Heterologous Expression of Termite Cellulases in Yeast

Abstract

The present invention provides for heterologous expression of termite and termite-associated symbiont cellulases. The cellulases can, for example, be codon-optimized and expressed in yeast host cells, such as the yeast Saccharomyces cerevisiae . The cellulases can also be co-expressed in host cells with other cellulases. The expression in such host cells of the termite and termite-associated symbiont cellulases, and variants and combinations thereof, result in yeast with improved cellulosic activity. Thus, such genes and expression systems are useful for efficient and cost-effective consolidated bioprocessing systems.

1 . An isolated polynucleotide comprising a nucleic acid fragment which encodes at least 50 contiguous amino acids of a mature cellulase, wherein said nucleic acid fragment is codon-optimized for expression in a yeast strain and wherein the cellulase is a termite cellulase or a termite-associated symbiont cellulase. 
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . The polynucleotide of  claim 1 , wherein the yeast is selected from the group consisting of  Saccharomyces cerevisiae, Kluveromyces lactus, Kluyveromyces marxianus, Schizzosaccharomyces pombe, Candida albicans, Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorphs, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe  and  Schwanniomyces occidentalis.   
     
     
         5 . (canceled) 
     
     
         6 . The polynucleotide of  claim 1 , wherein the cellulase has exogluconase activity or endogluconase activity. 
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . The polynucleotide of  claim 1 , wherein the cellulase has β-glucosidase activity. 
     
     
         10 . The polynucleotide of  claim 1 , wherein the cellulase is a protozoan cellulase or a metazoan cellulase. 
     
     
         11 . The polynucleotide of  claim 10 , wherein the cellulase is a  Holomastigotoides mirabile, Reticulitermes speratus  symbiont,  Coptotermes lacteus  symbiont,  Reticulitermes speratus  symbiont,  Cryptocercus punctulatus  symbiont,  Mastotermes darwiniensis  symbiont,  Pseudotrichonympha grassii, Reticulitermes flavipes  gut symbiont,  Hodotermopsis sjoestedti  symbiont or  Neotermes koshunensis  symbiont cellulase. 
     
     
         12 . (canceled) 
     
     
         13 . The polynucleotide of  claim 10 , wherein the cellulase is a  Coptotermes formosanus, Nasutitermes takasagoensis, Coptotermes acinaciformis, Mastotermes darwinensis, Reticulitermes speratus, Reticulitermes flavipes, Nasutitermes walkeri  or  Panesthia cribrata  cellulase. 
     
     
         14 . The polynucleotide of  claim 1 , wherein the cellulase is a bacterial cellulase. 
     
     
         15 . The polynucleotide of  claim 1 , wherein the cellulase is a fungal cellulase. 
     
     
         16 . The polynucleotide of  claim 1 , wherein the cellulase is a yeast cellulase and wherein said nucleic acid fragment is codon-optimized for expression in a heterologous yeast strain. 
     
     
         17 - 20 . (canceled) 
     
     
         21 . The polynucleotide of  claim 1 , wherein at least one nucleotide within a sequence of 4, 5, 6, 7, 8, 9, or 10 consecutive A, T, C or G nucleotides is replaced with a different nucleotide, wherein the nucleotide replacement does not alter the amino acid sequence encoded by the polynucleotide and wherein the nucleotide replacement creates a codon that is the second most frequently used codon to encode an amino acid in  Saccharomyces cerevisiae.   
     
     
         22 . The polynucleotide of  claim 1 , wherein at least one restriction enzyme site within the polynucleotide is removed by replacing at least one nucleotide within the restriction enzyme site with a different nucleotide, wherein the nucleotide replacement does not alter the amino acid sequence encoded by the polynucleotide and wherein the nucleotide replacement creates a codon that is the second most frequently used codon to encode an amino acid in  Saccharomyces cerevisiae.   
     
     
         23 . (canceled) 
     
     
         24 . The polynucleotide of  claim 1 , wherein one or more direct repeats, inverted repeats and mirror repeats with lengths of 10 bases or longer within said polynucleotide is altered by replacing at least one nucleotide within the repeat with a different nucleotide, wherein the nucleotide replacement does not alter the amino acid sequence encoded by the polynucleotide and wherein the nucleotide replacement creates a codon that is the second most frequently used codon to encode an amino acid in  Saccharomyces cerevisiae.   
     
     
         25 - 31 . (canceled) 
     
     
         32 . The polynucleotide of  claim 1 , wherein the nucleic acid fragment comprises a nucleic acid coding sequence selected from the group consisting of SEQ ID NOs: 1-20. 
     
     
         33 . A vector comprising a first polynucleotide, wherein said first polynucleotide is the polynucleotide of  claim 1 . 
     
     
         34 - 53 . (canceled) 
     
     
         54 . A host cell comprising a polynucleotide encoding at least 50 contiguous amino acids of a mature heterologous cellulase, wherein the heterologous cellulase is a termite cellulase or a termite-associated symbiont cellulase, wherein the host cell is a yeast cell and wherein the heterologous cellulase is expressed. 
     
     
         55 - 86 . (canceled) 
     
     
         87 . A host cell comprising the polynucleotide of  claim 1 . 
     
     
         88 - 92 . (canceled) 
     
     
         93 . A method for hydrolyzing a cellulosic substrate, comprising contacting said cellulosic substrate with a host cell according to  claim 54 . 
     
     
         94 . (canceled) 
     
     
         95 . A method of fermenting cellulose using the host cell of  claim 54 , said method comprising culturing said host cell in medium that contains crystalline cellulose under suitable conditions for a period sufficient to allow saccharification and fermentation of the cellulose. 
     
     
         96 - 100 . (canceled) 
     
     
         101 . A method for hydrolyzing a cellulosic substrate, comprising contacting said cellulosic substrate with a host cell according to  claim 87 .