at least about 3, or at least about 4 percentage points greater than the wt% of total products formed as a result of the reaction of the WT or reference or parent SHC with the same mixture containing EEH. For example, the wt% of total products formed as a result of the reaction of the enzyme variant may be up to about 40, or up to about 30, or up to about 20, or up to about 15, or up to about 10 percentage points greater than the wt% of total products formed as a result of the reaction of the WT or reference or parent SHC with the same mixture containing EEH. For example, the wt % of total product formed as a result of the reaction of the enzyme variant may be from about 1 to about 40 percentage points, or from about 2 to about 30 percentage points, or from about 3 to about 20 percentage points, or from about 4 to about 10 percentage points greater than the wt % of total product formed as a result of the reaction of WT or a reference or parent SHC with the same mixture containing EEH. The total product formed as a result of the reaction of the enzyme variant of WT SHC or of the reference or parent SHC may comprise, consist essentially of, or consist of the compound of formula (I) ((-)-ambrox) and the compound of formula (IV) described herein, for example, when a mixture containing at least two distinct substrate isomers of homofarnesol (the mixture containing EEH) is used as a substrate.In all of these embodiments, the mixture of homofarnesol substrate isomers, comprising at least two distinct substrate isomers of homofarnesol, comprises an EEH isomer and is selected from one or more of the following mixtures: [(3Z,7Z), (3E,7Z), (3Z,7E), and (3E,7E)], [(3Z,7E) and (3E,7E)], [(3Z,7E), (3E,7Z)], and / or [(3E,7E) and (3E,7Z)]. In embodiments, the mixture is [(3E,7E), (3Z,7E)], also designated [EE:EZ] or EEH:EZH. In embodiments, the ratio of EEH:EZH in this mixture may range from about 55:45, from about 50:50 to about 99:01, or from about 60:40 to about 99:1, or from about 70:30 to about 95:5, or from about 80:20 to about 95:5.

[0112] The total product formed as a result of the reaction of the WT or reference or parent SHC enzyme variant may comprise, consist essentially of, or consist of compounds of formula (X) and / or formula (XII) as described herein, for example, when bisEEH is used as a substrate.

[0113] For example, WT SHC / HAC (SEQ ID NOs: 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293,Those WTs represented by 294, 295, 296, 307, 308, 309, or 310 The wt % of (-)-ambrox formed using an enzyme variant of a WT or reference / variant SHC / HAC (such as those represented by SEQ ID NOs: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383) may be at least about 1 percentage point greater than the wt % of (-)-ambrox formed as a result of the reaction of a WT or reference / variant SHC with a mixture comprising at least two distinct substrate isomers of homofarnesol, the mixture including EEH. For example, the wt% of (-)-ambrox formed as a result of reaction of the WT or reference or parent SHC enzyme variant with the mixture containing EEH may be at least about 2, or at least about 3, or at least about 4 percentage points greater than the wt% of (-)-ambrox formed as a result of reaction of the WT or reference or parent SHC with the same mixture containing EEH. For example, the wt% of (-)-ambrox formed as a result of reaction of the WT or reference or parent SHC enzyme variant with the mixture containing EEH may be up to about 40, or up to about 30, or up to about 20, or up to about 15, or up to about 10 percentage points greater than the wt% of (-)-ambrox formed as a result of reaction of the WT or reference or parent SHC with the same mixture containing EEH. For example, the wt% of (-)-ambrox formed as a result of reaction of the WT or the enzyme variant of the parent or reference SHC with the mixture containing EEH may be from about 1 to about 40 percentage points, or from about 2 to about 30 percentage points, or from about 3 to about 20 percentage points, or from about 4 to about 10 percentage points greater than the wt% of (-)-ambrox formed as a result of reaction of the WT or the reference or parent SHC with the same mixture containing EEH.The mixture of homofarnesol substrate isomers, comprising at least two distinct substrate isomers of homofarnesol, includes the EEH isomer and is selected from one or more of the following mixtures: [(3Z,7Z), (3E,7Z), (3Z,7E), and (3E,7E)], [(3Z,7E) and (3E,7E)], [(3Z,7E), (3E,7Z)], and / or [(3E,7E) and (3E,7Z)]. In embodiments, the mixture is [(3E,7E), (3Z,7E)], also designated [EE:EZ] or EEH:EZH. In embodiments, the ratio of EEH:EZH in this mixture may range from about 55:45, from about 50:50 to about 99:01, or from about 60:40 to about 99:1, or from about 70:30 to about 95:5, or from about 80:20 to about 95:5.

[0114] Enzyme variants of WT SHC / HAC (SEQ ID NO: 1 or 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 05, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 1 36, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 7, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198 8, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229 , 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260 , 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291,292, 293, 294, 295, 296, 307, 308, 309, or 310), or their parent or reference SHC / HAC (SEQ ID NOs: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, The selectivity of a SHC / HAC enzyme (such as those represented by 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383) may also be compared to the selectivity of the corresponding WT SHC / HAC or corresponding reference / parent SHC / HAC enzyme by comparing the EEH:EZH conversion ratio (i.e., % conversion of EEH:% conversion of EZH) or bisEEH:bisEZH conversion ratio (i.e., % conversion of bisEEH:% conversion of bisEZH) of the reaction using each enzyme. This may be determined by measuring the amount of EEH and EZH or bisEEH and bisEZH remaining in the reaction mixture when the reaction is complete. Often, the reaction is complete after 18 or 20 hours. Alternatively, the selectivity of a WT ... Enzyme variants of SHC / HAC (SEQ ID NOs: 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 9, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 1 42, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174,175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 28, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281 , 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310), or reference or variant SHC / HAC (SEQ ID NOs: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, The selectivity of a bis-EEH (compounds represented by formulas I and IV) to EZH (compounds represented by formulas II and III), or a bis-EEH (compounds represented by formulas X and XII) to bis-EZH (compounds represented by formulas XI and XIII), may also be compared to the selectivity of the corresponding WT or reference or parent SHC / HAC by comparing the product ratios resulting from the conversion of EEH (compounds represented by formulas I and IV) to EZH (compounds represented by formulas II and III), respectively, or bis-EEH (compounds represented by formulas X and XII) to bis-EZH (compounds represented by formulas XI and XIII), respectively.

[0115] SHC / HAC enzyme variants of WT or reference or parent SHC / HAC provide an EEH:EZH conversion ratio of at least about 2.0, for example, in a process for producing (-)-ambrox from a mixture of homofarnesol containing at least two distinct substrate isomers of homofarnesol (the mixture including EEH and EZH). In embodiments, the mixture of homofarnesol substrate isomers containing at least two distinct substrate isomers of homofarnesol includes an EEH isomer and an EZH isomer and is selected from one or more of the following mixtures: [(3Z,7Z), (3E,7Z), (3Z,7E), and (3E,7E)], [(3Z,7E) and (3E,7E)], [(3Z,7E), (3E,7Z)], and / or [(3E,7E) and (3E,7Z)]. In embodiments, the mixture includes EEH and EZH. In embodiments, the mixture is [(3E,7E), (3Z,7E)], also designated as [EE:EZ] or EEH:EZH. In embodiments, the ratio of EEH:EZH in this mixture may range from about 55:45, from about 50:50 to about 99:01, or from about 60:40 to about 99:1, or from about 70:30 to about 95:5, or from about 80:20 to about 95:5.

[0116] For example, an enzyme variant of the WT or reference or parent SHC / HAC may provide an EEH:EZH conversion ratio of at least about 2.5, or at least about 3.0, or at least about 3.5 in a process for producing (-)-ambrox from a mixture as defined above. In embodiments, the mixture comprises EEH and EZH. For example, an enzyme variant of the WT SHC / HAC or the reference or parent SHC / HAC may provide an EEH:EZH conversion ratio of up to about 5.0, or up to about 4.5, or up to about 4.0 in a process for producing (-)-ambrox from a mixture as defined above. In embodiments, the mixture comprises EEH and EZH. For example, the WT or reference or parent SHC / HAC enzyme variant may provide an EEH:EZH conversion ratio ranging from about 2.0 to about 5.0, or from about 2.5 to about 4.5, or from about 3.0 to about 4.0 in a process for producing (-)-ambrox from a mixture as defined above. In some embodiments, the mixture comprises EEH and EZH. This may be in contrast to the conversion ratio provided by AacSHC in a process for producing (-)-ambrox from a mixture comprising EEH and EZH, which may be, for example, less than about 2.0.

[0117] In particular, if the WT SHC enzyme or WT SHC enzyme variant has a higher selectivity for EEH over other isomers of homofarnesol compared to WT AacSHC and / or variants of WT AacSHC, the wild-type SHC / HAC enzyme (e.g., from which the SHC / HAC enzyme variant may be derived) may be selected from TelSHC1, ApaSHC1, ZmoSHC1, ZmoSHC2, BjaSHC (or BjpSHC), GmoSHC BmeSHC, SalSHC, ApaSHCA. For example, if the WT SHC enzyme or WT SHC enzyme variant has a higher selectivity for EEH over other isomers of homofarnesol compared to WT AacSHC and / or a variant of WT AacSHC, the wild-type SHC / HAC enzyme (e.g., from which the SHC / HAC enzyme variant may be derived) may be selected from ZmoSHC1, BjaSHC (BjpSHC), GmoSHC, ApaSHC1, and BmeSHC.

[0118] In embodiments, the SHC / HAC enzyme variants described herein are identical to or contain the SHC / HAC enzyme variants of a reference SHC enzyme (e.g., a wild-type SHC / HAC enzyme, e.g., SEQ ID NOs: 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 200, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 232, 23 , 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 6, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157 , 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 2 20, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 1, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282,283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310), or a known SHC / HAC enzyme variant, or the parent SHC from which the variant is derived (SEQ ID NOs: 2, 3, 47, 48, 49, 50, 51, 52, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383. The term "conversion" refers to the amount of substrate (i.e., EEH or bisEEH) converted per gram of biocatalyst and per time unit (which may be calculated as a percent molar conversion). In this context, "increase" may mean an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 200%, or at least 400% over the conversion obtained using a reference SHC enzyme.

[0119] In embodiments, the SHC / HAC enzyme variants described herein are synthesized using a reference SHC enzyme (e.g., a wild-type SHC / HAC enzyme, e.g., SEQ ID NOs: 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 220, 221, 222, 232, 243, 252, 253, 254, 255, 256, 257, 258, 260, 261, 262, 163, 164, 165, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 4, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155 , 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 2 18, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249 9, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280,281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, or known SHC / HAC enzyme variants, or the parent SHC from which the variants are derived, e.g., SEQ ID NOs: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 7 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383). The term "substrate specificity" also refers to the amount of a particular converted substrate isomer (i.e., EEH or bisEEH) per gram of substrate initially present at a given time or throughout a "section" of an enzyme-catalyzed reaction. In particular, the selectivity may be observed over an "interval" corresponding to conversion of 1-99%, 2-95%, 3-90%, 5-85%, 10-80%, 15-75%, 20-70%, 25-65%, 30-60%, or 40-50% of the initial amount of substrate. In embodiments, the "interval" may be from 1 hour to 8 hours, or from 2 hours to 7 hours, or from 3 hours to 6 hours. In embodiments, the "interval" is 6 hours. In this context, "increase" may mean an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 200%, or at least 400% of the substrate specificity obtained using a reference SHC enzyme. ,

[0120] In additional embodiments, the SHC / HAC enzyme variants described herein can be synthesized from a reference SHC enzyme (e.g., wild-type AacSHC (SEQ ID NO: 1), or 215G2 AacSHC (SEQ ID NO: 3), or SHC#65 (SEQ ID NO: 2), or the parent SHC enzyme from which the variant is derived, e.g., those identified in Tables 2 or 3 or 16 or SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,The following wild-type SHs are represented by 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310: C / HAC or a variant such as any of the following SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383. The term "productivity" refers to the amount of recoverable product (i.e., (-)-ambrox or ambroxide) in grams per liter of reaction capacity per hour of bioconversion time (i.e., time after substrate is added). The term "productivity" also refers to the amount of recoverable product in grams per liter of reaction capacity per hour of bioconversion time (i.e., time after substrate is added) per gram of biocatalyst used in the reaction. In this context, "increase" may mean an increase of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 200%, or at least 400% over the productivity obtained using a reference SHC enzyme.

[0121] In embodiments, the SHC / HAC enzyme variants disclosed herein exhibit the following: Reference SHC enzymes (e.g., wild-type SHCs, e.g., SEQ ID NOs: 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 4, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135 , 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 1 98, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229 9, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260 , 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291,292, 293, 294, 295, 296, 307, 308, 309, 310, 215G2 AacSHC or SHC#65, or the parent SHC enzyme from which the variant is derived, e.g., represented by SEQ ID NOs: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383), - increased substrate specificity for EEH (or for bisEEH) when a homofarnesol or bis-homofarnesol substrate is used; - increased product selectivity when homofarnesol or bis-homofarnesol substrates are used relative to (-)-ambrox (or ambroxide); - increased specificity for specific isomers of the substrate when substrates other than homofarnesol or bis-homofarnesol are used (e.g., ethyl-homofarnesol, hydroxyfarnesylacetone, etc. - see e.g., WO2021 / 110858 and WO2021 / 209482, respectively); - increased productivity, and / or - an increased degree of conversion of EEH (or bisEEH) over the first 3 to 6 hours (or over the first 4, 5, or 6 hours) of the reaction, as well as an increased conversion rate of EEH (or bisEEH).

[0122] In embodiments, the SHC / HAC enzyme variants disclosed herein exhibit: Compared to a reference SHC enzyme (e.g., wild-type AacSHC, or 215G2 AacSHC, or SHC#65, or the parent SHC enzyme from which the variant is derived), - increased substrate specificity for EEH (or for bisEEH) when a homofarnesol or bis-homofarnesol substrate is used; - increased product selectivity when homofarnesol or bis-homofarnesol substrates are used relative to (-)-ambrox (or ambroxide); - increased specificity for specific isomers of the substrate when substrates other than homofarnesol or bis-homofarnesol are used (e.g., ethyl-homofarnesol, hydroxyfarnesylacetone, etc. - see, for example, WO2021 / 110858 and WO2021 / 209482, respectively); - increased productivity; - an increased degree of conversion of EEH (or bisEEH) over the first 3 to 6 hours (or over the first 4, 5, or 6 hours) of the reaction, as well as increased conversion.

[0123] In further embodiments, the SHC / HAC enzyme variants described herein are not limited to a reference SHC enzyme (e.g., wild-type AaC SHC (SEQ ID NO: 1) or SHC#65 (SEQ ID NO: 2), 215G2 AacSHC (SEQ ID NO: 3), ZmoSHC1 represented by SEQ ID NO: 13 or 384, ZmoSCH2 represented by SEQ ID NO: 15, BjpSHC represented by SEQ ID NO: 16, Burkhoderia ambifaria SHC represented by SEQ ID NO: 17 or 18, Bacillus anthracis SHC represented by SEQ ID NO: 19, Frankia alni SHC represented by SEQ ID NO: 20, Rhodopseudomonas palustris SHC represented by SEQ ID NO: 21, GmoSCO SHC represented by SEQ ID NO: 22, Tel SHC represented by SEQ ID NO: 23, or a combination thereof). SHC, ApaSHC1 represented by SEQ ID NO:24, BmeSHC represented by SEQ ID NO:25, SalSHC represented by SEQ ID NO:26, ApaSHCA represented by SEQ ID NO:27, BamSHC1 represented by SEQ ID NO:28, BamSHC2 represented by SEQ ID NO:29, PcaSHC2 (or Syntrophotalea carbinolica DSM 2380) represented by SEQ ID NO:30, RpaSHC1 represented by SEQ ID NO:31, ScoSHC represented by SEQ ID NO:32, SfuSHC represented by SEQ ID NO:33, TtuSHC represented by SEQ ID NO:34).

[0124] In preferred embodiments, the SHC / HAC enzyme variants described herein exhibit improved (i.e., increased) yield compared to the yield of the reference AacSHC (SEQ ID NO: 1) or SHC#65 (SEQ ID NO: 2), or 215G2 AacSHC (SEQ ID NO: 3), or ZmoSHC1 represented by SEQ ID NO: 13, or ZmoSCH2 represented by SEQ ID NO: 15, or Tel SHC represented by SEQ ID NO: 23, or ScoSHC represented by SEQ ID NO: 32.

[0125] In particular, the wild-type SHC / HAC enzyme (e.g., from which SHC / HAC enzyme variants may be derived) may be an Alicyclobacillus acidocaldarius (Aac) SHC / HAC enzyme, a Zymomonas mobilis (Zmo) SHC / HAC enzyme, a Bradyrhizobium japonicum (Bjp / Bja) SHC / HAC enzyme, an Acetobacter pasteurianus (Apa) SHC / HAC enzyme, a Bacillus megaterium (Bme) SHC / HAC enzyme, or a Gluconobacter morbifer (Gmo) SHC / HAC enzyme. In particular, the wild-type SHC / HAC enzyme (e.g., from which SHC / HAC enzyme variants may be derived) may be an Alicyclobacillus acidocaldarius (Aac) SHC / HAC enzyme.

[0126] The term "target yield factor" refers to the ratio between the obtained product concentration and the concentration of the SHC / HAC variant enzyme in the reaction medium (e.g., purified SHC / HAC enzyme variant or an extract from a recombinant host cell producing the SHC / HAC enzyme variant). In various embodiments, the SHC / HAC enzyme variants disclosed herein are identical to or contain a reference SHC protein (e.g., SEQ ID NOs: 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, , 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 1 44, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 2 8, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252,253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276 , 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310 or 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383). This increase in activity may be at least: 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, and / or 100 fold.

[0127] As used herein, the term "amino acid modification" refers to the insertion of one or more amino acids between two amino acids, the deletion of one or more amino acids, or the substitution of one or more amino acids with one or more different amino acids (which may be conservative or non-conservative) compared to the amino acid sequence of a reference amino acid sequence. A substitution replaces amino acids in the reference sequence with the same number of amino acids in the variant sequence. The reference amino acid sequence can be, for example, a wild-type (WT) amino acid sequence (e.g., SEQ ID NO: 1, or SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 2, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279 9, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310), or may be, for example, the sequence of an SHC / HAC enzyme variant (e.g., an Aac variant - SEQ ID NO: 2, or 3, or 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383) itself. The word "substitute" is synonymous with the word "replace."

[0128] Amino acid modifications can be readily identified by comparison of the amino acid sequence of the SHC / HAC enzyme variant with that of a reference amino acid sequence.

[0129] Conservative amino acid substitutions may be made, by way of illustration, on the basis of similarity in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or the amphipathic nature of the amino acid residues involved. The 20 naturally occurring amino acids, as outlined above, can be grouped into the following six standard amino acid groups: (1) Hydrophobic: Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr; Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe.

[0130] Consequently, as used herein, the term "conservative substitution" refers to the replacement of an amino acid with another amino acid listed in the same group of the six standard amino acid groups shown above. For example, replacing Asp with Glu maintains a negative charge in the resulting polypeptide. In addition, glycine and proline may be substituted for each other based on their ability to disrupt alpha-helices. Some preferred conservative substitutions within the above six groups are within the following subgroups: (i) Ala, Val, Leu, and Ile; (ii) Ser and Thr; (ii) Asn and Gln; (iv) Lys and Arg; and (v) Tyr and Phe. Given the known genetic code and recombinant and synthetic DNA techniques, skilled scientists can easily construct DNA encoding conservative amino acid variants.

[0131] In a preferred embodiment (as demonstrated in Example 11), the conserved amino acids in the amino acid sequence of wild-type SHC, particularly wild-type AacSHC of SEQ ID NO: 1, are L22, Q26, G30, W32, A44, L48, Q72, G76, W78, Y95, L98, G102, A113 ... I117, G121, G122, F129, T130, L134, A135, G138, W142, P146, W169, A170, R171, F217, D222, R2 37, I261, P263, P281, S309, P310, W312, D313, T314, A320, W339, Q344, G349, D350, W351, G361 , G362, A364, F365, N369, Y372, P373, D374, D376, D377, W406, Q411, G415, A419, P433, D436, D442, P443, D447, V448, Q479, G483, W485, G487, R488, W489, G490, N492, Y495, G496, T497, L5 Includes 04, W522, Q527, G531, G532, W533, G534, E535, S539, Y540, G547, T552, T556, W558, A559, A565, L581, Q585, G589, W591, G600, F601, P602, F605, Y609, Y612, F616, P617, A620, and R623.

[0132] In a preferred embodiment (as demonstrated in Example 14), conserved amino acid sequences are presented in the amino acid sequences of five wild-type SHCs, specifically wild-type AacSHC of SEQ ID NO: 1, wild-type ZmoSHC1 of SEQ ID NO: 13, wild-type ZmoSHC2 of SEQ ID NO: 15, wild-type TelSHC of SEQ ID NO: 23, and wild-type ScoSHC of SEQ ID NO: 32.

[0133] As used herein, a "non-conservative substitution" or "non-conservative amino acid exchange" is defined as the replacement of an amino acid with another amino acid listed in a different group of the six standard amino acid groups (1) to (6) as set forth above. Typically, the SHC / HAC enzyme variants described herein are prepared using non-conservative substitutions that alter the biological function (e.g., HAC activity) of the disclosed SHC / HAC enzyme variant. For ease of reference, the one-letter amino acid symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission are indicated as follows: A, B, C, D, E, F, G, G, H, H, I, I, II, III, IV ...

[0134] Table 1: Amino acid nomenclature [Table 1]

[0135] Amino acid modifications, such as amino acid substitutions, may be introduced using known protocols of recombinant genetic technology, including PCR, gene cloning, site-directed mutagenesis of cDNA, transfection of host cells, and in vitro transcription, which may be used to introduce such changes into a reference sequence to yield SHC / HAC enzyme variants. The enzyme variants may then be screened for SHC / HAC functional activity.

[0136] Suitable sources of SHC / HAC enzymes are identified in Tables 2, 3, or 16. In embodiments, suitable sources include, for example, Alicyclobacillus acidocaldarius (Aac), Zymomonas mobilis (Zmo), Bradyrhizobium japonicum (Bjp), Gluconobacter morbifer (Gmo), Burkholderia ambifaria, Bacillus anthracis, Methylococcus capsulatus, Frankia alni, Acetobacter pasteurianus(Apa), Thermosynechococcus elongatus(Tel), Streptomyces coelicolor(Sco), Rhodopseudomonas palustris(Rpa), Teredinibacter turnerae(Ttu), Pelobacter carbinolicus(Pca) or Syntrophotalea carbinolica DSM 2380, Bacillus megaterium(Bme), Streptomyces albolongus (Sal), and Tetrahymena pyriformis (see, e.g., WO 2010 / 139719, US 2012 / 01345477, WO 2012 / 066059, the contents of which are incorporated herein by reference).

[0137] In particular, the SHC / HAC enzyme (e.g., an SHC / HAC enzyme from which an SHC / HAC enzyme variant may be derived) may be an Alicyclobacillus acidocaldarius (Aac) SHC / HAC enzyme, a Zymomonas mobilis SHC / HAC (ZmoSHC1) enzyme, a Bradyrhizobium japonicum (Bjp or Bja) SHC / HAC enzyme, a Gluconobacter morbifer (Gmo) SHC / HAC enzyme, an Acetobacter pasteurianus SHC / HAC (ApaSHC1) enzyme, or a Bacillus megaterium (Bme) SHC / HAC enzyme. In particular, the SHC / HAC enzyme (e.g., an SHC / HAC enzyme from which an SHC / HAC enzyme variant may be derived) may be an Alicyclobacillus acidocaldarius (Aac) SHC / HAC enzyme. This enzyme is represented by SEQ ID NO: 1.

[0138] For ease of reference, the designation "AacSHC" may be used to refer to the Alicyclobacillus acidocaldarius (Aac) SHC / HAC enzyme, "ZmoSHC" may be used to refer to the Zymomonas mobilis (Zmo) SHC / HAC enzyme, "BjpSHC" or "BjaSHC" may be used to refer to the Bradyrhizobium japonicum (Bjp) SHC / HAC enzyme, "ApaSHC" may be used to refer to the Acetobacter pasteurianus (Apa) SHC / HAC enzyme, "BmeSHC" may be used to refer to the Bacillus megaterium (Bme) SHC / HAC enzyme, "SalSHC" may be used to refer to the Streptomyces albolongus (Sal) SHC / HAC enzyme, and "GmoSHC" may be used to refer to the Gluconobacter morbifer (Gmo) SHC / HAC enzyme.

[0139] The enzyme sequences for AacSHC, ZmoSHC, and BjpSHC are disclosed in BASF WO 2010 / 139719, US 2012 / 01345477A1, Seitz et al. (cited above), and Seitz (2012 PhD thesis, cited above). Two different sequences have been disclosed for ZmoSHC, designated ZmoSHC1 and ZmoSHC2. The Gmo SHC / HAC enzyme sequence is disclosed in WO 2018 / 157021. The SalSHC enzyme is disclosed in Liu et al. (2020): A Novel Soluble Squalene-Hopene Cyclase and Its Application in Efficient Synthesis of Hopene, Frontiers in Bioengineering and Biotechnology, vol 8, article 426, https: / / doi.org / 10.3389 / fbioe.2020.00426.

[0140] Table 2. Sources and accession numbers of wild-type (WT) SHC enzymes. [Table 2-1]

[0141] [Table 2-2]

[0142] [Table 2-3]

[0143] [Table 2-4]

[0144] [Table 2-5]

[0145] [Table 2-6]

[0146] Table 3. Sources and accession numbers of preferred wild-type (WT) SHC enzymes. [Table 3-1]

[0147] [Table 3-2]

[0148] The sequences of wild-type AacSHC, wild-type ZmoSHC1, wild-type ZmoSHC2, wild-type BjpSHC, wild-type GmoSHC, wild-type TelSHC, and wild-type ApaSHC1, wild-type BmeSHC, wild-type SalSHC, and wild-type ApaSHCA are also disclosed herein in Table 3.

[0149] The most preferred wild-type SHCs are: AacSHC (SEQ ID NO: 1), or ZmoSHC1 represented by SEQ ID NO: 13, or ZmoSCH2 represented by SEQ ID NO: 15, or Tel SHC represented by SEQ ID NO: 23, or ScoSHC represented by SEQ ID NO: 32.

[0150] Alignment of WT SHC sequences prepared by Hoshino and Sato (2002, cited above) indicates that multiple motifs were detected and consist of the core sequence Gln-XXX-Gly-X-Trp, which was found six times in the SHC sequences of both Z. mobilis and A. acidocaldarius (see Figure 3 in Reipen et al 1995, Microbiology 141, 155-161), where X can be any amino acid. Hoshino and Sato (2002, cited above) reported that aromatic amino acids are unusually abundant in SHC and noted two characteristic motifs in SHC: one is the QW motif, represented by a specific amino acid motif [(K / R)(G / A)X2-3(F / Y / W)(L / IV)3X3QX2-5GXW], and the other is the DXDDTA motif (SEQ ID NO: 314), which Wendt et al. (1997, Science 277, 1811-1815 and 1999, J Mol Biol 286, 175-187) reported in their X-ray structural analysis of SHC (X can be any amino acid). The DXDDTA motif appears to correlate with the SHC active site.

[0151] As used herein, a reference AacSHC protein may refer to the wild-type AacSHC protein as disclosed in SEQ ID NO: 1. AacSHC has the activity of a homofarnesol ambrox cyclase (HAC), which is useful for the production of ambrox derivatives through the biocatalytic reaction of SHC with a homofarnesol substrate. The primary reaction of AacSHC is the cyclization of a linear or non-linear substrate, such as homofarnesol, to produce ambrox.

[0152] Another reference AacSHC protein is the SHC / AHC enzyme 215G2 SHC or 215G2 SHC as disclosed in SEQ ID NO: 3. SEQ ID NO: 3 corresponds to SEQ ID NO: 1 with the mutations M132R, A224V, and I432T.

[0153] Another reference AacSHC protein is SHC / AHC enzyme variant #65 as disclosed in SEQ ID NO: 2. SEQ ID NO: 2 corresponds to SEQ ID NO: 1 with the substitutions M132R, A224V, I432T, A557T, and R613S, and is sometimes referred to herein as SHC / HAC enzyme variant #65 or SHC#65 variant.

[0154] The term "functional homologue" may be replaced by "functional equivalent" or by "homologue". Functional homologs of the wild-type SHC / HAC enzymes or SHC / HAC enzyme variants described herein are also suitable for use in cyclization reactions, e.g., to produce (-)-ambrox, e.g., in a recombinant host. Thus, the recombinant host may include one or more heterologous nucleic acid(s) encoding a functional homolog of the above-described polypeptides and / or a heterologous nucleic acid encoding a SHC / HAC derivative enzyme as described herein.

[0155] A functional homolog is a polypeptide that has sequence identity, and optionally sequence similarity, with a reference polypeptide and performs one or more of the biochemical or physiological functions of the reference polypeptide. The functional homolog and the reference polypeptide may be naturally occurring polypeptides, and the sequence identity, and optionally sequence similarity, may result from convergent or divergent evolutionary events. Therefore, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs. Variants of naturally occurring functional homologs, such as polypeptides encoded by mutants of a wild-type coding sequence, may themselves be functional homologs. Functional homologs can also be created for a given polypeptide through site-directed mutagenesis of the coding sequence, or for different naturally occurring polypeptides by combining domains from the coding sequences ("domain swapping"). Techniques for improving genes encoding functional homologs described herein are known and include, among others, directed evolution, site-directed mutagenesis, and random mutagenesis techniques, and may be useful to increase a specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or improve polypeptide:polypeptide interactions in a desired manner. Such improved polypeptides are considered functional homologs. The term "functional homolog" is sometimes applied to nucleic acids encoding functionally homologous polypeptides.

[0156] Functional homologs can be identified by analyzing nucleotide and polypeptide sequence alignments. For example, performing a query on a nucleotide or polypeptide sequence database can identify homologs of nucleic acid sequences encoding SHC derivative polypeptides, etc.

[0157] Hybridization can also be used to identify functional homologs and / or to identify homology between two nucleic acid sequences. Nucleic acid sequences encoding any of the proteins disclosed herein, or portions thereof, can be used as hybridization probes according to standard hybridization techniques. Hybridization of the probe to DNA or RNA from a test source (e.g., mammalian cells) is indicative of the presence of the relevant DNA or RNA in the test source. Hybridization conditions are known to those skilled in the art and can be found in *Current Protocols in Molecular Biology*, John Wiley & Sons, NY, 6.3.1-6.3.6, 1991. Moderate hybridization conditions are defined as equivalent to hybridization in 2x sodium chloride / sodium citrate (SSC) at 30°C, followed by a wash in 1x SSC, 0.1% SDS at 50°C. Highly stringent conditions are defined as equivalent to hybridization in 6x sodium chloride / sodium citrate (SSC) at 45°C, followed by a wash in 0.2x SSC, 0.1% SDS at 65°C. Sequence analysis to identify functional homologs can also involve BLAST, reciprocal BLAST, or PSI-BLAST analysis of non-redundant databases using related amino acid sequences as reference sequences. Amino acid sequences are, in some cases, deduced from nucleotide sequences. Those polypeptides in the database with greater than 40% sequence identity are candidates for further evaluation for suitability for use in SHC / HAC biotransformation reactions. Amino acid sequence similarity allows for conservative amino acid substitutions, such as the substitution of one hydrophobic residue for another, or one polar residue for another. If desired, manual inspection of such candidates can be performed to narrow the number of candidates to be further evaluated. Manual inspection can be performed, for example, by selecting those candidates that appear to have conserved functional domains.

[0158] Typically, polypeptides exhibiting at least about 30% amino acid sequence identity are useful for identifying conserved regions. Conserved regions of related polypeptides exhibit at least 30%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, or 69% amino acid sequence identity. In some embodiments, the conserved region exhibits at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity. Sequence identity can be determined as described above and below. Often, in this context, the region may contain 5 to 50 amino acids, or 10 to 150, or 10 to 80, or 10 to 100 amino acids.

[0159] The SHC / HAC enzymes or enzyme variants described herein and used in the methods described herein include, for example, those set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123 , 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 1 86, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217 7, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248 ,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279,280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, or the amino acid sequence of any of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, or a variant, homologue, mutant, derivative, or fragment thereof.

[0160] Additionally, the reference SHC enzyme produced may be based on an amino acid sequence produced from E. coli.

[0161] "Percent (%) identity" with respect to the nucleotide sequence of a gene is defined as the percentage of nucleotides in a candidate DNA sequence that are identical to the nucleotides in the DNA sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotide sequence identity can be achieved in a variety of ways that are within the skill in the art, illustratively using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms required to achieve maximal alignment over the full length of the sequences being compared. The terms "polypeptide" and "protein" are used interchangeably herein and refer to any peptide chain of amino acids, regardless of length or post-translational modification.

[0162] As used herein, the term "derivative" includes, but is not limited to, variants. The terms "derivative" and "variant" are used interchangeably herein.

[0163] In preferred embodiments, variant enzymes usable in the present disclosure exhibit a total of up to 200 changes (modifications) (i.e., replacements, insertions, deletions, N-terminal truncations, and / or C-terminal truncations) in the amino acid sequence (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200). The amino acid exchanges may be conservative and / or non-conservative. In a preferred embodiment, the variants usable in the present disclosure differ from the protein or domain from which the variant is derived by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid exchanges (preferably conservative amino acid changes). The variants may additionally or alternatively contain amino acid deletions, which may be N-terminal truncations, C-terminal truncations, or internal deletions, or any combination thereof. Such variants containing N-terminal truncations, C-terminal truncations, and / or internal deletions are referred to in the context of this application as "deletion variants" or "fragments." The terms "deletion variants" and "fragments" are used interchangeably herein. Deletion variants can be naturally occurring (for example, splice variants), or can be artificially constructed, preferably by genetic engineering means.Typically, the protein or protein domain from which deletion variants are derived is a wild-type protein.However, the deletion variants of the present disclosure can also be derived from homologs, orthologs, or paralogs of parent polypeptides or from artificially constructed variants, provided that the deletion variants exhibit at least one biological activity of parent polypeptides.Preferably, a deletion variant (or fragment) has a deletion of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acids at its N-terminus, and / or at its C-terminus, and / or internally, compared to the parent polypeptide.

[0164] In some embodiments, the SHC / HAC enzyme variants described herein include only substitutions and do not include any deletions or insertions.

[0165] "Variant," as used herein, may alternatively or additionally be characterized by a degree of sequence identity or similarity with the parent polypeptide from which it is derived. Variants of the WT / reference SHC / HAC or SHC / HAC derivatives of the present disclosure share at least 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 1 It may have a sequence identity of 5%, 76%, 77%, 78%, 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.

[0166] The expression "at least 30%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, The terms "3%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity" are used throughout this specification in reference to sequence comparisons of polypeptides and polynucleotides. Polynucleotides belonging to any of the families of enzymes or proteins disclosed herein can be identified based on their similarity to related genes or proteins, respectively. For example, identification can be based on sequence identity. In a preferred embodiment, the present disclosure provides a method for the preparation of a SHC / HAC enzyme comprising: (a) a wild-type SHC / HAC enzyme disclosed herein (e.g., SEQ ID NO: 1, SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128 , 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159,160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 2 22, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 3, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284 , 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, or any other SHC / HAC disclosed in Tables 2, 3, or 16 disclosed herein), (b) the nucleotide sequence of SEQ ID NO: 35, and (c) at least 30 (e.g., at least 30, 40, 50, 60, 80, 100, 125, 150, 175, 200, 250, 300, 400, 500, 600, 700, 800, 850, 900, 950, 1000, 1100, 1250, 1300, 1400, 1500, 1600, 1750, 1800, 1900, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3100, 3200, 3300, 3400, 3500, 3600, 3700, 3800, 3900, 4100, 4200, 4300, 4400, 4500, 4600, 4700, 4800, 4900, 5100, 5200, 5300, 5400, 5500, 5600, 5700, 5800 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 1010, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 200%, 2010The invention features isolated nucleic acid molecules that are 79%, 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the original.

[0167] Preferably, the subject polypeptide and the reference polypeptide exhibit the indicated sequence identity or similarity over a continuous stretch of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids. Preferably, the subject polynucleotide and the reference polynucleotide exhibit the indicated sequence identity over a continuous stretch of 60, 90, 120, 135, 150, 180, 210, 240, 270, 300 or more nucleotides. In cases where two sequences are compared and the reference sequence is not specified in comparison with the sequence for which the sequence identity percentage is to be calculated, unless otherwise specifically indicated, the sequence identity should be calculated with reference to the longer of the two sequences to be compared. Where a reference sequence is indicated, sequence identity is assumed to be the sequence of the reference sequence (e.g., SEQ ID NOs: 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 17 16, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 209, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2020, 2021, 2022, 2023, 202 6, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, and any SHC / HAC enzyme as disclosed in Tables 2, 3, or 16.

[0168] For example, a peptide sequence consisting of 130 amino acids may exhibit a maximum percentage sequence identity of 20.6% (130 / 631x100) when compared to the full-length amino acid of wild-type AacSHC, which has 631 amino acid residues, while a sequence with a length of 300 amino acids may exhibit a maximum percentage sequence identity of 47.5% (300 / 631x100).

[0169] The identity of nucleotide and amino acid sequences, i.e., the percentage of sequence identity, can be determined through sequence alignment. Such alignments include, for example, https: / / www.ebi.ac.uk / Tools / msa / clustalo / or the GAP program (a mathematical algorithm from University of Iowa), or the mathematical algorithm of Myers and Miller (1989-Cabios 4:11-17), or several algorithms known in the art available on Clone Manager 9, preferably the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), hmmalign (HMMER package, http: / / hmmer.wustl.edu / ), or the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80). Preferred parameters used are: https: / / www.ebi.ac.uk / Tools / msa / clustalo / These are the initial setting parameters as set above.

[0170] The grade of sequence identity (sequence matching) may be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms are incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215, 403-410. BLAST polynucleotide searches are performed with the BLASTN program, score=100, word length=12, to obtain polynucleotide sequences homologous to those nucleic acids encoding related proteins.

[0171] BLAST protein searches are performed using the BLASTP program, score = 50, word length = 3, to obtain amino acid sequences homologous to the SHC polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques, such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov random fields. When percentages of sequence identity are mentioned in this application, these percentages are calculated with respect to the full length of the longer sequence, unless otherwise specifically indicated.

[0172] In a specific embodiment, the percent identity between two sequences is determined using CLUSTAL O (version 1.2.4).

[0173] In some embodiments, "similarity" refers to the degree of sequence relatedness between such sequences, as determined by the match between the strings of amino acid sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of another polypeptide. "Identity" and "similarity" can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).

[0174] Preferred parameters for comparing polypeptide sequences include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison Matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from the Genetics Computer Group, Madison, WI. The aforementioned parameters are the default parameters for amino acid comparisons (as well as no penalty for end gaps).

[0175] Optionally, in determining the degree of amino acid similarity, one skilled in the art may also take into account so-called "conservative" amino acid substitutions, as would be apparent to one skilled in the art. The "similarity" between two amino acid sequences is determined by comparing the amino acid sequence of one polypeptide and its conserved amino acid substitutes with the sequence of another polypeptide. As used herein, "conservative" amino acid substitutions refer to the interchangeability of residues with similar side chains. Examples of classes of amino acid residues for conservative substitutions are provided in the table below.

[0176] Table 4: Classes of amino acid residues [Table 4]

[0177] Table 5: Alternative conservative amino acid residue substitution classes [Table 5]

[0178] Table 6: Physical and functional classification of amino acid residue alternatives [Table 6]

[0179] For example, the group of amino acids with aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; the group of amino acids with aliphatic-hydroxyl side chains is serine and threonine; the group of amino acids with amide-containing side chains is asparagine and glutamine; the group of amino acids with aromatic side chains is phenylalanine, tyrosine, and tryptophan; the group of amino acids with basic side chains is lysine, arginine, and histidine; and the group of amino acids with sulfur-containing side chains is cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitution variants of the amino acid sequences disclosed herein are variants in which at least one residue in the disclosed sequence is removed and a different residue is inserted in its place. Preferably, the amino acid changes are conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to Ser; Arg to Lys; Asn to Gln or His; Asp to Glu; Cys to Ser or Ala; Gln to Asn; Glu to Asp; Gly to Pro; His to Asn or Gln; Ile to Leu or Val; Lys to Arg; Gln or Glu; Met to Leu or Ile; Phe to Met, Leu, or Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp or Phe; and Val to Ile or Leu.

[0180] Particular SHC / HAC enzymes and enzyme variants that may be used in the methods described herein are described further below.

[0181] SHC / HAC variants with novel mutations Surprisingly, the present invention relates to the use of SHC / HAC enzymes derived from the Aac SHC / HAC enzyme (SEQ ID NO: 1), or variants of the Aac SHC / HAC enzyme (SEQ ID NO: 2 or SEQ ID NO: 3), or other wild-type SHCs as identified herein (such as those identified in Tables 2, 3, or 16, in particular SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 200, 210, 220, 230, 240, 250, 251, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145 , 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205 , 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265,266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310) or other variants (SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53, 54) and Table 14 of WO2016 / 170099 and / or WO2 SHC / HAC enzyme variants derived from those disclosed in Table 6 of US Pat. No. 021 / 110848 (as represented by SEQ ID NOs: 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 as provided below) exhibit improved / optimized activity / biological activity as previously defined herein and as further demonstrated in the experimental part.

[0182] SEQ ID NO:3 corresponds to SEQ ID NO:1 with the amino acid modifications (substitutions) M132R, A224V, and I432T. SEQ ID NO:2 corresponds to SEQ ID NO:1 with the amino acid modifications (substitutions) M132R, A224V, I432T, A557T, and R613S.

[0183] In embodiments, the new SHC / HAC enzyme variants derived from SEQ ID NO: 2 or 3 still contain amino acid modifications (substitutions) when compared to SEQ ID NO: 1. These amino acid modifications are as follows: - variants derived from SEQ ID NO: 3 may comprise the following amino acid modifications (substitutions) compared to SEQ ID NO: 1: M132R, A224V, and I432T, - variants derived from SEQ ID NO: 2 may comprise the following amino acid modifications (substitutions) compared to SEQ ID NO: 1: M132R, A224V, I432T, A557T, and R613S, - a variant derived from SEQ ID NO: 2 may comprise the following amino acid modifications (substitutions) compared to SEQ ID NO: 1: M132R and I432T, - variants derived from SEQ ID NO: 2 may comprise the following amino acid modifications (substitutions) compared to SEQ ID NO: 1: M132R and I432T, A557T and R613S.

[0184] Variants derived from any one of the sequences listed in Table 14 of WO2016 / 170099 include, but are not limited to, SEQ ID NOs: 5, 7, 9, 11, 13, 15, 17, 19, 171, 23, 25, 27, 29, 31, 33, 35, 37, or 39 (corresponding to SEQ ID NOs: 361-368, 378, 3, 369-377, respectively, of the present application) (see Table 7 below and sequences disclosed at the end of the experimental part).

[0185] Variants derived from any one of the sequences listed in Table 6 of WO2021 / 110848 include, but are not limited to, SEQ ID NOs: 5, 17, 18, 2, or 4 (see Table 8 below and the sequences disclosed at the end of the experimental part).

[0186] Table 7: Corresponds to Table 14 of WO 2016 / 170099: AacSHC derivative amino acid and nucleotide sequence numbers [Table 7]

[0187] Table 8: Corresponding to Table 6 of WO2021 / 110848. Mutations in selected new SHC variant enzymes. [Table 8] Note: These mutations appear to be in addition to the mutations present in 215G2 SHC: M132R, A224V, and I432T. SEQ ID NOs: 2, 3, 4, 5, 17, and 18 in Table 8 correspond to SEQ ID NOs: 379, 2, 380, 381, 382, ​​and 383.

[0188] The sequences identified in Tables 7 and 8 above are represented by any of SEQ ID NOs: 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, as indicated in the sequence listing and at the end of the experimental part.

[0189] In some embodiments, the SHC / HAC enzyme variants are selected from the group consisting of SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 14 3, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 , 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 1 97, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228 8, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259 , 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383. For example, the SHC / HAC enzyme variants are selected from the group consisting of SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 , 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193 2, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192 , 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242,243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276 6, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, may have less than about 25 amino acid modifications, or less than about 20 amino acid modifications, or less than about 15 amino acid modifications, or less than about 10 amino acid modifications, or less than about 9 amino acid modifications, or less than about 8 amino acid modifications, or less than about 7 amino acid modifications, or less than about 6 ... For example, SHC / HAC enzyme variants can be selected from the group consisting of SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157,158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 3, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 31 49, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294 , 295, 296, 307, 308, 309, 310, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383. The amino acid modifications may be, for example, insertions, deletions, and / or substitutions as described above.

[0190] In one embodiment, SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244 7, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138 , 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 2 01, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232 2, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263 , 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294,The only amino acid modifications in the SHC / HAC enzyme variants compared to 295, 296, 307, 308, 309, 310, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 are substitutions (i.e., no insertions or deletions).

[0191] Amino acid alterations are defined relative to a reference sequence. An amino acid alteration relative to a reference sequence means that the amino acid sequence of a variant sequence differs from the reference sequence.

[0192] Amino acids in a reference sequence and a variant sequence may be assigned numbers, where the numbering begins with the amino acid at the N-terminus of the polypeptide (i.e., the amino acid at the N-terminus of a polypeptide is numbered 1, the next amino acid is numbered 2, etc.). A "position" in a reference sequence refers to a particular amino acid residue present in the reference sequence, as identified by a particular numbered amino acid in the reference sequence. A "position" in a variant sequence refers to a particular amino acid residue present in the variant sequence, as identified by a particular numbered amino acid in the variant sequence.

[0193] Because a variant sequence may contain deletions or insertions compared to a reference sequence, amino acids in the variant sequence may be numbered differently than the same amino acids in the reference sequence. As an example, if an amino acid is inserted between amino acids 131 and 132 of SEQ ID NO: 1, the amino acid will retain the number 132 in the reference sequence, while the amino acid following the insertion will have the number 133 in the variant sequence. In this example, the position in the variant sequence that corresponds to position 132 in the reference sequence is position 133. Thus, an amino acid in a variant sequence that is retained from a reference sequence may be defined by referring to the "corresponding position" in the reference sequence. In other words, a "position" in a variant sequence may be defined by referring to the "corresponding position" in the reference sequence. Notably, a substitution in a variant sequence compared to a reference sequence may be defined by referring to the "corresponding position" in the reference sequence, despite any insertions and / or deletions in the reference sequence. If an amino acid in the reference sequence is deleted, there will be no "corresponding position" in the variant sequence. If there are no insertions or deletions (i.e., only substitutions) compared to the reference sequence, the "corresponding position" of the reference sequence will be the same as the position in the variant sequence.

[0194] For example, position 169 in AacSHC represented by sequence number 1 corresponds to position 222 in ZmoSHC1 (sequence number 13 or 14), position 177 in ZmoSHC2 (sequence number 15), position 172 in TelSHC (sequence number 23), and position 196 in ScoSH1 (sequence number 32).

[0195] For example, position 306 in AacSHC represented by sequence number 1 corresponds to position 368 in ZmoSHC1 (sequence number 13 or 14), position 321 in ZmoSHC2 (sequence number 15), position 311 in TelSHC (sequence number 23), and position 335 in ScoSH1 (sequence number 32).

[0196] For example, position 600 in AacSHC represented by sequence number 1 corresponds to position 667 in ZmoSHC1 (sequence number 13 or 14), position 619 in ZmoSHC2 (sequence number 15), position 609 in TelSHC (sequence number 23), and position 629 in ScoSH1 (sequence number 32).

[0197] In embodiments, the SHC / HAC enzyme variant is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103 , 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 66, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 1 97, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228 8, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259 , 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, wherein the amino acid sequence of the SHC / HAC enzyme variant is set forth in SEQ ID NOs: 1, 2, 3, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 3, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111 11, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 1 42, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 3, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204 , 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 2 3, or at positions corresponding to W169, A306, and / or G600 of SEQ ID NO: 1, 2, 3, compared to 96, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 in the amino acid sequence of a wild-type SHC / HAC.

[0198] The amino acid sequences of the SHC / HAC enzyme variants are SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, and 103. , 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 66, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 1 97, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228 8, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259 , 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 at positions corresponding to W169, A306, and / or G600 of SEQ ID NO: 1, 2, or 3 in the amino acid sequence of a wild-type SHC / HAC.

[0199] In embodiments, the SHC / HAC enzyme or enzyme variant (also designated as Type 1 variant) is selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO Column number 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, and / or SEQ ID NOs: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 23 19, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 1 50, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 1, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212 , 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 66, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 and at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 1 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity to SEQ ID NO: 1, 2, or 3, and at positions corresponding to positions W169, A306, and / or G600 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, and / or 54 ... SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, corresponding to positions W169, A306, and / or G600 at 48, 49, 50, 51, 52, 53, and / or 54;68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310.

[0200] In a preferred embodiment, the SHC / HAC enzyme or enzyme variant has at least 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 1, 2, 3. The present invention relates to a method for the preparation of a human ovarian cancer cell line comprising administering to a mammalian animal or mammalian patient an amino acid sequence having 0%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity or similarity to the present invention, and having amino acid modifications at positions corresponding to W169, A306, and / or G600 of SEQ ID NOs: 1, 2, 3, relative to SEQ ID NOs: 1, 2, 3.

[0201] In embodiments, the SHC / HAC enzyme variants (also designated Type 2 variants) are selected from the group consisting of SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 1 0, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 1 55, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 1 86, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217 7, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248 ,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279,280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and having an amino acid sequence having at least about 30.0% identity or similarity to SEQ ID NOs: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 378, 379, 380, 381, 382, ​​or 383, and 4, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 at a position corresponding to position W169 of SEQ ID NOs: 1, 2, 3, or 4, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 379, 380, 381, 382, ​​or 383 of SEQ ID NOs: 1, 2, 3, or 4 ...7, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 5, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 1 05, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136 6, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167 , 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198,199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258 , 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 47, 48, 49, 50, 51, 52, 53, or 54.

[0202] In this context, identity or similarity is at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 1 It may be 4%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0203] In embodiments, the SHC / HAC enzyme variants (also designated Type 3 variants) are selected from the group consisting of SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 1 0, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 1 55, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 1 86, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217 7, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248 ,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279,280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and having an amino acid sequence having at least about 30.0% identity or similarity to SEQ ID NOs: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 378, 379, 380, 381, 382, ​​or 383, and 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 at positions corresponding to W169 and G600M in SEQ ID NOs: 1, 2, 3, or corresponding to W169 and G600M in SEQ ID NOs: 1, 2, 3, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110 01, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 2, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163 , 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194,195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 , 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310.

[0204] In this context, identity or similarity is at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 1 It may be 4%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0205] In embodiments, the SHC / HAC enzyme variants (also designated Type 4 variants) are selected from the group consisting of SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 1 0, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 1 55, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 1 86, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217 7, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248 ,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279,280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and having an amino acid sequence having at least about 30.0% identity or similarity to SEQ ID NOs: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 at positions corresponding to W169, A306, and G600 of SEQ ID NO: 1, 2, or 3, or at positions corresponding to W169, A306, and G600 of SEQ ID NO: 1, 2, or 3 , 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127 7, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158 , 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 22 0, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 2 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310.

[0206] In this context, identity or similarity is at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 1 It may be 4%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0207] All combinations of modified positions in the above-identified SHC / AHC enzyme variants are encompassed by the present disclosure: (when referring to SEQ ID NO: 1) - W169 - W169, A306 - W169、A306、G600 - W169、G600 - A306、G600 - A306 - G600。

[0208] The new amino acid (X) at the position corresponding to position 169 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 can be G, A, or V, or functional equivalents thereof. The functional equivalent of G is P. A and V are functional equivalents. M, L, and I are also functional equivalents of A and V. SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19 at position 169 of SEQ ID NOs: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, or corresponding to 169 of SEQ ID NOs: 1, 2, or 3 , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148 , 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195,196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255 , 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, the preferred new amino acids are G, A, or V. SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63 at position 169 of SEQ ID NOs: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, or corresponding to 169 of SEQ ID NOs: 1, 2, or 3 , 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 1 05, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137,138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 26, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 3 The most preferred new amino acid at the position in the amino acid sequence of the wild-type SHC / HAC enzyme as follows: 70, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 is G.

[0209] For example, position 169 in AacSHC represented by sequence number 1 corresponds to position 222 in ZmoSHC1 (sequence number 13), position 177 in ZmoSHC2 (sequence number 15), position 172 in TelSHC (sequence number 23), and position 196 in ScoSH1 (sequence number 32).

[0210] The new amino acid (X) at the position corresponding to position 306 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 can be V or a functional equivalent thereof. Functional equivalents of V are A, M, L, and I. at position 306 of SEQ ID NOs: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, or corresponding to 306 of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 200, 210, 220, 230, 240, 250, 260, 270, 271, 272, 273, 274, 275, 276, 277, 5, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 , 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181 , 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, A preferred new amino acid at a position in the amino acid sequence of the wild-type SHC / HAC enzyme as 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 is V.

[0211] For example, position 306 in AacSHC represented by sequence number 1 corresponds to position 368 in ZmoSHC1 (sequence number 13), position 321 in ZmoSHC2 (sequence number 15), position 311 in TelSHC (sequence number 23), and position 335 in ScoSH1 (sequence number 32).

[0212] The new amino acid (X) at the position corresponding to position 600 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 can be A, V, L, I, or M, or functional equivalents thereof. All of these amino acids are functional equivalents. SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 at position 600 of SEQ ID NOs: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, or corresponding to 600 of SEQ ID NOs: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54 , 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130 , 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179 , 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228,229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, At positions in the amino acid sequence of the wild-type SHC / HAC enzyme as 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, preferred new amino acids are A, V, L, I, or M. at position 600 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, or corresponding to 600 of SEQ ID NO: 1, 2, or 3 SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 29, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169,170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242 42, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 2 The most preferred new amino acid at the position in the amino acid sequence of the wild-type SHC / HAC enzyme as follows: 78, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 is M.

[0213] For example, position 600 in AacSHC represented by sequence number 1 corresponds to position 667 in ZmoSHC1 (sequence number 13), position 619 in ZmoSHC2 (sequence number 15), position 609 in TelSHC (sequence number 23), and position 629 in ScoSH1 (sequence number 32).

[0214] Table 9: Positions of the three preferred mutated amino acids (AA) in four different wild-type SHC / HAC [Table 9]

[0215] In embodiments, the SHC / HAC enzyme variants (also designated Type 5 variants) are selected from the group consisting of SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 8, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 61, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 1 92, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 3, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254 ,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and having an amino acid sequence having at least about 30.0% identity or similarity to SEQ ID NOs: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and 66, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 at positions corresponding to W169, A306, and / or G600 of SEQ ID NO: 1, 2, or 3, or at positions corresponding to W169, A306, and / or G600 of SEQ ID NO: 1, 2, or 3 , 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 1 00, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131 1, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162 , 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193,194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, wherein the amino acid modification is as follows: - W at position 169 is replaced by G, A, or V, or a functional equivalent thereof, preferably wherein W at position 169 is replaced by G; - A in position 306 is replaced by V or its functional equivalent, and / or - G at position 600 is replaced by A, V, L, I, or M, or functional equivalents thereof, preferably wherein G at position 600 is replaced by M.

[0216] In this context, identity or similarity is at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 1 It may be 4%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0217] In embodiments, the SHC / HAC enzyme variants (also designated Type 6 variants) are selected from the group consisting of SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 8, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 61, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 1 92, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 3, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254 ,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, and having an amino acid sequence having at least about 30.0% identity or similarity to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, at a position corresponding to W169 or corresponding to W169 in SEQ ID NOs: 1, 2, or 3 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 1 32, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163 3, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194 , 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 3, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, wherein the amino acid modification is a W replaced by a G at position 169.

[0218] In embodiments, the W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by G.

[0219] In this context, identity or similarity is at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 1 It may be 4%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0220] In embodiments, the SHC / HAC enzyme variants (also designated Type 7 variants) are selected from the group consisting of SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 8, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 61, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 1 92, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 3, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254 ,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and 6, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 at positions corresponding to W169 and G600M in SEQ ID NOs: 1, 2, or 3; , 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 1 06, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137 7, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168 , 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, wherein the amino acid modification is as follows: - W at position 169 is replaced by G, and - The G at position 600 is replaced by an M.

[0221] In embodiments, the W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by G. and the G at position 600 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by M.

[0222] In this context, identity or similarity is at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 1 It may be 4%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0223] In embodiments, the SHC / HAC enzyme variant is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103 , 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 66, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 1 97, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228 8, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259 , 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and 71, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 at positions corresponding to positions W169 and A306 of SEQ ID NOs: 1, 2, or 3; ,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,1 11, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142 2, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173 , 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204,205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260 and having an amino acid modification at a position in the amino acid sequence of the wild-type SHC / HAC enzyme as follows: 0, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, wherein the amino acid modification is as follows: - W at position 169 is replaced by G, and - A at position 306 is replaced by V.

[0224] In embodiments, the W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by G, and - an A at position 306 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is replaced by a V.

[0225] In this context, identity or similarity is at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 1 It may be 4%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0226] In embodiments, the SHC / HAC enzyme variants (also designated Type 8 variants) are selected from the group consisting of SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 8, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161 61, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 1 92, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 3, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254 ,255,256,257,258,259,260,261,262,263,264,265,266,267,268,269,270,271,272,273,274,275,276,277,278,279,280,281,282,283,284,285,286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and having an amino acid sequence having at least about 30.0% identity or similarity to SEQ ID NOs: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and 66, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 at positions corresponding to W169, A306, and G600 of SEQ ID NOs: 1, 2, or 3; , 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 1 03, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 4, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165 , 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, wherein the amino acid modification is: - W at position 169 is replaced by G, - A at position 306 is replaced by V, and - The G at position 600 is replaced by an M.

[0227] In embodiments, the W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by G. - A at position 306 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by V; and - G at position 600 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is replaced by M.

[0228] In this context, identity or similarity is at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 1 It may be 4%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0229] In embodiments, the SHC / HAC enzyme variant has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and is selected from the group consisting of SEQ ID NOs:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383. 37, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and have amino acid modifications at positions corresponding to positions W169 and G600 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, wherein the amino acid modifications are as follows: - W at position 169 is replaced by G, and - The G at position 600 is replaced by an M.

[0230] In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0231] In embodiments, the SHC / HAC enzyme variant has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and is selected from the group consisting of SEQ ID NOs:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383. 37, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and have amino acid modifications at positions corresponding to positions W169 and A306 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, wherein the amino acid modifications are as follows: - W at position 169 is replaced by G, and - A at position 306 is replaced by V.

[0232] In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0233] In embodiments, the SHC / HAC enzyme variant (also designated as Type 9 variant) has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and is selected from the group consisting of SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, with amino acid modifications at positions corresponding to W169, A306, and / or G600 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, wherein the amino acid modifications are as follows: - W at position 169 is replaced by G, - A at position 306 is replaced by V, and - The G at position 600 is replaced by an M.

[0234] In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0235] In a preferred embodiment, the SHC / HAC enzyme variant (also designated a Type 10 variant) has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, 305, or 306, and is identical to SEQ ID NO:1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, 305, or 306, and have an amino acid modification at a position corresponding to position W169 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, or 54, wherein the amino acid modification is W replaced by G at position 169.

[0236] In embodiments, each of the SHC / HAC enzyme variants (Type 10 variants) having at least 70.0% identity or similarity to SEQ ID NO:2, 3, 47, 48, 49, 50, 51, 53, 54, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 305, or 306 still have the same mutation(s) but which differ from SEQ ID NO: 1. Each of these mutations has been previously defined herein, at least in the section entitled "Sequence Summary." In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0237] In a preferred embodiment, the SHC / HAC enzyme variant (ZmoSHC1 P1 variant, SEQ ID NO: 312) (also designated as the Type 10 variant) has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO: 13 or 14, and has an amino acid modification at position W222 of SEQ ID NO: 13 or 14 relative to SEQ ID NO: 13, where the amino acid modification is a W replaced by a G at position 222.

[0238] In a preferred embodiment, this enzyme variant is represented by an amino acid sequence having at least 70% similarity to SEQ ID NO: 312 and still having W222G. In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0239] In a preferred embodiment, the SHC / HAC enzyme variant (ZmoSHC1 P2 variant, SEQ ID NO: 353) (also designated as the Type 10 variant) has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO: 13 and has amino acid modifications at positions W222 and G667 of SEQ ID NO: 13 relative to SEQ ID NO: 13, where W is replaced by G at position 222 and G is replaced by M at position 667.

[0240] In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0241] In a preferred embodiment, the SHC / HAC enzyme variant (ZmoSHC1 P2 variant, SEQ ID NO: 353) (also designated as the Type 10 variant) has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO: 13 and has amino acid modifications at positions W222 and A368 of SEQ ID NO: 13 relative to SEQ ID NO: 13, where W is replaced by G at position 222 and A is replaced by V at position 368.

[0242] In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0243] In a preferred embodiment, the SHC / HAC enzyme variant (ZmoSHC1 P3 variant, SEQ ID NO: 354) (also designated as the Type 10 variant) has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO: 13 and has amino acid modifications at positions W222, A368, and G667 of SEQ ID NO: 13 relative to SEQ ID NO: 13, such amino acid modifications being W replaced by G at position 222, A replaced by V at position 368, and G replaced by M at position 667.

[0244] In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0245] In a preferred embodiment, the SHC / HAC enzyme variant (ZmoSHC1 P2 variant, SEQ ID NO: 313) (also designated as the Type 10 variant) has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO: 15 and has an amino acid modification at position W177 of SEQ ID NO: 15, where W is replaced by G at position 177.

[0246] In a preferred embodiment, this enzyme variant is represented by an amino acid sequence that has at least 70% similarity to SEQ ID NO: 313 and still has W177G. In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0247] In a preferred embodiment, the SHC / HAC enzyme variant (ZmoSHC2 P2 variant, SEQ ID NO: 355) (also designated as the Type 10 variant) has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO: 15 and has amino acid modifications at positions W177 and G619 of SEQ ID NO: 15 relative to SEQ ID NO: 15, where W is replaced by G at position 177 and G is replaced by M at position 619.

[0248] In a preferred embodiment, the enzyme variant is represented by an amino acid sequence that has at least 70% similarity to SEQ ID NO: 313 or 355 and still has W177G and G619M. In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0249] In a preferred embodiment, the SHC / HAC enzyme variant has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO: 15 and has amino acid modifications relative to SEQ ID NO: 15 at positions W177 and 321 of SEQ ID NO: 15, such amino acid modifications being a W replaced by a G at position 177 and an A replaced by a V at position 321.

[0250] In a preferred embodiment, this enzyme variant is represented by an amino acid sequence that has at least 70% similarity to SEQ ID NO: 313 and still has W177G and A321V. In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0251] In a preferred embodiment, the SHC / HAC enzyme variant (ZmoSHC2 P3 variant, SEQ ID NO:356) (also designated as the Type 10 variant) has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO:15 and has amino acid modifications at positions W177, A321, and G619 of SEQ ID NO:15 relative to SEQ ID NO:15, where W is replaced by G at position 177, A is replaced by V at position 321, and G is replaced by M at position 619.

[0252] In a preferred embodiment, this enzyme variant is represented by an amino acid sequence that has at least 70% similarity to SEQ ID NO: 313 and still has W177G, A321V and G619M. In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0253] In a preferred embodiment, the SHC / HAC enzyme variant (Tel SHC variant P1, SEQ ID NO: 304) (also designated as the Type 10 variant) has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO: 23 and has an amino acid modification, relative to SEQ ID NO: 23, at position W172 of SEQ ID NO: 23, where such amino acid modification is a W replaced by a G at position 172.

[0254] In a preferred embodiment, this enzyme variant is represented by an amino acid sequence having at least 70% similarity to SEQ ID NO: 304 and still having W172G. In a preferred embodiment, this enzyme variant is represented by an amino acid sequence having at least 70% similarity to SEQ ID NO: 302 and still having W172G.

[0255] In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0256] In a preferred embodiment, the SHC / HAC enzyme variant (Tel SHC variant P2, SEQ ID NO:359) (also designated as the Type 10 variant) has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO:23 and has amino acid modifications at positions W172 and G609 of SEQ ID NO:23, relative to SEQ ID NO:23, where W is replaced by G at position 172 and G is replaced by M at position 609.

[0257] In a preferred embodiment, this enzyme variant is represented by an amino acid sequence that has at least 70% similarity to SEQ ID NO: 359 and still has W172G and G609M. In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0258] In a preferred embodiment, the SHC / HAC enzyme variant has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO:23 and has amino acid modifications at positions W172 and A311 of SEQ ID NO:23, relative to SEQ ID NO:23, where W is replaced by G at position 172 and A is replaced by V at position 311.

[0259] In a preferred embodiment, this enzyme variant is represented by an amino acid sequence that has at least 70% similarity to SEQ ID NO: 360 and still has W172G and A311V. In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0260] In a preferred embodiment, the SHC / HAC enzyme variant (Tel SHC variant P3, SEQ ID NO:360) (also designated as the Type 10 variant) has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO:23 and has amino acid modifications at positions W172, A311, and G609 of SEQ ID NO:23, relative to SEQ ID NO:23, where W is replaced by G at position 172, A is replaced by V at position 311, and G is replaced by M at position 609.

[0261] In a preferred embodiment, this enzyme variant is represented by an amino acid sequence that has at least 70% similarity to SEQ ID NO: 360 and still has W172G, A311V, and G609M. In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0262] In a preferred embodiment, the SHC / HAC enzyme variant (ScoSHC1 P1 variant, SEQ ID NO:311) (also designated as the Type 10 variant) has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO:32, and has an amino acid modification, relative to SEQ ID NO:32, at position W196 of SEQ ID NO:32, where such amino acid modification is a W replaced by a G at position 196.

[0263] In a preferred embodiment, this enzyme variant is represented by an amino acid sequence that has at least 70% similarity to SEQ ID NO: 311 and still has W196G.

[0264] In a preferred embodiment, the SHC / HAC enzyme variant (ScoSHC1 P2 variant, SEQ ID NO:357) (also designated as the Type 10 variant) has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO:32 and has amino acid modifications at positions W196 and G629 of SEQ ID NO:32, relative to SEQ ID NO:32, where W is replaced by G at position 196 and G is replaced by M at position 629.

[0265] In a preferred embodiment, this enzyme variant is represented by an amino acid sequence that has at least 70% similarity to SEQ ID NO: 311 and still has W196G and G629M.

[0266] In a preferred embodiment, the SHC / HAC enzyme variant has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO: 32 and has amino acid modifications relative to SEQ ID NO: 32 at positions W196 and A335 of SEQ ID NO: 32, where such amino acid modifications are W replaced by G at position 196 and A replaced by V at position 335.

[0267] In a preferred embodiment, the enzyme variant is represented by an amino acid sequence that has at least 70% similarity to SEQ ID NO: 311 and still has W196G and A335V. In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0268] In a preferred embodiment, the SHC / HAC enzyme variant (ScoSHC1 P3 variant, SEQ ID NO:358) (also designated as the Type 10 variant) has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO:32 and has amino acid modifications at positions W196, A335, and G629 of SEQ ID NO:32, relative to SEQ ID NO:32, where W is replaced by G at position 196, A is replaced by V at position 335, and G is replaced by M at position 629.

[0269] In a preferred embodiment, this enzyme variant is represented by an amino acid sequence that has at least 70% similarity to SEQ ID NO: 311 and still has W196G, A335V, and G629M. In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0270] In a preferred embodiment, the enzyme variant is: - the SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 1, 4, 5, 6, or 350 and has at least one of the following mutations: W169G, A306V, and G600M; or - the SHC / HAC enzyme variant has an amino acid sequence with at least 90.0% identity to SEQ ID NO: 7, 8, 9, or 386 and has the following mutations: M132R, A224V, I432T, A557T, R613S, and has at least one of the following mutations: W169G, A306V, and G600M; or - the SHC / HAC enzyme variant has an amino acid sequence with at least 90.0% identity to SEQ ID NO: 10, 11, 12, or 385, and has the following mutations: M132R, A224V, I432T, and at least one of the following mutations: W169G, A306V, and G600M; or - the SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 23, 304, 359, or 360 and has at least one of the following mutations: W172G, A311V, and G609M (Tel SHC / HAC variant); or - the SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 32, 311, 357, or 358 and has the following mutations: W196G, A335V, and G629M (Sco SHC / HAC variant); or - the SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 13, 312, 353, or 354 and has at least one of the following mutations: W222G, A368V, and G667M (Zmo SHC1 variant); or - the SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 15, 313, 355, or 356 and has at least one of the following mutations: W177G, A321V, and G619M (Zmo SHC2 variant); or - the SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 1 and has at least the following mutations: W169G, G600M, M132R, and I432T; or - the SHC / HAC enzyme variant has an amino acid sequence with at least 90.0% identity to SEQ ID NO: 1 and has the following mutations: W169G, G600M, M132R, and I432T; or - the SHC / HAC enzyme variant has an amino acid sequence with at least 95.0% identity to SEQ ID NO: 1 and has the following mutations: W169G, G600M, M132R, and I432T; or - the SHC / HAC enzyme variant has an amino acid sequence with at least 96.0%, 97.0%, 99.0%, 99.0% identity to SEQ ID NO: 1 and has the following mutations: W169G, G600M, M132R, and I432T.

[0271] In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0272] In a preferred embodiment, the SHC / HAC enzyme variant (also designated a Type 11 variant) has an amino acid sequence having at least 70.0% identity or similarity to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, and is identical to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364 , 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, with amino acid modifications at positions corresponding to positions W169 and G600M in SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, or 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, wherein the amino acid modifications are as follows: - W at position 169 is replaced by G, and - The G at position 600 is replaced by an M. In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0273] In embodiments, the SHC / HAC enzyme variant (also designated as a Type 12 variant) has an amino acid sequence having at least 70.0% identity to SEQ ID NO:1 and has at least one of the following mutations relative to SEQ ID NO:1: W169G, A306V, and G600M.

[0274] In embodiments, the SHC / HAC enzyme variant (also designated as a Type 13 variant) has an amino acid sequence having at least 70.0% identity to SEQ ID NO:4 and has at least the following mutation relative to SEQ ID NO:1: W169G.

[0275] In embodiments, the SHC / HAC enzyme variant (also designated as a Type 14 variant) has an amino acid sequence having at least 70.0% identity to SEQ ID NO:5 and has at least the following mutations relative to SEQ ID NO:1: W169G and G600M.

[0276] In embodiments, the SHC / HAC enzyme variant has an amino acid sequence having at least 70.0% identity to SEQ ID NO:350 and has at least the following mutations relative to SEQ ID NO:1: W169G and A306V.

[0277] In embodiments, the SHC / HAC enzyme variant (also designated as a Type 15 variant) has an amino acid sequence having at least 70.0% identity to SEQ ID NO:6 and has at least the following mutations relative to SEQ ID NO:1: W169G, A306V, and G600M.

[0278] In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0279] In embodiments, the SHC / HAC enzyme variant (also designated a Type 16 variant) has an amino acid sequence having at least 70.0% identity to SEQ ID NO:3, and has the following mutations relative to SEQ ID NO:1: M132R, A224V, I432T, and at least one of the following mutations relative to SEQ ID NO:3: W169G, A306V, and G600M.

[0280] In embodiments, the SHC / HAC enzyme variant (also designated as a Type 17 variant) has an amino acid sequence having at least 70.0% identity to SEQ ID NO: 10, and has the following mutations relative to SEQ ID NO: 1: M132R, A224V, I432T, and relative to SEQ ID NO: 3: W169G.

[0281] In embodiments, the SHC / HAC enzyme variant (also designated as a Type 18 variant) has an amino acid sequence having at least 70.0% identity to SEQ ID NO:11 and has the following mutations relative to SEQ ID NO:1: M132R, A224V, I432T, and relative to SEQ ID NO:3: W169G and G600M.

[0282] In embodiments, the SHC / HAC enzyme variant (also designated as a Type 32 variant) has an amino acid sequence having at least 70.0% identity to SEQ ID NO:385 and has the following mutations relative to SEQ ID NO:1: M132R, A224V, I432T, and relative to SEQ ID NO:3: W169G and A306V.

[0283] In embodiments, the SHC / HAC enzyme variant (also designated as the Type 19 variant) has an amino acid sequence having at least 70.0% identity to SEQ ID NO: 12 and has the following mutations relative to SEQ ID NO: 1: M132R, A224V, I432T, and relative to SEQ ID NO: 3: W169G, A306V, and G600M.

[0284] In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0285] In embodiments, the SHC / HAC enzyme variant (also designated a Type 20 variant) has an amino acid sequence having at least 70.0% identity to SEQ ID NO:2 and has the following mutations relative to SEQ ID NO:1: M132R, A224V, I432T, A557T, R613S, and at least one of the following mutations relative to SEQ ID NO:2: W169G, A306V, and G600M.

[0286] In embodiments, the SHC / HAC enzyme variant (also designated as a Type 21 variant) has an amino acid sequence having at least 70.0% identity to SEQ ID NO:7 and has the following mutations relative to SEQ ID NO:1: M132R, A224V, I432T, A557T, R613S, and relative to SEQ ID NO:2: W169G.

[0287] In embodiments, the SHC / HAC enzyme variant (also designated as a Type 22 variant) has an amino acid sequence having at least 70.0% identity to SEQ ID NO:8 and has the following mutations relative to SEQ ID NO:1: M132R, A224V, I432T, A557T, R613S, and relative to SEQ ID NO:2: W169G and G600M.

[0288] In embodiments, the SHC / HAC enzyme variant (also designated as a Type 33 variant) has an amino acid sequence having at least 70.0% identity to SEQ ID NO: 386 and has the following mutations relative to SEQ ID NO: 1: M132R, A224V, I432T, A557T, R613S, and relative to SEQ ID NO: 2: W169G and A306V.

[0289] In embodiments, the SHC / HAC enzyme variant (also designated as a Type 23 variant) has an amino acid sequence having at least 70.0% identity to SEQ ID NO:9 and has the following mutations relative to SEQ ID NO:1: M132R, A224V, I432T, A557T, R613S, and relative to SEQ ID NO:2: W169G, A306V, and G600M.

[0290] In this context, identity or similarity may be at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0291] In embodiments, the SHC / AHC enzyme variant (also designated a Type 34 variant) has an amino acid sequence having at least 30.0% identity or similarity to SEQ ID NOs: 1-12, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47-54, 55-296, 301-313, 315-353, 354-383, wherein W at or corresponding to position 169 of SEQ ID NO: 1 is replaced by G; wherein A at or corresponding to position 306 of SEQ ID NO: 1, 2, or 3 is replaced by V; and / or wherein G at or corresponding to position 600 of SEQ ID NO: 1, 2, or 3 is replaced by M.

[0292] In this context, identity or similarity is at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 1 It may be 4%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0293] In embodiments, the SHC / AHC enzyme variant (also designated as the Type 24 variant) has an amino acid sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 4, 5, 6, 7, 8, 9, 10, 11, 12, 304, 311, 312, 313, 305, 306, 302, 359, 357, 353, 355, 360, 358, 354, 356.

[0294] In embodiments, the SHC / AHC enzyme variant (also designated as a Type 25 variant) has an amino acid sequence comprising, consisting of, or consisting essentially of SEQ ID NOs: 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 350, 351, 352.

[0295] In embodiments, each of the SHC / HAC enzyme variants defined herein (Types 1-34 variants) may have the following additional amino acid modifications, wherein the (new) amino acid (X) at a position corresponding to position 168 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 may be S or a functional equivalent thereof. Functional equivalents of S are C, T, N, or Q. SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 at position 168 of SEQ ID NOs: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, or corresponding to position 168 of SEQ ID NOs: 1, 2, 3, 45, 46, 47, 48, 49, 50, 51, 52, 7, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97 , 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144 3, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188,189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 2 20, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251 A preferred new amino acid at a position in the amino acid sequence of a wild-type SHC / HAC enzyme as follows: 1, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 is S.

[0296] In a preferred embodiment, the SHC / HAC enzyme variant has an amino acid sequence having at least 30.0% identity or similarity to SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383; and - SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 at positions corresponding to position W169 of SEQ ID NOs: 1, 2, 3, or relative to W169 of SEQ ID NOs: 1, 2, 3, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 1 50, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 1 81, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212 2, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243 , 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,an amino acid modification at a position in the amino acid sequence of a wild-type SHC / HAC enzyme as follows: 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, wherein the amino acid modification is a W replaced by a G at position 169; and at position 168 with respect to SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383, at a position corresponding to S168 in SEQ ID NO: 1, 2, 3 or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 corresponding to S168 in SEQ ID NO: 1, 2, 3 , 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 9 4, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 1 58, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189 9, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220 , 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251,252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310.

[0297] In this context, identity or similarity is at least 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 1 It may be 4%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0298] In embodiments, any SHC / HAC enzyme variant is referred to as follows by reference to the wild-type Aac SHC / HAC having SEQ ID NO: 1, or any variant derived from SEQ ID NO: 1 and having SEQ ID NO: 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383: Conserved amino acids L22, Q26, G30, W32, A44, L48, Q72, G76, W78, Y95, L98, G102, A113, I117, G121, G122, F129, T130, L134, A135, G138, W142, P146, W169, A170, R171, F217, D222, R237, I261, P263, P281, S309, P310, W312, D313, T 314, A320, W339, Q344, G349, D350, W351, G361, G362, A364, F365, N369, Y372, P373, D374, D376, D377, W406, Q411, G415, A419, P433, D436, D442, P443, D447, V448, Q479, G483, W485, G487, R488, W489, G490, N492, Y495, It may contain at least one of G496, T497, L504, W522, Q527, G531, G532, W533, G534, E535, S539, Y540, G547, T552, T556, W558, A559, A565, L581, Q585, G589, W591, G600, F601, P602, F605, Y609, Y612, F616, P617, A620, and R623. The variants may also be any wild-type SHC / HAC enzyme as defined herein (e.g., SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 195, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150 , 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252 05, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259 , 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310), where the corresponding positions are identified corresponding to those identified above for SEQ ID NO: 1. The variant may be derived from any of the variant types previously defined herein.

[0299] Thus, provided herein are processes for producing (-)-ambrox by enzymatically converting EEH to (-)-ambrox. Also provided herein are processes for producing ambroxide by enzymatically converting E,E-bishomofarnesol to ambroxide. These processes may use any of the SHC / HAC enzyme variants described herein (particularly types 1-25).

[0300] "Percent (%) identity" with respect to a polypeptide sequence or nucleotide sequence is defined as the percentage of amino acids or nucleotides in a candidate sequence that are identical to those in a reference sequence, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, but does not consider any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in a variety of ways that are within the skill in the art, illustratively using publicly available computer software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The terms "polypeptide" and "protein" are used interchangeably herein and refer to any peptide chain of amino acids, regardless of length or post-translational modification.

[0301] Nucleotide and amino acid sequence similarity, i.e., percentage of sequence identity, can be determined through sequence alignment. Such alignments include, for example, https: / / www.ebi.ac.uk / Tools / msa / clustalo / The algorithm can be implemented with several algorithms known in the art, such as those available in the GAP program (a mathematical algorithm from the University of Iowa), or the mathematical algorithm of Myers and Miller (1989-Cabios 4:11-17), preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877), with hmmalign (HMMER package, http: / / hmmer.wustl.edu / ), or with the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80). The preferred parameters used are: https: / / www.ebi.ac.uk / Tools / msa / clustalo / These are the initial setting parameters as set above.

[0302] Percentage sequence identity may be calculated using, for example, BLAST, BLAT, or BlastZ (or BlastX). Similar algorithms are incorporated into the BLASTN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215, 403-410. BLAST polynucleotide searches are performed with the BLASTN program, score = 100, word length = 12, to obtain polynucleotide sequences homologous to those nucleic acids encoding related proteins. BLAST protein searches are performed with the BLASTP program, score = 50, word length = 3, to obtain amino acid sequences homologous to polypeptides.

[0303] To obtain gapped alignments for comparison purposes, Gapped BLAST is used as described in Altschul et al. (1997) Nucleic Acids Res. 25, 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1:154-162) or Markov Random Fields. When percentages of sequence identity are mentioned in this application, these percentages are calculated with respect to the full length of the longer sequence unless otherwise specifically indicated.

[0304] In a specific embodiment, the percent identity between two sequences is determined using CLUSTAL O (version 1.2.4).

[0305] Additional amino acid modifications may also be present in the SHC / AHC variants disclosed herein. In embodiments, SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, or ... 7, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 1 35, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254,Examples of additional amino acid modifications to 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 are set forth in SEQ ID NOs: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or ... 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 , 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 12 0, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151 , 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182,183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214 4, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 32 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 in the amino acid sequence of the wild-type SHC / HAC enzyme.

[0306] Type 26 variants: In embodiments, SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67 , 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 1 38, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 1 69, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 20 0, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,Examples of additional amino acid modifications to 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 are set forth in SEQ ID NOs: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, or ..., 2, or 3; 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 15, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146 6, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177 , 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208,209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 59, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 in the amino acid sequence of the wild-type SHC / HAC enzyme.

[0307] Type 27 variants: In embodiments, SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106 6, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137 , 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 2 00, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 1, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,Examples of additional amino acid modifications to 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 are set forth in SEQ ID NOs: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or ..., 2, or 3; 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 13, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144 4, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175 , 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 58, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 in the amino acid sequence of the wild-type SHC / HAC enzyme.

[0308] Type 28 variants: In embodiments, SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106 6, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137 , 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 2 00, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 1, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,Examples of additional amino acid modifications to 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 are set forth in SEQ ID NOs: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 361, 362, 363, 364, 36 , 379, 380, 381, 382, ​​or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 8 8, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122 2, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153 , 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184,185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 2 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310,

[0309] Type 29 variants: In embodiments, SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106 6, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137 , 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 2 00, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 1, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,Examples of additional amino acid modifications to 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 are at positions corresponding to positions 77, 92, 129, 579, 601, 605 of SEQ ID NOs: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, and / or 54, or correspond to 77, 92, 129, 579, 601, and 605 of SEQ ID NO: 1, 2, or 3, respectively, and SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100 , 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 63, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194 4, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225,226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267 , 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 in the amino acid sequence of the wild-type SHC / HAC enzyme.

[0310] at a position corresponding to position 81 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 1, 2 An amino acid modification (at a position in the amino acid sequence of a wild-type SHC / HAC enzyme as SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310, corresponding to 81 of SEQ ID NO: 1, or 3) may be, for example, Y81X. This refers to the substitution of amino acid Y at position 81 of SEQ ID NO: 1 with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 81.

[0311] at a position corresponding to position 81 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 39, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383) The new amino acid (X) (at a position in the amino acid sequence of the wild-type SHC / HAC enzyme as follows: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, or Phe. For example, an amino acid modification at a position corresponding to position 81 of SEQ ID NO: 1 can be a substitution of the amino acid of SEQ ID NO: 1 (i.e., Y) with a basic amino acid (i.e., His, Lys, or Arg). For example, at a position corresponding to position 81 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 39, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme) may substitute the amino acid of SEQ ID NO: 1 (i.e., Y) with histidine (i.e., the amino acid modification at the position corresponding to position 81 of SEQ ID NO: 1 is Y81H).

[0312] at a position corresponding to position 431 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 1, 2, or 3431 in the amino acid sequence of the wild-type SHC / HAC enzyme as SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310, may be, for example, H431X. This is SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 at position 431 (or SEQ ID NO: 1, 2, if " refers to the substitution of any different amino acid (X) for the amino acid H (at a position in the amino acid sequence of a wild-type SHC / HAC enzyme as SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310, corresponding to positions 431-4381 or 439-5401 in the wild-type SHC / HAC enzyme). As noted above, variant SHC / HAC enzymes may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 431.

[0313] at a position corresponding to position 431 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 39, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383); , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme) can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, at a position corresponding to position 431 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at a position corresponding to position 81 of SEQ ID NO: 1, 2, or 3, or at a position corresponding to position 82 of SEQ ID NO: 1, 2, or 3, , 15, 16, 17, 18, 19, or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme) may substitute a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, or Ile) for the amino acid of SEQ ID NO: 1 (i.e., Y).For example, at a position corresponding to position 431 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 39, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383). , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme) may substitute an amino acid (i.e., H) of SEQ ID NO: 1 with leucine (i.e., the amino acid modification at the position corresponding to position 431 of SEQ ID NO: 1 is H431L).

[0314] at a position corresponding to position 90 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 1, 2 , or 3, 90, at positions in the amino acid sequence of the wild-type SHC / HAC enzyme as SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310, which correspond to positions 55-296, 307-310, may be, for example, T90X. This includes a sequence similar to that of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 at position 90 (or SEQ ID NO: 1, 2, or refers to the substitution of any different amino acid (X) for the amino acid T (at a position in the amino acid sequence of a wild-type SHC / HAC enzyme as SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310, corresponding to 90 of 3). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 90.

[0315] at a position corresponding to position 90 of SEQ ID NOs: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383); The new amino acid (X) (at positions 0, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme) can be, for example, Met, Ala, Val, Leu, Ile, Cys, Ser, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, at a position corresponding to position 90 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13 corresponding to position 90 of SEQ ID NO: 1, 2, or 3). , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme) may substitute a hydrophobic amino acid (i.e., Met, Ala, Val, Leu, Ile) for the amino acid of SEQ ID NO: 1 (i.e., T).For example, at a position corresponding to position 90 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 39, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme) may substitute an amino acid of SEQ ID NO: 1 (i.e., T) with an alanine (i.e., an amino acid modification at a position corresponding to position 90 of SEQ ID NO: 1 is T90A).

[0316] at a position corresponding to position 172 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 1, 2, or 3172 (at positions in the amino acid sequence of the wild-type SHC / HAC enzyme as SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310, corresponding to positions 172-172) may be, for example, A172X. This includes a sequence similar to that of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 at position 172 (or SEQ ID NO: 1, 2, or refers to the substitution of any different amino acid (X) for the amino acid T (at a position in the amino acid sequence of a wild-type SHC / HAC enzyme as SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310, corresponding to 172 of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 172.

[0317] at a position corresponding to position 172 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or corresponding to position 172 of SEQ ID NO: 1, 2, or 3); The new amino acid (X) (at positions 9, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme) can be, for example, Met, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, at a position corresponding to position 172 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at a position corresponding to position 172 of SEQ ID NO: 13, , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme) may substitute an amino acid of SEQ ID NO: 1 (i.e., A) with a neutral hydrophilic amino acid (i.e., Cys, Ser, Thr, Asn, Gln).For example, at a position corresponding to position 172 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 39, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13, 14, 15, 16, 17, 18, 20, 21, 22, 23 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme) may substitute the amino acid of SEQ ID NO: 1 (i.e., A) with threonine (i.e., the amino acid modification at the position corresponding to position 172 of SEQ ID NO: 1 is A172T).

[0318] at a position corresponding to position 277 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 1, 2, The amino acid modification (at a position in the amino acid sequence of the wild-type SHC / HAC enzyme as SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310, corresponding to position 3277) may be, for example, M277X. This includes a sequence similar to that of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 at position 277 (or SEQ ID NO: 1, 2, or refers to the substitution of amino acid M (at a position in the amino acid sequence of a wild-type SHC / HAC enzyme as SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310, corresponding to 277 in SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 277.

[0319] at a position corresponding to position 277 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 39, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383; , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme) can be, for example, Ala, Val, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, at a position corresponding to position 277 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or at a position corresponding to position 277 of SEQ ID NO: 1, 2, or 3). The amino acid modification (at positions in the amino acid sequence of the wild-type SHC / HAC enzyme as SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) may substitute a basic amino acid (i.e., His, Lys, Arg) for the amino acid of SEQ ID NO: 1 (i.e., M).For example, at a position corresponding to position 277 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 39, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383). The amino acid modification (at positions 5, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme) may substitute an amino acid (i.e., M) of SEQ ID NO: 1 with a lysine (i.e., the amino acid modification at the position corresponding to position 277 of SEQ ID NO: 1 is M277K).

[0320] at a position corresponding to position 37 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 1, 2 , or 3 of 37) in the amino acid sequence of the wild-type SHC / HAC enzyme as SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310, may be, for example, L37X. This includes a sequence similar to that of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 at position 37 (or SEQ ID NO: 1, 2, or refers to the substitution of an amino acid L (at a position in the amino acid sequence of a wild-type SHC / HAC enzyme as SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310, corresponding to 37 of SHC / HAC enzyme) with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 37.

[0321] at a position corresponding to position 37 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 39, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383) , 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme) can be, for example, Met, Ala, Val, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. For example, at a position corresponding to position 37 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 39, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383) , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme) may substitute a neutral hydrophilic amino acid (i.e., Cys, Ser, Thr, Asn, or Gln) for the amino acid of SEQ ID NO: 1 (i.e., L).For example, at a position corresponding to position 37 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 39, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme) may substitute the amino acid of SEQ ID NO: 1 (i.e., L) with glutamine (i.e., the amino acid modification at the position corresponding to position 37 of SEQ ID NO: 1 is L37Q).

[0322] at a position corresponding to position 174 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 1, 2, or 3, 174, at positions in the amino acid sequence of the wild-type SHC / HAC enzyme as SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310, corresponding to positions 174, ... This includes a sequence similar to that of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 at position 174 (or SEQ ID NO: 1, 2, or refers to the substitution of amino acid V (at a position in the amino acid sequence of a wild-type SHC / HAC enzyme as SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310, corresponding to 277 of SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) with any different amino acid (X). As noted above, SHC / HAC enzyme variants may additionally contain insertions and / or deletions, and the numbering of the new amino acid (X) in the new SHC / HAC enzyme variant may not be 174.

[0323] at a position corresponding to position 174 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or corresponding to position 174 of SEQ ID NO: 1, 2, or 3); The new amino acid (X) (at positions 9, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme) can be, for example, Met, Ala, Leu, Ile, Cys, Ser, Thr, Asn, Gln, Asp, Glu, His, Lys, Arg, Gly, Pro, Trp, Tyr, or Phe. at a position corresponding to position 174 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or corresponding to 174 of SEQ ID NO: 1, 2, or 3 The new amino acid (X) (at a position in the amino acid sequence of the wild-type SHC / HAC enzyme as SEQ ID NOs: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310) may be, for example, a hydrophobic amino acid (i.e., Met, Ala, Leu, or Ile).For example, at a position corresponding to position 174 of SEQ ID NO: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 (or SEQ ID NO: 13, 14, 15, 16, 17, 18, 29, 29, 30, 31, 32 The amino acid modification (at positions 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55-296, 307-310 in the amino acid sequence of the wild-type SHC / HAC enzyme) ma...

Claims

1. 1. A process for preparing (-)-ambrox or a mixture containing (-)-ambrox, the process comprising enzymatically converting (3E,7E)-homofarnesol (EEH) or a mixture of homofarnesol isomers containing EEH to (-)-ambrox or a mixture containing (-)-ambrox using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 0, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 , or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 ,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 1 26, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 7, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188 , 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 2 51, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282 2, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, and wherein the amino acid sequence of the SHC / HAC enzyme variant is selected from the group consisting of: 1, 2, 3, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310. 9, 380, 381, 382, ​​or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89 9, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 1 23, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154 4, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185,186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 1, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 at a position corresponding to position W169G of SEQ ID NO: 1, 2, or 3, or at a position in the amino acid sequence of a wild-type SHC / HAC enzyme corresponding to W169G of SEQ ID NO: 1, 2, or 3.

2. 1. A process for preparing (-)-ambrox or a mixture containing (-)-ambrox, the process comprising enzymatically converting (3E,7E)-homofarnesol (EEH) or a mixture of homofarnesol isomers containing EEH to (-)-ambrox or a mixture containing (-)-ambrox using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant has an amino acid sequence having at least 30.0% identity or similarity to SEQ ID NO: 1, 13, 15, 23, 32, and wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid modification relative to SEQ ID NO: 1 at a position corresponding to position W169G of SEQ ID NO: 1 or at a position in the amino acid sequence of a wild-type SHC / HAC enzyme corresponding to W169G of SEQ ID NO:

1.

3. 1. A process for preparing an ambroxide or a mixture containing an ambroxide, the process comprising enzymatically converting (2,E)-bishomofarnesol (bisEEH) or a mixture of bishomofarnesol isomers containing bisEEH to an ambroxide or a mixture containing an ambroxide using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant is selected from the group consisting of SEQ ID NOs: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 106, 107, 108, 109, 110, 111, 112, , 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 38 3, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 1 28, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 9, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190 , 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221,222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 2 53, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310, and wherein the amino acid sequence of the SHC / HAC enzyme variant is 1, 382, ​​or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 14 25, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156 6, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187,188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 at a position corresponding to position W169G of SEQ ID NO: 1, 2, or 3, or at a position in the amino acid sequence of a wild-type SHC / HAC enzyme corresponding to W169G of SEQ ID NO: 1, 2, or 3.

4. A process for preparing amblyoxide or a mixture containing amblyoxide, the process comprising enzymatically converting (2,E)-bishomofarnesol (bisEEH) or a mixture of bishomofarnesol isomers containing bisEEH into amblyoxide or a mixture containing amblyoxide using an SHC / HAC enzyme variant, wherein the SHC / HAC enzyme variant has an amino acid sequence having at least 30.0% identity or similarity to SEQ ID NO: 1, 13, 15, 23, 32, and wherein the amino acid sequence of the SHC / HAC enzyme variant has an amino acid modification relative to SEQ ID NO: 1 at a position corresponding to position W169G of SEQ ID NO: 1 or at a position in the amino acid sequence of a wild-type SHC / HAC enzyme corresponding to W169G of SEQ ID NO:

1.

5. - W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is replaced by G; - an A at position 306 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is replaced by a V or a functional equivalent thereof; and / or 2. The process of claim 1, wherein the G at position 600 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by A, V, L, I, or M, or a functional equivalent thereof.

6. 2. The process of claim 1, wherein W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by G.

7. - W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by G; and 2. The process of claim 1, wherein an A at position 306 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by a V.

8. - W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by G; and 2. The process of claim 1, wherein G at position 600 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by M.

9. - W at position 169 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382 or 383 is replaced by G; - A at position 306 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by V; and 2. The process of claim 1, wherein G at position 600 of SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383 is replaced by M.

10. SEQ ID NO: 1, 2, 3, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 47, 48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72 , 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110 , 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 1 73, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204 4, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235 , 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266,267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 is S, or 1, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 1 33, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164 4, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195 , 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226,227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270 2. The process of claim 1, wherein the amino acid at position 168 of the amino acid sequence of wild-type SHC corresponding to positions 0, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, or 310 is S.

11. - the SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 1, 4, 5, 6, or 350, and has the following mutations: W169G in SEQ ID NO: 4, W169G and G600M in SEQ ID NO: 5, W169G, G600M and A306V in SEQ ID NO: 6, and W169G and A306V in SEQ ID NO: 350; or - the SHC / HAC enzyme variant has an amino acid sequence with at least 70.0% identity to SEQ ID NO: 7, 8, 9, or 386 and has the following mutations: M132R, A224V, I432T, A557T, R613S, and, in addition, has the following mutations: W169G in SEQ ID NO: 7, W169G and G600M in SEQ ID NO: 8, W169G, G600M and A306V in SEQ ID NO: 9, and W169G and A306V in SEQ ID NO: 386; or 2. The process of claim 1, wherein the SHC / HAC enzyme variant has an amino acid sequence having at least 70.0% identity to SEQ ID NO: 10, 11, or 12 and has the following mutations: M132R, A224V, I432T, and additionally has the following mutations: W169G in SEQ ID NO: 10, W169G and G600M in SEQ ID NO: 11, and W169G, G600M and A306V in SEQ ID NO:

12.

12. 12. The process according to any one of claims 1 to 11, wherein W at position 169 is replaced by G and A at position 306 is replaced by V.

13. 12. The process of any one of claims 1 to 11, wherein W at position 169 is replaced by G and G at position 600 is replaced by M.

14. 12. The process of any one of claims 1 to 11, wherein W at position 169 is replaced by G, A at position 306 is replaced by V, and G at position 600 is replaced by M.

15. The SHC / HAC enzyme variants disclosed herein exhibit: Reference SHC enzymes (e.g., wild-type SHCs, e.g., SEQ ID NOs: 1, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, , 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 200 7, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 25 15, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 307, 308, 309, 310, or those represented by 215G2 compared to the parent SHC enzyme from which AacSHC, or SHC#65, or variants thereof, are derived, e.g., those represented by SEQ ID NOs: 2, 3, 47-48, 49, 50, 51, 52, 53, 54, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​or 383), - increased substrate specificity for EEH (or for bisEEH) when a homofarnesol or bis-homofarnesol substrate is used; - increased product selectivity when homofarnesol or bis-homofarnesol substrates are used relative to (-)-ambrox (or ambroxide); - specificity for specific isomers of the substrate, which is increased when substrates other than homofarnesol or bis-homofarnesol are used (e.g., ethyl-homofarnesol, hydroxyfarnesylacetone); - increased productivity, and / or - an increased degree of conversion of EEH (or BisEEH) over the first 3 to 6 hours (or over the first 4, 5, or 6 hours) of the reaction, as well as an increased conversion rate of EEH (or BisEEH), according to the process of claims 1 to 11.

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