Methods for Production of Xylitol in Microorganisms
AssigneeBiotechnology Research and Development Corporation
InventorsPaul Taylor, Ian Fotheringham, Nathan Wymer, Badal Saha, David Demirjian, Yoshikiyo Sakaibara, Francis Michael Racine
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Abstract
The invention provides biosynthetic routes to xylitol production that do not require pure D-xylose for synthesis and that can utilize inexpensive substrates such as hemicellulose hydrolysates.
Claims65
A recombinant bacterium that expresses proteins comprising:
- (a) xylose reductase;
- (b) L-arabitol dehydrogenase or ribitol dehydrogenase or both;
- and
- (c) L-xylulose reductase;
- wherein the recombinant bacterium can produce an end-product of xylitol from substrates comprising:
- D-xylose;
- or L-arabinose;
- or L-arabinose and D-xylose;
- or L-arabinose, D-xylose and other sugars;
- and wherein no substantial amount of L-arabitol is produced as an end-product.
A method of producing xylitol from a substrate comprising D-xylose;
- or L-arabinose;
- or L-arabinose and D-xylose;
- or L-arabinose, D-xylose and other sugars;
- comprising contacting the substrate with one or more isolated bacteria that comprise:
- (a) xylose reductase activity;
- (b) L-arabitol dehydrogenase activity or ribitol dehydrogenase activity or a combination of both;
- and
- (c) L-xylulose reductase activity;
- wherein the substrate is converted to an end-product of xylitol and wherein substantially no L-arabitol is produced as an end-product.
A process for producing xylitol from a substrate comprising D-xylose;
- or L-arabinose;
- or L-arabinose and D-xylose;
- or L-arabinose, D-xylose and other sugars;
- comprising contacting the substrate with:
- (a) xylose reductase;
- (b) L-arabitol dehydrogenase or ribitol dehydrogenase, or both;
- and
- (c) L-xylulose reductase;
- wherein the substrate is converted to an end-product of xylitol and wherein substantially no L-arabitol is produced as an end-product.
An isolated microorganism comprising xylose specific reductase activity, wherein the xylose specific reductase activity does not convert L-arabinose to L-arabitol.
A purified xylose specific reductase comprising SEQ ID NO:43.
A purified P. stipitis xylose reductase comprising a Ser233Pro mutation and a Phe286Leu mutation.
A process for producing xylitol comprising contacting a substrate comprising D-xylose;
- or L-arabinose;
- or L-arabinose and D-xylose;
- or L-arabinose, D-xylose and other sugars;
- with a xylose-specific reductase.
A recombinant E. coli that comprises a nucleic acid sequence encoding xylitol dehydrogenase, wherein the E. coli produces substantially no xylose isomerase.
A recombinant E. coli comprising L-xylulose kinase activity, L-xylulose 5-phosphate epimerase activity, and L-ribulose 5-phosphate 4-epimerase activity.
A recombinant E. coli strain comprising a deleted or inactive yiaJ gene.
A purified xylose reductase that is active at 37° C.
A method for screening for bacteria that cannot utilize L-arabinose comprising:
- transforming bacteria that do not have xylose isomerase activity or an araBAD operon and that have xylitol dehydrogenase activity and L-ribulokinase activity, with a nucleic acid encoding a xylose reductase, wherein if the xylose reductase is a xylose-specific reductase the transformed bacteria cannot utilize L-arabinose and will grow on media comprising L-arabinose and D-xylose, and wherein if the xylose reductase is not a xylose-specific reductase, the transformed bacteria can utilize L-arabinose and will not grow on media comprising L-arabinose and D-xylose.
An isolated microorganism comprising a recombinant operon comprising a nucleic acid encoding a xylitol dehydrogenase and a nucleic acid encoding a xylose isomerase.
A purified E. coli xylose isomerase (xylA) wherein the amino acid sequence comprises the following mutations:
- (a) F9L, L213Q, F283Y, K311R, H420N;
- (b) F9L, Q11K, L213Q, F283Y, K311R, H420N;
- (c) F9L, Q11K, S20L, L213Q, F283Y, K311R, H420N;
- or
- (d) F9L, Q11K, S20L, L213Q, F283Y, K311R, H420N, H439Q.
A recombinant microorganism comprising a mutated E. coli xylose isomerase coding sequence, wherein the microorganism can grow in the presence of about 1% or more of xylitol.
Dependencies are inferred from the claim text.