Engineering Fungi for the Utilisation of L-Arabinose

Abstract

A fungal microorganism can be engineered by means of genetic engineering to utilise L-arabinose. The genes of the L-arabinose pathway, which were unknown, i.e. L-arabinitol 4-dehydrogenase and L-xylulose reductase, were identified. These genes, together with the known genes of the L-arabinose pathway, form a functional pathway. This pathway can be introduced to a fungus, which is completely or partially lacking this pathway.

1 . A method for producing ethanol, lactic acid or xylitol using fungal biofermentation of L-arabinose, which comprises
 (a) providing a first fungus that is unable to use or is inefficient in metabolising L-arabinose;   (b) providing a nucleic acid that
 (1) encodes a fungal enzyme selected from the group consisting of LA4D and LXR wherein said LA4D converts L-arabinitol and NAD to L-xylulose and NADH and wherein said LXR converts xylitol and NADP to L-xylulose and NADPH; 
 (2) is contained within the genomic DNA of a second fungus that is able to efficiently metabolise L-arabinose; and 
 (3) is amplifiable from said genomic DNA by PCR, using the primers of SEQ ID NOS:13 and 14 when said nucleic acid encodes LA4D and the primers of SEQ ID NOS:15 and 16 when said nucleic acid encodes LXR; 
   (c) expressing said nucleic acid in said first fungus; and   (d) performing biofermentation by growing said first fungus in the presence of L-arabinose to produce ethanol, lactic acid or xylitol.   
     
     
         2 . The method of  claim 1 , wherein said first fungus is selected from the group consisting of  Aspergillus  species,  Trichoderma  species,  Neurospora  species,  Fusarium  species,  Penicillium  species,  Humicola  species,  Tolypocladium geodes, Trichoderma reesei  ( Hypocrea jecorina ),  Mucor  species,  Trichoderma longibrachiatum, Aspergillus nidulans, Aspergillus niger  and  Aspergillus awamori.    
     
     
         3 . The method of  claim 1 , wherein said second fungus is selected from the group consisting of  Saccharomyces  species,  Schizosaccharomyces  species,  Kluveromyces  species,  Pichia species, Candida  species and  Pachysolen  species. 
     
     
         4 . The method of  claim 3 , wherein said second fungus is  Saccharomyces cerevisiae.    
     
     
         5 . The method of  claim 1 , wherein said nucleic acid encodes SEQ ID NO:17. 
     
     
         6 . The method of  claim 1 , wherein said nucleic acid encodes SEQ ID NO:18. 
     
     
         7 . A method for producing ethanol, lactic acid or xylitol using fungal biofermentation of L-arabinose, which comprises
 (a) providing a fungus that is unable to use or is inefficient in metabolising L-arabinose;   (b) providing a nucleic acid that encodes a protein sequence selected from the group consisting of SEQ ID NOs:17 and 18;   (c) expressing said nucleic acid in said fungus; and   (d) performing biofermentation by growing said fungus in the presence of L-arabinose to produce ethanol, lactic acid or xylitol.   
     
     
         8 . A method of  claim 7 , wherein said fungus selected from the group consisting of  Aspergillus  species,  Trichoderma  species,  Neurospora  species,  Fusarium  species,  Penicillium  species,  Humicola  species,  Tolypocladium geodes, Trichoderma reesei  ( Hypocrea jecorina ),  Mucor  species,  Trichoderma longibrachiatum, Aspergillus nidulans, Aspergillus niger  and  Aspergillus awamori.    
     
     
         9 . A method of modifying the L-arabinose metabolic pathway of a fungus to allow metabolic conversion of L-arabinose to ethanol, lactic acid or xylitol, which comprise transferring to said fungus an LA4D-encoding nucleic acid from  Penicillium chrysogenum  or  Aspergillus niger , an LXR-encoding nucleic acid from  Erwinia uredovora  or  Aspergillus niger , or both. 
     
     
         10 . The method of  claim 9 , wherein said fungus is selected from the group consisting of  Aspergillus  species,  Trichoderma  species,  Neurospora  species,  Fusarium  species,  Penicillium  species,  Humicola  species,  Tolypocladium geodes, Trichoderma reesei  ( Hypocrea jecorina ),  Mucor  species,  Trichoderma longibrachiatum, Aspergillus nidulans, Aspergillus niger  and  Aspergillus awamori.    
     
     
         11 . A method of producing a useful product from biomass containing L-arabinose, which comprises:
 (a) providing a genetically modified fungus which is transformed with a DNA encoding an enzyme selected from the group consisting of L-arabinitol 4-dehydrogenase (LA4D) and L-xylulose reductase (LXR), each of the fungal L-arabinose pathway, wherein said fungus has an increased ability to utilise L-arabinose; and   (b) fermenting said fungus under conditions suitable to produce said useful product,   
       wherein the DNA is selected from the group consisting of nucleic acids that encode SEQ ID NOs:17 and 18.