Method for the enzymatic production of D-erythrose and acetyl phosphate
Described is a method for the production of D-erythrose and acetyl phosphate comprising the enzymatic conversion of D-fructose into D-erythrose and acetyl phosphate by making use of a phosphoketolase. The produced D-erythrose can further be converted into glycolaldehyde by a method for the production of glycolaldehyde comprising the enzymatic conversion of D-erythrose into glycolaldehyde by making use of an aldolase, wherein aldolase is a 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4) or a fructose-bisphosphate aldolase (EC 4.1.2.13). The produced glycolaldehyde can finally be converted into acetyl phosphate by the enzymatic conversion of the thus produced glycolaldehyde into acetyl phosphate by making use of a phosphoketolase or a sulfoacetaldehyde acetyltransferase.
1. A method of producing D-erythrose and acetyl phosphate comprising enzymatically converting D-fructose and phosphate into D-erythrose and acetyl phosphate by a phosphoketolase (EC 4.1.2.9) or a fructose-6-phosphate phosphoketolase (EC 4.1.2.22) wherein the phosphoketolase or fructose-6-phosphate phosphoketolase is obtained from fungi or bacteria.
2. The method of claim 1 , further comprising enzymatically converting D-erythrose into glycolaldehyde by an aldolase, wherein said aldolase is a 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4) or a fructose-bisphosphate aldolase (EC 4.1.2.13).
3. The method of claim 2 , wherein the method further comprises: enzymatically converting glycolaldehyde into acetyl phosphate by a phosphoketolase (EC 4.1.2.9), a fructose-6-phosphate phosphoketolase (EC 4.1.2.22), or a sulfoacetaldehyde acetyltransferase (EC 2.3.3.15).
4. The method of claim 1 , wherein the method further comprises enzymatically converting D-glucose into said D-fructose by a glucose-fructose isomerase.
5. The method of claim 4 , wherein said glucose-fructose isomerase is a xylose isomerase (EC 5.3.1.5).
6. The method of claim 1 , wherein said method is carried out in vitro.
7. The method of claim 2 , wherein said method is carried out in vitro).
8. The method of claim 1 , wherein the method is carried out in a microorganism expressing the phosphoketolase (EC 4.1.2.9) or the fructose-6-phosphate phosphoketolase (EC 4.1.2.22).
9. The method of claim 2 , wherein the method is carried out in a microorganism expressing the 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4) or the fructose-bisphosphate aldolase (EC 4.1.2.13).
10. The method of claim 1 , wherein the method further comprises enzymatically converting the acetyl phosphate into acetyl-CoA by a phosphotransacetylase in the presence of co-enzyme A (CoA).
11. The method of claim 2 , wherein the method further comprises enzymatically converting the acetyl phosphate into acetyl-CoA by a phosphotransacetylase in the presence of co-enzyme A (CoA).
12. The method of claim 3 , wherein the method further comprises enzymatically converting the acetyl phosphate into acetyl-CoA by a phosphotransacetylase in the presence of co-enzyme A (CoA).
13. The method of claim 6 , wherein the method further comprises enzymatically converting the acetyl phosphate into acetyl-CoA by a phosphotransacetylase in the presence of co-enzyme A (CoA).
14. The method of claim 4 , wherein the method further comprises enzymatically converting the acetyl phosphate into acetyl-CoA by a phosphotransacetylase in the presence of co-enzyme A (CoA).
15. The method of claim 5 , wherein the method further comprises enzymatically converting the acetyl phosphate into acetyl-CoA by a phosphotransacetylase in the presence of co-enzyme A (CoA).
16. The method of claim 7 , wherein the method further comprises enzymatically converting glycolaldehyde into acetyl phosphate by a phosphoketolase (EC 4.1.2.9) or the fructose-6-phosphate phosphoketolase (EC 4.1.2.22) or a sulfoacetaldehyde acetyltransferase (EC 2.3.3.15).
17. The method of claim 4 , wherein said method is carried out in vitro.
18. The method of claim 9 , wherein the method is carried out in a recombinant microorganism expressing:
(a) a recombinant phosphoketolase (EC 4.1.2.9) or the fructose-6-phosphate phosphoketolase (EC 4.1.2.22) and/or a recombinant sulfoacetaldehyde acetyltransferase (EC 2.3.3.15); and
(b) a recombinant 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4) or a recombinant fructose-bisphosphate aldolase (EC 4.1.2.13);
and optionally
(c) a recombinant glucose-fructose isomerase.
19. The method of claim 18 , wherein the recombinant microorganism expresses both the recombinant phosphoketolase (EC 4.1.2.9) or the fructose-6-phosphate phosphoketolase (EC 4.1.2.22) and the recombinant sulfoacetaldehyde acetyltransferase (EC 2.3.3.15).
20. The method of claim 4 , wherein said method is carried out in a microorganism expressing the recombinant glucose-fructose isomerase.
21. The method of claim 4 , wherein said glucose-fructose isomerase is a xylose isomerase (EC 5.3.1.5).
22. The method of claim 18 , wherein the recombinant microorganism is genetically modified to overexpress the recombinant phosphoketolase, the recombinant sulfoacetaldehyde acetyltransferase (EC 2.3.3.15), the recombinant 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), the recombinant fructose-bisphosphate aldolase (EC 4.1.2.13) and/or the recombinant glucose-fructose isomerase, wherein the genetic modification is selected from:
(a) operably associating a heterologous promoter with a polynucleotide encoding the phosphoketolase, the sulfoacetaldehyde acetyltransferase (EC 2.3.3.15), the 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), the fructose-bisphosphate aldolase (EC 4.1.2.13) and/or the glucose-fructose isomerase;
(b) transforming a heterologous polynucleotide encoding the phosphoketolase, the sulfoacetaldehyde acetyltransferase (EC 2.3.3.15), the 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), the fructose-bisphosphate aldolase (EC 4.1.2.13) and/or the glucose-fructose isomerase into the recombinant microorganism; and/or
(c) introducing a mutation in the promoter of a polynucleotide encoding the phosphoketolase, the sulfoacetaldehyde acetyltransferase (EC 2.3.3.15), the 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), the fructose-bisphosphate aldolase (EC 4.1.2.13), and/or the glucose-fructose isomerase wherein said mutation results in overexpression of the polynucleotide.
23. The method of claim 18 , wherein the recombinant microorganism is genetically modified to alter or improve the enzymatic activity of the recombinant phosphoketolase, the recombinant sulfoacetaldehyde acetyltransferase (EC 2.3.3.15), the recombinant 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), the recombinant fructose-bisphosphate aldolase (EC 4.1.2.13) and/or the recombinant glucose-fructose isomerase, wherein the genetic modification is selected from:
(a) transforming a heterologous polynucleotide encoding the phosphoketolase, the sulfoacetaldehyde acetyltransferase (EC 2.3.3.15), the 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), the fructose-bisphosphate aldolase (EC 4.1.2.13), and/or the glucose-fructose isomerase into the recombinant microorganism; and/or
(b) introducing a mutation into a polynucleotide encoding the phosphoketolase, the sulfoacetaldehyde acetyltransferase (EC 2.3.3.15), the 2-deoxyribose-5-phosphate aldolase (EC 4.1.2.4), the fructose-bisphosphate aldolase (EC 4.1.2.13), and/or the glucose-fructose isomerase wherein said mutation alters or improves enzymatic activity.
24. The method of claim 3 , wherein the phosphoketolase is different than the phosphoketolase used in converting D-fructose into D-erythrose and acetyl phosphate.
25. The method of claim 10 , wherein the method is carried out in vitro.
26. The method of claim 10 , wherein the method is carried out in a recombinant microorganism.
27. The method of claim 3 , wherein the method is carried out in vitro.
28. The method of claim 3 , wherein the method is carried out in a recombinant microorganism.