Pentose fermentation by a recombinant microorganism

Abstract

The present invention provides recombinant nucleic acid constructs comprising a xylose isomerase polynucleotide, a recombinant fungal host cell comprising a recombinant xylose isomerase polynucleotide, and related methods.

We claim: 
     
         1 . An isolated xylose isomerase variant, wherein said variant is a mature form having xylose isomerase activity and comprising a substitution at one or more positions selected from 2, 6, 13, 16, 18, 29, 62, 64, 67, 70, 71, 74, 75, 78, 81, 91, 106, 111, 116, 127, 128, 139, 156, 164, 182, 199, 201, 206, 211, 223, 237, 233, 236, 244, 248, 250, 274, 277, 281, 284, 325, 328, 329, 330, 339, 342, 356, 360, 371, 372, 373, 375, 378, 380, 382, 386, 389, 390, 391, 393, 397, 398, 399, 400, 404, 407, 414, 423, 424, 426, 427, 431, 433, 434, 435, and/or 436, wherein the positions are numbered by correspondence with the amino acid sequence of SEQ ID NO:2. 
     
     
         2 . The isolated xylose isomerase variant of  claim 1 , wherein said variant is a mature form having xylose isomerase activity and comprising a substitution at one or more positions selected from E2, N6, Q13, K16, T18, E29, G62, T64, T67, Q70, S71, A74, A75, K78, V81, L91, S106, K111, Q116, K127, Q128, A139, S156, A164, Y182, M199, K201, M206, K211, K223, K233, T236, K237, T244, V247, L248, F250, H274, Q277, 8281, 8284, A325, F328, T329, N330, A339, G342, G356, F360, I371, E372, D373, R375, K378, V380, D382, S386, T389, G390, I391, A393, A397, G398, K399, A400, S404, K407, E414, R423, Q424, M426, V431, N433, V434, L435, and/or F436, wherein the positions are numbered by correspondence with the amino acid sequence set forth in SEQ ID NO:2. 
     
     
         3 . An isolated xylose isomerase variant of  claim 2 , wherein said variant is a mature form having xylose isomerase activity and comprising a substitution at one or more positions selected from E2S, N6G, N6H, Q13K, K16E, T18C, T18K, T18L, T18M, E29N, G62F, T64Q, T67S, Q70E, S71L, A74G, A75T, K78R, V81I, L91M, S109D, K111A, K111L, Q116C, K127I, K127R, Q128A, A139G, S156T, A164V, Y182C, M199A, M199V, L201H, M206T, K211H, K223T, K233C, T236A, T236L, K237A, T244S, V247A, L248S, F250C, F250V, H274R, Q277R, R281L, R284H, A325R, A325S, F328H, T329S, N330G, N330H, N330L, N330W, N330Y, A339R, G342P, G342V, G356A, F360M, I371G, I371L, I371Q, I371R, I371T, E372G, E372T, D373G, R375Q, R375T, R375V, K378A, K378D, V380W, D382G, D382N, S386K, T389H, G390M, I391A, I391L, A393T, A397L, A397S, G398E, K399E, K399T, K399V, A400G, S404Y, K407E, K407L, K407R, E414A, R423G, Q424H, M426R, V431E, N433A, N433H, N433R, V434Q, V434S, L435S, and/or F436G, wherein the positions are numbered by correspondence with the amino acid sequence set forth in SEQ ID NO:2. 
     
     
         4 . An isolated xylose isomerase variant of  claim 1 , wherein said variant is a mature form having xylose isomerase activity and comprising at least one substitution set selected from N6G/E372G/F436G; K16E/K111A/E372G; K16E/K111A/E372G/K399T; E29N/E372G; T64Q/S71L/Q116C/M199A/F360M/E372G/K407R; T64Q/S71L/Q116C/K233C/F360M/E372G/K407L/Q424H; T64Q/S71L/M199A/K233C/E372G/I391L; T64Q/S71L/K233C/F360M/E372G; T64Q/L91M/A139G/A164V/K233C/E372G; T64Q/Q116C/M199A/F360M/E372G/K407L; T64Q/Q116C/K233C/E372G; T64Q/M199A/K233C/E372G; T64Q/M199A/K233C/E372G/K407L/Q424H; T64Q/K233C/F250C/E372G; T64Q/K233C/F360M/E372G/K407L/Q424H; T64Q/F360M/E372G; T67S/Q70E/A75T/E372G; T67S/Q70E/S 109D/T236A/E372G/S386K; T67S/Q70E/S109D/T236L/E372G/I391L/G398E/V434S; T67S/Q70E/S109D/R281L/E372G; T67S/Q70E/S109D/R281L/E372G/S404Y; T67S/Q70E/S109D/E372G/S386K; T67S/Q70E/S109D/E372G/I391L/S404Y; T67S/Q70E/S109D/E372G/V431E; T67S/Q70E/S109D/E372G/V434S; T67S/Q70E/T236A/E372G; T67S/Q70E/T236L/E372G/S386K; T67S/Q70E/T236L/E372G/V431E; T67S/Q70E/R281L/E372G; T67S/Q70E/R281L/E372G/S404Y; T67S/Q70E/A325 S/E372G; T67S/Q70E/E372G/S386K; T67S/Q70E/E372G/G390M; T67S/S109D/R281L/E372G; T67S/S109D/E372G/G398E/V434S; T67S/R281L/A325R/E372G; Q70E/S109D/T236A/E372G/I391L; Q70E/S109D/T236A/E372G/V434S; Q70E/S109D/T236L/E372G/S386K/S404Y; Q70E/S109D/E372G; Q70E/S109D/E372G/G398E; Q70E/S109D/E372G/V431E; Q70E/T236A/E372G; Q70E/T236A/E372G/G398E; Q70E/T236A/R281L/A325S/E372G; Q70E/T236L/E372G/G398E; Q70E/E372G/V434S; Q70E/E372G/G398E/V434S; S71L/M199A/K233C/E372G/K407L; S71L/E372G; K78R/Y182C/G356A/E372G; K78R/V247A/L248S/G356A/E372G; K78R/V247A/E372G; K78R/G356A/E372G; K78R/E372G/K399E/R423G; K78R/D373G; S109D/T236A/R281L/E372G; S109D/T236L/R281L/A325R/E372G; S109D/R281L/E372G; Q116C/M199A/K233C/E372G/K407L; Q116C/M199A/F360M/E372G; K127R/G356A/E372G; K127R/E372G/D373G; Y182C/V247A/G356A; L201H/E372G; M206T/L248S/H274R/K399E; M206T/L248S/E372G; K211H/E372G/K407E; K233C/F360M/E372G/V380W/Q424H; K233C/E372G/V380W; K233C/E372G/K407L; K223T/K237A/E372G/K399T/K407E; V247A/L248S/G356A/E372G; R281L/A325S/E372G/A397S; R284H/E372G; T329S/N330H/E372G/R375V; N330Y/E372G/F436G; G356A/E372G; G356A/E372G/K399E/R423G; G356A/D373G; F360M/E372G/Q424H; I371G/E372G/N433A; E372G/K378D; E372G/K378D/K399T/K407E; E372G/I391L/S404Y/V434S; E372G/K399T; E372G/K399T/K407E; E372G/K407E; E372G/K407R; and/or E372G/L435S, wherein the positions are numbered by correspondence with the amino acid sequence set forth in SEQ ID NO:2. 
     
     
         5 . A recombinant fungal host cell comprising a polynucleotide sequence that encodes a polypeptide that is capable of catalyzing the isomerization of D-xylose directly to D-xylulose, wherein the polynucleotide is a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 70% identity to SEQ ID NO:2, or a polynucleotide that hybridizes under stringent hybridization conditions to the complement of a polynucleotide that encodes a polypeptide having the amino acid sequence of SEQ ID NO:2. 
     
     
         6 . The host cell of  claim 5 , wherein the polynucleotide is integrated into the host cell genome. 
     
     
         7 . The host cell of  claim 5 , wherein the host cell is a yeast cell. 
     
     
         8 . The host cell of  claim 5 , wherein the host cell has had one or more native genes deleted from its genome. 
     
     
         9 . The host cell of  claim 8 , wherein the deletion results in one or more phenotypes including increased transport of xylose into the host cell, increased xylulose kinase activity, increased flux through the pentose phosphate pathway, decreased sensitivity to catabolite repression, increased tolerance to ethanol, increased tolerance to acetate, increased tolerance to increased osmolarity, increased tolerance to low pH, and/or reduced production of by products, wherein comparison is made with respect to the corresponding host cell without the deletion(s). 
     
     
         10 . The host cell of  claim 5 , wherein the host cell is altered to overexpress one or more polynucleotides. 
     
     
         11 . The host cell of  claim 10 , wherein overexpression results in one or more phenotypes, including increased transport of xylose into the host cell, increased xylulose kinase activity, increased flux through the pentose phosphate pathway, decreased sensitivity to catabolite repression, increased tolerance to ethanol, increased tolerance to acetate, increased tolerance to increased osmolarity, increased tolerance to low pH, and/or reduced product of by products, wherein comparison is made to the corresponding unaltered host cell. 
     
     
         12 . The host cell of  claim 5 , wherein the host cell is capable of growth in a xylose-based culture medium. 
     
     
         13 . The host cell of  claim 5 , wherein the host cell is capable of fermentation in a xylose-based culture medium. 
     
     
         14 . The host cell of  claim 5 , wherein the host cell is capable of faster growth in a xylose-based culture medium as compared to wild-type  Saccharomyces cerevisiae.    
     
     
         15 . The host cell of  claim 5 , wherein the xylose-based culture medium is a product from a cellulosic saccharification process and/or a hemicellulosic feedstock.