Engineered beta-glucosidases and glucosylation methods

Abstract

The present invention provides engineered β-glucosidase (BGL) enzymes, polypeptides having BGL activity, and polynucleotides encoding these enzymes, as well as vectors and host cells comprising these polynucleotides and polypeptides. The present invention also provides compositions comprising the enzymes, and methods of using the engineered BGL enzymes to make products with β-glucose linkages.

1 . A non-naturally occurring beta-glucosidase variant having a polypeptide sequence comprising at least 80% sequence identity to SEQ ID NO:12, wherein the polypeptide sequence comprises one or more substitutions in its polypeptide sequence. 
     
     
         2 . The non-naturally occurring beta-glucosidase variant of  claim 1 , wherein said variant comprises at least one substitution or substitution set at positions 31, 32, 34, 36, 37, 38, 57/91, 58, 59, 60, 61, 62, 62/403, 66, 70, 89, 89/187, 91/595, 124, 124/297, 125, 126/188, 133, 138/296, 147, 150, 184, 186, 187, 230, 231, 233, 266, 296, 297, 403, 405, 449, 450, 492, 590, or 601, wherein said positions are numbered with reference to SEQ ID NO:12. 
     
     
         3 . The non-naturally occurring beta-glucosidase variant of  claim 1 , wherein said variant comprises at least one substitution or substitution set 31C/G, V32G/P/S, 34I/P, 36P, 37K/W, 38E, 57R/91P, 58P, 59R, 60C/R/V, 61R, 62N, 62P/403R, 66G/N, 70W, 89L, 89S/187R, 91G/595V, 124D/297P, 125G, 126V/188R, 133G/R, 138G/296S, 147L, 150H/L/M/P/T, 184A/R, 186E, 187G/N/Y, 230F/H/I, 231G/R, 233L/P/Q, 266F/G, 296R/T, 297R, 403E/P, 405R, 449G/P, 450G, 492P, 590W, 601A/E/L, 601E, or 601L, wherein said positions are numbered with reference to SEQ ID NO:12. 
     
     
         4 . The non-naturally occurring beta-glucosidase variant of  claim 1 , wherein said variant comprises at least one substitution or substitution set P31C/G, V32G/P/S, G34I/P, 136P, T37K/W, T38E, G57R/191P, L58P, F59R, N60C/R/V, L61R, K62N, K62P/W403R, R66G/N, V70W, D89L, D89S/H187R, 191G/G595V, A124D/V297P, A124D, S125G, A126V/F188R, F133G/R, D138G/M296S, R147L, E150H/L/M/P/T, C184A/R, K186E, H187G/N/Y, M230F/H/I, A231G/R, F233L/P/Q, Y266F/G, M296R/T, V297R, W403E/P, V405R, M449G/P, F450G, S492P, R590W, or F601A/E/L, wherein said positions are numbered with reference to SEQ ID NO:12. 
     
     
         5 . The non-naturally occurring beta-glucosidase variant of  claim 1 , wherein said variant comprises at least one substitution at or substitution set at positions 32, 34, 36, 57/91, 58, 59, 60, 62, 62/403, 66, 89, 89/187, 91/595, 124/297, 126/188, 133, 138/296, 147, 150, 184, 186, 187, 230, 231, 233, 266, 296, 297, 403, 405, 449, 492, 590, and 601, wherein said positions are numbered with reference to SEQ ID NO:12. 
     
     
         6 . The non-naturally occurring beta-glucosidase variant of  claim 1 , wherein said variant comprises at least one substitution or substitution set 32G/P/S, 34I/P, 36P, 57R/91P, 58P, 59R, 60C/R/V, 62N, 62P/403R, 66N, 89L, 89S/187R, 91G/595V, 124D/297P, 126V/188R, 133G/R, 138G/296S, 147L, 150H/L/M/P/T, 184R, 186E, 187G, 187N/Y, 230F/H/I, 231G/R, 233P/Q, 266F/G, 296R/T, 297R, 403E/P, 405R, M449G/P, 492P, 590W, or 601A/E/L, wherein said positions are numbered with reference to SEQ ID NO:12. 
     
     
         7 . The non-naturally occurring beta-glucosidase variant of  claim 1 , wherein said variant comprises at least one substitution or substitution set V32G/P/S, G34I/P, I36P, G57R/I91P, L58P, F59R, N60C/R/V, K62N, K62P/W403R, R66N, D89L, D89S/H187R, 191G/G595V, A124D/V297P, A126V/F188R, F133G/R, D138G/M296S, R147L, E150H/L/M/P/T, C184R, K186E, H187G/N/Y, M230F/H/I, A231G/R, F233P/Q, Y266F/G, M296R/Y, V297R, W403E/P, V405R, M449G/P, S492P, R590W, or F601A/E/L, wherein said positions are numbered with reference to SEQ ID NO:12. 
     
     
         8 . (canceled) 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . The non-naturally occurring beta-glucosidase variant of  claim 1 , wherein said variant comprises the polypeptide sequence of SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, or 172. 
     
     
         12 . A recombinant polynucleotide encoding at least one non-naturally occurring beta-glucosidase variant provided in  claim 1 . 
     
     
         13 . The recombinant polynucleotide of  claim 12 , wherein said polynucleotide comprises the sequence of SEQ ID NO: 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, or 171. 
     
     
         14 . The recombinant polynucleotide of  claim 12 , wherein said polynucleotide encodes a polypeptide comprising the polypeptide sequence of SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, or 172. 
     
     
         15 . A vector comprising at least one recombinant polynucleotide as set forth in  claim 12 . 
     
     
         16 . The vector of  claim 15 , wherein said vector further comprises at least one control sequence. 
     
     
         17 . A host cell comprising the vector provided in  claim 15 . 
     
     
         18 . The host cell of  claim 17 , wherein said host cell is selected from eukaryotic and prokaryotic organisms. 
     
     
         19 . The host cell of  claim 17 , wherein said host cell is  E. coli.    
     
     
         20 . A method for producing at least one non-naturally occurring beta-glucosidase variant, comprising culturing the host cell of  claim 17  under conditions such that said non-naturally occurring beta-glucosidase variant is produced by said host cell. 
     
     
         21 . The method of  claim 20 , further comprising the step of recovering said non-naturally occurring beta-glucosidase variant. 
     
     
         22 . A composition comprising at least one non-naturally occurring beta-glucosidase variant of  claim 1 . 
     
     
         23 . A method for converting a glycosyl group acceptor substrate to a beta-glucosylated product, comprising: providing at least one glycosyl group acceptor substrate, at least one glycosyl group donor co-substrate, and at least one beta-glucosidase of  claim 1 ; contacting said glycosyl group acceptor substrate, glycosyl group donor co-substrate, and beta-glucosidase under conditions such that said substrate is glucosylated by said beta-glucosidase to provide a beta-glucosylated product. 
     
     
         24 . The method of  claim 23 , wherein said glycosyl group donor co-substrate is selected from disaccharides, trisaccharides, oligosaccharides, cellobiose, gentiobiose, laminaribiose, and cellulose. 
     
     
         25 . The method of  claim 23 , wherein said glycosyl group acceptor substrate is glycosylated. 
     
     
         26 . The method of  claim 25 , wherein said glycosyl group acceptor substrate is a naturally-occurring glycosylated substrate. 
     
     
         27 . The method of  claim 26 , wherein said naturally-occurring glycosylated glycosyl group acceptor substrate is selected from stevioside, rebaudioside A, or rebaudioside D. 
     
     
         28 . The method of  claim 25 , wherein said glycosyl group acceptor substrate is a non-naturally occurring glycosylated substrate. 
     
     
         29 . The method of  claim 23 , wherein said glycosyl group acceptor substrate is an aglycosylated natural substance. 
     
     
         30 . The method of  claim 23 , wherein said glycosyl group acceptor substrate is a non-naturally occurring aglycosylated substance. 
     
     
         31 . (canceled) 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . The non-naturally occurring beta-glucosidase variant of  claim 1 , wherein the beta-glucosidase variant has at least 90% sequence identity to SEQ ID NO: 14. 
     
     
         36 . The non-naturally occurring beta-glucosidase variant of  claim 1 , wherein the beta-glucosidase variant has at least 95% sequence identity to SEQ ID NO: 14. 
     
     
         37 . The non-naturally occurring beta-glucosidase variant of  claim 1 , wherein the beta-glucosidase variant has at least 98% sequence identity to SEQ ID NO: 14. 
     
     
         38 . The non-naturally occurring beta-glucosidase variant of  claim 1 , wherein the beta-glucosidase variant has at least 99% sequence identity to SEQ ID NO: 14. 
     
     
         39 . The method of  claim 23 , wherein the beta-glucosidase comprises the polypeptide sequence of SEQ ID NO: 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, or 172.