Trichoderma reesei host cells expressing a glucoamylase from aspergillus fumigatus and methods of use thereof

Abstract

Fungal glucoamylases from Aspergillus fumigatus —expressed in Trichoderma reesei host cells (AfGATR) are provided. Trichoderma reesei host cells express AfGATRs at higher, or at least comparable, levels to natively expressed AfGA Aspergillus fumigatus . AfGATRs, including AfGA1TR and AfGA2TR, exhibit high activity at elevated temperatures and at low pH, so AfGATRs can be used efficiently in a process of saccharification in the presence of alpha-amylase, such as Aspergillus kawachii alpha-amylase (AkAA). AfGATRs advantageously catalyze starch saccharification to an oligosaccharide composition significantly enriched in DP1 (i.e., glucose) compared to the products of saccharification catalyzed by Aspergillus niger glucoamylase (AnGA) or native AfGA expressed in Aspergillus fumigatus . AfGATRs such as AfGA1TR, AfGA2TR or a variant thereof can be used at a lower dosage than AnGA and natively expressed AfGAs to produce comparable levels of glucose.

1 . A recombinant  Trichoderma reesei  host cell expressing an AfGATR, or variant thereof, having at least 80% sequence identity to SEQ ID NO: 12 or 13, wherein said  Trichoderma reesei  host cell expresses the AfGATR or variant at a comparable level to a  Aspergillus fumigatus  host cell that expresses an AfGA, or variant thereof, having the same amino acid sequence of the AfGATR, or variant thereof, under identical conditions. 
     
     
         2 . A recombinant  Trichoderma reesei  host cell expressing an AfGATR, or variant thereof, having at least 80% sequence identity to SEQ ID NO: 12 or 13, wherein the AfGATR, or variant thereof, is more thermostable than an AfGA, or variant thereof, having the same amino acid sequence of AfGATR, or variant thereof, and wherein the AfGA, or variant thereof, is expressed in an  A. fumigatus  host cell. 
     
     
         3 . A recombinant AfGATR, or variant thereof, produced by the host cell of  claim 2 . 
     
     
         4 . A recombinant AfGATR, or variant thereof, produced by the host cell of  claim 1 . 
     
     
         5 . The recombinant AfGATR, or variant thereof, of  claim 4 , wherein said AfGATR, or variant thereof, has at least 70% activity at 74° C. at pH 5.0 over 10 min. 
     
     
         6 . The recombinant AfGATR, or variant thereof, of  claim 5 , wherein said AfGATR, or variant thereof, is an AfGA1TR, or variant thereof. 
     
     
         7 . The recombinant AfGA1TR, or variant thereof, of  claim 6 , wherein said AfGA1TR, or variant, thereof has at least 70% activity over a temperature range of 55° to 74° C. at pH 5.0 over 10 min. 
     
     
         8 . The recombinant AfGA1TR, or variant thereof, of  claim 7 , wherein said AfGATR, or variant thereof, has an optimum temperature of about 68° C. 
     
     
         9 . The recombinant AfGATR, or variant, thereof of  claim 5 , wherein said AfGATR, or variant thereof, is an AfGA2TR, or variant thereof. 
     
     
         10 . The recombinant AfGA2TR, or variant thereof, of  claim 9 , wherein said AfGA2TR, or variant thereof, has at least 70% activity over a temperature range of 61° to 74° C. at pH 5.0 over 10 min. 
     
     
         11 . The recombinant AfGA2TR, or variant thereof, of  claim 10 , wherein said AfGA2TR, or variant thereof, has an optimum temperature of about 69° C. 
     
     
         12 . The recombinant AfGATR, or variant thereof, of  claim 4 , wherein said AfGATR, or variant thereof, comprises an amino acid sequence with at least 90%, 95%, or 99% amino acid sequence identity to SEQ ID NO: 12. 
     
     
         13 . The recombinant AfGATR, or variant thereof, of  claim 12 , wherein said AfGATR, or variant, thereof comprises SEQ ID NO: 12. 
     
     
         14 . The recombinant AfGATR, or variant thereof, of  claim 4 , wherein said AfGATR, or variant thereof, consists of an amino acid sequence with at least 80%, 90%, 95%, or 99% amino acid sequence identity to SEQ ID NO: 12. 
     
     
         15 . (canceled) 
     
     
         16 . The recombinant AfGATR, or variant thereof, of  claim 4 , wherein said AfGATR, or variant thereof, comprises an amino acid sequence with at least 90%, 95%, or 99% amino acid sequence identity to SEQ ID NO: 13. 
     
     
         17 . The recombinant AfGATR, or variant thereof, of  claim 16 , wherein said AfGATR, or variant, thereof comprises SEQ ID NO: 13. 
     
     
         18 . The recombinant AfGATR, or variant thereof, of  claim 4 , wherein said AfGATR, or variant thereof, consists of an amino acid sequence with at least 80%, 90%, 95%, or 99% amino acid sequence identity to SEQ ID NO: 13. 
     
     
         19 . (canceled) 
     
     
         20 . A method of saccharifying a composition comprising starch to produce a composition comprising glucose, wherein said method comprises:
 (i) contacting a starch composition with the isolated AfGATR, or variant thereof, of  claim 4 ; and   (ii) saccharifying the starch composition to produce said glucose composition;   wherein said AfGA1TR, or variant, thereof catalyzes the saccharification of the composition comprising starch to a composition comprising glucose.   
     
     
         21 . The method of  claim 20 , wherein said composition comprising glucose enriched in DP1 compared to a second composition comprising DP1 produced by AnGA after 24 hours of saccharification under the same conditions. 
     
     
         22 . The method of  claim 20 , wherein said composition comprising glucose is enriched in DP1 compared to a second composition comprising DP1 produced by a wild-type AfGA under the same conditions. 
     
     
         23 . The method of  claim 20 , wherein said AfGATR, or variant thereof, is AfGATR2 and wherein said composition comprising glucose is enriched in DP1 compared to a second composition comprising DP1 produced by AfGA1TR under the same conditions. 
     
     
         24 . The method of  claim 20 , wherein the AfGA1TR, or variant thereof, is dosed at about 40%-50% the dose of AnGA, to produce the same DP1 yield after 24 hours of saccharification under the same conditions. 
     
     
         25 - 30 . (canceled) 
     
     
         31 . The method of  claim 20 , wherein the method further comprises contacting a starch composition with an alpha-amylase. 
     
     
         32 . The method of  claim 31 , wherein the alpha-amylase is AkAA. 
     
     
         33 . The method of  claim 20 , wherein the method further comprises contacting a starch composition with a pullulanase. 
     
     
         34 . The method of  claim 20 , further comprising fermenting the glucose composition to produce an End of Fermentation (EOF) product. 
     
     
         35 - 40 . (canceled) 
     
     
         41 . The method of  claim 34 , wherein the EOF product comprises a metabolite. 
     
     
         42 - 45 . (canceled) 
     
     
         46 . The method of  claim 20 , wherein said isolated AfGATR, or variant thereof, is secreted by said  Trichoderma reesei  host cell. 
     
     
         47 . The method of  claim 46 , wherein said host cell further expresses and secretes an alpha-amylase. 
     
     
         48 . The method of  claim 47 , wherein said host cell further expresses and secretes a pullulanase. 
     
     
         49 . The method of  claim 46 , wherein said composition comprising starch is contacted with said host cell. 
     
     
         50 - 74 . (canceled)