Metabolic engineering of arabinose-fermenting yeast cells

Abstract

The invention relates to an eukaryotic cell expressing nucleotide sequences encoding the ara A, ara B and ara D enzymes whereby the expression of these nucleotide sequences confers on the cell the ability to use L-arabinose and/or convert L-arabinose into L-ribulose, and/or xylulose 5-phosphate and/or into a desired fermentation product such as ethanol. Optionally, the eukaryotic cell is also able to convert xylose into ethanol.

1 . A eukaryotic cell capable of expressing the following nucleotide sequences, wherein the expression of these nucleotide sequences confers on the cell the ability to use L-arabinose and/or to convert L-arabinose into L-ribulose, and/or xylulose 5-phosphate and/or into a desired fermentation product:
 (a) a nucleotide sequence encoding an arabinose isomerase (araA), wherein said nucleotide sequence is selected from the group consisting of:
 i. nucleotide sequences encoding an araA, said araA comprising an amino acid sequence that has at least 55% sequence identity with the amino acid sequence of SEQ ID NO:1, 
 ii. nucleotide sequences comprising a nucleotide sequence that has at least 60% sequence identity with the nucleotide sequence of SEQ ID NO:2, 
 iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic add molecule of sequence of (i) or (ii); 
 iv, nucleotide sequences the sequences of which differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy of the genetic; code, 
   (b) a nucleotide sequence encoding a L-ribulokinase (araB), wherein said nucleotide sequence is selected from the group consisting of:
 i. nucleotide sequences encoding an araB, said araB comprising an amino acid sequence that has at least 20% sequence identity with the amino acid sequence of SEQ ID NO:3, 
 ii. nucleotide sequences comprising a nucleotide sequence that has at least 50% sequence identity with the nucleotide sequence of SEQ ID NO:4, 
 iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence of (i) or (ii); 
 iv. nucleotide sequences the sequences of which differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy of the genetic code, 
   (c) a nucleotide sequence encoding an L-ribulose-5-P-4-epimerase (araD), wherein said nucleotide sequence is selected from the group consisting of:
 i. nucleotide sequences encoding an araD, said araD comprising an amino acid sequence that has at least 60% sequence identity with the amino acid sequence of SEQ ID NO:5, 
 ii. nucleotide sequences comprising a nucleotide sequence that has at least 60% sequence identity with the nucleotide sequence of SEQ ID NO:6, 
 iii. nucleotide sequences the complementary strand of which hybridizes to a nucleic acid molecule of sequence of (i) or (ii); 
 iv. nucleotide sequences the sequences of which differs from the sequence of a nucleic acid molecule of (iii) due to the degeneracy of the genetic code. 
   
     
     
         2 . A cell according to  claim 1 , wherein one, two or three of the araA, araB and araD nucleotide sequences originate from a  Lactobacillus  genus, preferably a  Lactobacillus plantarum  species. 
     
     
         3 . A cell according to  claim 1 , wherein the cell is a yeast cell, preferably belonging to one of the genera:  Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces  or  Yarrowia.    
     
     
         4 . A cell according to  claim 3 , wherein the yeast cell belongs to one of the species:  S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus  or  K. fragilis.    
     
     
         5 . A cell according to  claim 1 , wherein the nucleotide sequences encoding the araA, araB and/or araD are operably linked to a promoter that causes sufficient expression of the corresponding nucleotide sequences in the cell to confer to the cell the ability to use L-arabinose and/or to convert L-arabinose into L-ribulose, and/or xylulose 5-phosphate and/or into a desired fermentation product. 
     
     
         6 . A cell according to  claim 1 , wherein the cell exhibits the ability to directly isomerise xylose into xylulose. 
     
     
         7 . A cell according to  claim 6 , wherein the cell comprises a genetic modification that increases the flux of the pentose phosphate pathway. 
     
     
         8 . A cell according to  claim 6 , wherein the genetic modification comprises overexpression of at least one gene of the non-oxidative part of the pentose phosphate pathway. 
     
     
         9 . A cell according to  claim 8 , wherein the gene is selected from the group consisting of the genes encoding ribulose-5-phosphate isomerase, ribulose-5-phosphate epimerase, transketolase and transaldolase. 
     
     
         10 . A cell according to  claim 8 , wherein the genetic modification comprises overexpression of at least the genes coding for a transketolase and a transaldolase. 
     
     
         11 . A cell according to  claim 1 , wherein the cell further comprises a genetic modification that increases the specific xylulose kinase activity. 
     
     
         12 . A cell according to  claim 11 , wherein the genetic modification comprises overexpression of a gene encoding a xylulose kinase. 
     
     
         13 . A cell according to  claim 8 , wherein the gene that is overexpressed is endogenous to the cell. 
     
     
         14 . A cell according to  claim 5 , wherein the cell comprises a genetic modification that reduces unspecific aldose reductase activity in the cell. 
     
     
         15 . A cell according to  claim 14 , wherein the genetic modification reduces the expression of, or inactivates a gene encoding an unspecific aldose reductase. 
     
     
         16 . A cell according to  claim 15 , wherein the gene is inactivated by deletion of at least part of the gene or by disruption of the gene. 
     
     
         17 . A cell according to  claim 14 , wherein the expression of each gene in the cell that encodes an unspecific aldose reductase is reduced or inactivated. 
     
     
         18 . A cell according to  claim 1 , wherein the fermentation product is selected from the group consisting of ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, butanol, a β-lactam antibiotic and a cephalosporin. 
     
     
         19 . A nucleic acid construct comprising a nucleic acid sequence encoding an araA, a nucleic acid sequence encoding an araB and/or a nucleic acid sequence encoding an araD all as defined in  claim 1 . 
     
     
         20 . A process for producing a fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, butanol, a β-lactam, antibiotic and a cephalosporin, whereby the process comprises:
 (a) fermenting a medium containing a source of arabinose and optionally xylose with a modified cell as defined in  claim 1 , whereby the cell ferments arabinose and optionally xylose to the fermentation product; and optionally,   (b) recovering the fermentation product.   
     
     
         21 . A process for producing a fermentation product selected from the group consisting of ethanol, lactic acid, 3-hydroxy-propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, malic acid, fumaric acid, an amino acid, 1,3-propane-diol, ethylene, glycerol, butanol, a β-lactam antibiotic and a cephalosporin, wherein the process comprises:
 (a) fermenting a medium containing at least a source of L-arabinose and a source of xylose with a cell as defined in  claim 1  and a cell able to use xylose and/or exhibiting the ability to directly isomerise xylose into xylulose, whereby each cell ferments L-arabinose and/or xylose to the fermentation product; and optionally,   (b) recovering the fermentation product.   
     
     
         22 . A process according to  claim 20 , wherein the medium also contains a source of glucose. 
     
     
         23 . A process according to  claim 20 , wherein the fermentation product is ethanol. 
     
     
         24 . A process according to  claim 23 , wherein the volumetric ethanol productivity is at least 0.5 g ethanol per litre per hour. 
     
     
         25 . A process according to  claim 23 , wherein the ethanol yield is at least 30%. 
     
     
         26 . A process according to  claim 20 , wherein the process is anaerobic. 
     
     
         27 . A process according to  claim 20 , wherein the process is aerobic, preferably performed under oxygen limited conditions.