- Document Number:
20240240215
- Appl. No:
18/492264
- Application Filed:
October 23, 2023
- نبذة مختصرة :
Disclosed are components and methods for preparing tagatose from galactose via isomerization reactions using engineered components. The engineered components include microbial cells and methods for preparing microbial cells that have been engineered to catalyze isomerization of galactose to tagatose, in which the microbial cells express cytoplasmically an exogenous L-arabinose isomerase enzyme. The disclosed microbial cells may further be modified for use in methods for preparing tagatose from galactose via isomerization reactions where the microbial cells are treated with reagents that permeabilize the cells. The disclosed methods enable isomerization reactions of galactose to tagatose at relatively high rates, high conversions, and elevated temperatures.
- Claim:
1-20. (canceled)
- Claim:
21. A microbial cell comprising: a recombinant exogenous L-arabinose isomerase enzyme, the microbial cell expressing the enzyme cytoplasmically such that the enzyme is effective to cytoplasmically catalyze the isomerization of D-galactose to tagatose.
- Claim:
22. The microbial cell of claim 21, wherein the enzyme has at least 90% sequence identity with SEQ ID NO: 1.
- Claim:
23. The microbial cell of claim 21, wherein the enzyme is Lactobacillus sakei L-arabinose isomerase, or a variant thereof having at least 90% identity with Lactobacillus sakei L-arabinose isomerase.
- Claim:
24. The microbial cell of claim 23, wherein the enzyme is engineered to exhibit increased catalytic efficiency for D-galactose substrate.
- Claim:
25. The microbial cell of claim 21, further comprising an exogenous galactose, an exogenous disaccharide comprising galactose or an exogenous oligosaccharide comprising galactose.
- Claim:
26. The microbial cell of claim 21, wherein the microbial cell is a gram positive bacterial cell selected from Lactobacillus spp., Bacillus spp., Corynebacterium spp., and Brevibacterium spp.
- Claim:
27. The microbial cell of claim 21, wherein the microbial cell is Lactobacillus plantarum.
- Claim:
28. The microbial cell of claim 21, wherein the microbial cell is a gram negative bacterial cell selected from Escherichia spp., Rhodobacter spp., Zymomonas spp., Vibrio spp, Agrobacterium spp., Paracoccus spp., and Pseudomonas spp.
- Claim:
29. The microbial cell of claim 21, wherein the microbial cell is Escherichia coli.
- Claim:
30. The microbial cell of claim 21, wherein the microbial cell is permeabilized.
- Claim:
31. The microbial cell of claim 21, wherein the microbial cell further comprises one or more genetic modifications that increase expression of an exogenous sugar transporter for D-galactose.
- Claim:
32. The microbial cell of claim 21, wherein the microbial cell further comprises one or more genetic modifications that increase the membrane permeability of the microbial cell for D-galactose.
- Claim:
33. A method for catalyzing isomerization of galactose to tagatose, comprising: providing a feedstock comprising galactose to a culture of the microbial cell of claim 21, and recovering tagatose from the culture.
- Claim:
34. The method of claim 33, wherein the feedstock comprises a disaccharide or oligosaccharide comprising galactose.
- Claim:
35. The method of claim 33, wherein the culture is maintained at a temperature between 37° C. and 50° C.
- Claim:
36. A microbial cell comprising: a recombinant exogenous L-arabinose isomerase enzyme, the microbial cell expressing the enzyme cytoplasmically such that the enzyme is effective to cytoplasmically catalyze the isomerization of D-galactose to tagatose at a temperature greater than 37° C.
- Claim:
37. The microbial cell of claim 36, wherein the microbial cell further comprises one or more genetic modifications that increase one or more of (1) expression of an exogenous sugar transporter for D-galactose, and (2) membrane permeability of the microbial cell for D-galactose.
- Claim:
38. The microbial cell of claim 36, wherein the microbial cell further comprises one or more genetic modifications that decrease conversion of galactose to glucose.
- Claim:
39. The microbial cell of claim 36, wherein the microbial cell further comprises a partial inactivation of at least one enzyme selected from galactokinase, galactose-1-phosphate uridinyltransferase, and UDP-galactose-4′ epimerase.
- Claim:
40. A method for preparing a microbial cell comprising engineering the microbial cell to express cytoplasmically an exogenous L-arabinose isomerase enzyme at a temperature between 37° C. and 50° C.
- Current International Class:
12; 12; 12
- الرقم المعرف:
edspap.20240240215
Processing Request
No Comments.