Pentose fermentation by a recombinant microorganism

Abstract

The present invention provides methods and compositions suitable for use in the conversion of xylose to xylitol and xylulose, including nucleic acid constructs, recombinant fungal host cells, and related materials.

We claim: 
     
         1 . A recombinant fungal host cell comprising at least one polynucleotide sequence encoding at least one xylose reductase and/or xylitol dehydrogenase variant, wherein said variant is has xylose reductase activity and comprises a substitution at one or more positions selected from 2, 3, 7, 11, 14, 17, 19, 20, 23, 24, 33, 36, 46, 47, 49, 56, 62, 63, 68, 86, 89, 97, 102, 107, 108, 114, 116, 123, 132, 134, 143, 152, 155, 157, 162, 168, 184, 206, 219, 224, 225, 226, 227, 228, 246, 231, 232, 233, 236, 240, 242, 245, 246, 249, 252, 255, 261, 264, 266, 267, 270, 271, 272, 275, 276, 279, 281, 282, 283, 285, 297, 299, 301, 302, 303, and/or 318, wherein the positions are numbered by correspondence with the amino acid sequence set forth in SEQ ID NO:2. 
     
     
         2 . The recombinant fungal host cell of  claim 1 , wherein said variant has xylose reductase activity and further comprises a substitution at one or more positions selected from P2T, S3H, S3R, S3W, S3X, N7L, D11K, A14V, F17W, C19L, W20X, D23E, V24G, R33L, R33V, K36Q, E46C, E46K, D47G, D47N, A49G, K52R, A56E, A56Y, I62V, D63X, K68G, K68M, K68R, D86N, E89N, E89V, S97R, S97T, D102T, F107Y, L108Y, T114S, K116Q, K123C, K132A, K132N, D134E, D134H, D134V, I143L, K152A, K152E, K152H, K152Q, K155A, K155D, K155I, K155R, K155Y, G157R, I162L, P168S, S184A, R206S, R206V, Q219H, Q219L, Q219T, L224A, L224S, L224V, N225D, N225E, N225K, N225S, N225Y, Q226D, Q226E, Q226S, Q226V, G227D, R228T, N231G, N231H, N231L, N231S, T232A, T232C, T232S, T232V, S233C, S233F, S233G, S233I, S233K, S233V, S233X, F236L, T240V, K242L, A245S, A246L, A246S, G249D, P252C, V255I, S261A, S261C, S261N, S261T, G264D; A266V, A266C, I267V, I267X, K270R, S271G, N272P, P275A, R276M, R276F, R276W, W276M, E279Q, K281L, K281V, D282C, D282G, D282R, V283H, S285E, S285T, A297H, A297S, L299X, I301C, I301Y, N302D, N302G, N302S, L303I, L303V, and V318C, wherein the positions are numbered by correspondence with the amino acid sequence set forth in SEQ ID NO:2. 
     
     
         3 . The recombinant fungal host cell of  claim 1 , wherein said variant has xylose reductase activity and comprises at least one of the following substitutions and/or substitution sets P2T/S3H/A49G/P168S/S233K/R276W; P2T/S3X/A49G/S233X/1267X/R276W/L299X; P2T/C19L/E46C/A49G/F107Y/K132N/S233K/1267V/R276W; P2T/C19L/E46C/A49G/K132N/S233K/1267V/R276W; P2T/W20X/A49G/D63X/R276W; P2T/V24G/A49G; P2T/V24G/A49G/R276W; P2T/K36Q/A49G/G227D/R276W; P2T/E46C/A49G/F107Y/K132N/S233K/I267V/R276W; P2T/E46C/A49G/K132N/V164I/S233K/I267V/R276W; P2T/A49G; P2T/A49G/K52R/K152E/S233K/I267V/R276W; P2T/A49G/K52R/R206S/I267V/R276W; P2T/A49G/K52R/R276W; P2T/A49G/K68G/S261N/R276W/V318C; P2T/A49G/K68G/S261N/V318C; P2T/A49G/D86N/K132N/R276W; P2T/A49G/K132N; P2T/A49G/K132N/P168S/S233K/I267V/R276W; P2T/A49G/K132N/G227D/P252C/R276W; P2T/A49G/K132N/G227D/R276W; P2T/A49G/K132N/S233K/I267V/R276W; P2T/A49G/K132N/S233K/R276W; P2T/A49G/K132N/I267V/R276W; P2T/A49G/K132N/R276W; PT2/A49G/K152E; P2T/A49G/K152E/P168S/S233V/I267V/R276W; P2T/A49G/K152E/I267V/R276W; P2T/A49G/K152E/R276W; P2T/A49G/P168S/R206S/I267V/R276W; P2T/A49G/P168S/S233K/R276W; P2T/A49G/P168S/R276W; P2T/A49G/R206S/S233K/I267V/R276W; P2T/A49G/R206S; P2T/A49G/R206S/R276W; P2T/A49G/G227D/P252C/G264D/R276W; P2T/A49G/G227D/P252C/R276W; P2T/A49G/G227D/I267V/R276W; P2T/A49G/G227D/R276W; P2T/A49G/S233K/R276W; P2T/A49G/S233K/I267V/R276W; P2T/A49G/S233V/A246L/R276W/N302S; P2T/A49G/S233V/A246L/N302S; P2T/A49G/S233V/I267V/R276W; P2T/A49G/S233V/R276W; P2T/A49G/P252C/R276W; P2T/A49G/S261N; P2T/A49G/S261N/R276W; P2T/A49G/I267V/R276W; P2T/A49G/R276W; P2T/A46G/R276W/N302S; P2T/A49G/R276W/V318C; P2T/A49G/N302S; P2T/A49G/V319C; E46K/D47N; A49G/K132A; A49G/K132A/K152E; A49G/K132A/K152E/R276W; A49G/K132A/R206S; A49G/K132A/R276W; A49G/K132A/R206S/R276W; A49G/K132N/K152E/R276W; A49G/K132N/K152E/R276W/N302S; A49G/K132N/K152E/N302S; A49G/K132N/R206S/S233V/W276M/N302S; A49G/K132N/R206S/S233V/R276M/N302S; A49G/K152E; A49G/K152E/R276W; A49G/K152E; and A49G/R276W, wherein the positions are numbered by correspondence with the amino acid sequence set forth in SEQ ID NO:2. 
     
     
         4 . The recombinant fungal host cell of  claim 1 , wherein, said variant comprises the polypeptide sequence set forth in SEQ ID NOS:55, 57, or 59. 
     
     
         5 . A recombinant fungal host cell comprising at least one polynucleotide sequence encoding at least one xylose reductase, wherein said polynucleotide sequence comprises SEQ ID NOS:1, 3, 4, 40, 42, 44, and/or 46; and at least one polynucleotide encoding at least one xylitol dehydrogenase, wherein said polynucleotide sequence comprises SEQ ID NOS:5, 7, 8, and/or 48. 
     
     
         6 . The recombinant fungal host cell of  claim 5 , further comprising at least one polynucleotide encoding at least one xylulokinase, wherein said polynucleotide comprises SEQ ID NO: 11 and/or 12.