Methods for production of xylitol in microorganisms
View Patent ↗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.
1. A recombinant bacterium that expresses proteins comprising:
(a) xylose specific reductase, wherein the xylose specific reductase is a Pichia stipitis reductase with Ser233Pro and Phe286Leu mutations;
(b) L-arabitol dehydrogenase or ribitol dehydrogenase or both; and
(c) L-xylulose reductase;
that have a biochemical activity that produces xylitol from substrates comprising D-xylose; or L-arabinose; or L-arabinose and D-xylose, such that the recombinant bacterium produces and accumulates xylitol, and accumulates no substantial amount of L-arabitol; wherein the xylose reductase protein, or L-arabitol dehydrogenase protein, or ribitol dehydrogenase protein, or L-xylulose reductase protein, or a combination thereof are expressed from a recombinant polynucleotide.
2. The recombinant bacterium of claim 1 , wherein the recombinant bacterium produces and accumulates xylitol from substrates comprising a xylan hydrolysate or a hemicellulose hydrolysate.
3. The recombinant bacterium of claim 1 , wherein the bacterium further expresses a ribitol transporter protein.
4. The recombinant bacterium of claim 1 , wherein the bacterium is Escherichia coli.
5. The recombinant bacterium of claim 1 , wherein the bacterium does not have a ptsG gene or has an inactive ptsG gene.
6. The recombinant bacterium of claim 1 , wherein the recombinant bacterium produces L-arabitol as an intermediate to the xylitol end-product.
7. The recombinant bacterium of claim 1 , wherein the recombinant bacterium produces L-xylulose as an intermediate to the xylitol end-product.
8. The recombinant bacterium of claim 1 , wherein the recombinant bacterium comprises one or more recombinant polynucleotides encoding aldose reductase, L-xylose reductase, L-arabitol dehydrogenase, ribitol dehydrogenase, ribitol transporter, or L-xylulose reductase.
9. The recombinant bacterium of claim 8 , wherein the recombinant polynucleotide encoding xylose reductase is a Pichia stipitis polynucleotide.
10. The recombinant bacterium of claim 8 , wherein the recombinant polynucleotide encoding ribitol dehydrogenase is a Klebsiella pneumoniae or Klebsiella aerogenes polynucleotide.
11. The recombinant bacterium of claim 8 , wherein the recombinant polynucleotide encoding L-xylulose reductase is an Ambrosioyma monospora polynucleotide.
12. The recombinant bacterium of claim 8 , wherein the recombinant polynucleotide encoding L-xylose reductase comprises a yafB or yajO polynucleotide from Escherichia coli.
13. The recombinant bacterium of claim 7 , wherein the recombinant polynucleotide encoding L-arabitol dehydrogenase is a Trichoderma reesei polynucleotide.
14. The recombinant bacterium of claim 8 , wherein the recombinant polynucleotide encoding L-xylose reductase is a T. reesei polynucleotide.
15. A method for producing a xylitol end-product comprising fermenting a substrate comprising D-xylose; or L-arabinose; or L-arabinose and D-xylose; or L-arabinose, D-xylose and other sugars with the recombinant bacterium of claim 1 .
16. The method of claim 15 , wherein L-arabitol is produced as an intermediate to the xylitol end-product.
17. The method of claim 15 , wherein L-xylulose in produced as an intermediate to the xylitol end-product.
18. The method of claim 15 , wherein in the method does not require separation of L-arabitol from the xylitol end-product.
19. A method for producing L-xylulose comprising fermenting a substrate comprising D-xylose; or L-arabinose; or L-arabinose and D-xylose; or L-arabinose, D-xylose and other sugars with the recombinant bacterium of claim 1 , and collecting L-xylulose before it is converted to xylitol.
20. 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 the isolated recombinant bacteria of claim 1 ,
wherein the substrate is converted to an end-product of xylitol and wherein substantially no L-arabitol is produced as an end-product.
21. The method of claim 20 , wherein the isolated recombinant bacteria comprise ribitol transporter activity.
22. An isolated recombinant microorganism that expresses a recombinant xylose specific reductase protein, wherein the xylose specific reductase protein is a Pichia stipitis reductase protein with Ser233Pro and Phe286Leu mutations.
23. The isolated recombinant microorganism of claim 22 , wherein the microorganism can produce an end-product of xylitol from a substrate comprising D-xylose; or L-arabinose; or L-arabinose and D-xylose; or L-arabinose, D-xylose and other sugars.
24. The isolated recombinant microorganism of claim 22 , wherein the microorganism produces no substantial amount of L-arabitol as an end-product.
25. The isolated recombinant microorganism of claim 22 , wherein the microorganism does not substantially produce L-arabitol from a substrate of xylan hydrolysate or hemicellulose hydrolysate.
26. The isolated recombinant microorganism of claim 22 , wherein the microorganism is E. coli.
27. The isolated recombinant microorganism of claim 22 , wherein the microorganism does not have a ptsG gene or has an inactive ptsG gene.
28. The isolated recombinant microorganism of claim 22 , wherein the recombinant microorganism is non-pathogenic.
29. The isolated recombinant microorganism of claim 22 , wherein the microorganism is a bacteria, fungus or yeast.
30. The isolated recombinant microorganism of claim 22 , wherein the xylose specific reductase protein has an amino acid sequence comprising SEQ ID NO:43.
31. A method for producing xylitol comprising fermenting a substrate comprising D-xylose; or L-arabinose; or L-arabinose and D-xylose; or L-arabinose, D-xylose and other sugars; or xylan hydrolysate; or hemicellulose hydrolysate with the isolated microorganism of claim 22 .
32. The method of claim 31 , wherein the method does not require separation of L-arabitol from xylitol.
33. A purified xylose specific reductase comprising SEQ ID NO:43.
34. A purified P. stipitis xylose reductase comprising a Ser233Pro mutation and a Phe286Leu mutation.
35. The recombinant bacterium of claim 1 , wherein: the L-arabitol dehydrogenase protein and the L-xylulose reductase protein are expressed from a recombinant yeast polynucleotide.