Compositions and methods for producing chemicals and derivatives thereof
The present invention provides methods for producing a product of one or more enzymatic pathways. The pathways used in the methods of the invention involve one or more conversion steps such as, for example, an enzymatic conversion of guluronic acid into D-glucarate (Step 7); an enzymatic conversion of 5-ketogluconate (5-KGA) into L-Iduronic acid (Step 15); an enzymatic conversion of L-Iduronic acid into Idaric acid Step 7b); and an enzymatic conversion of 5-ketocluconate into 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 16). In some embodiments the methods of the invention produce 2,5-furandicarboxylic acid (FDCA) as a product. The methods include both enzymatic and chemical conversions as steps. Various pathways are also provided for converting glucose into 5-dehdyro-4-deoxy-glucarate (DDG), and for converting glucose into 2,5-furandicarboxylic acid (FDCA). The methods also involve the use of engineered enzymes that perform reactions with high specificity and efficiency. Additional products that can be produce include metabolic products such as, but not limited to, guluronic acid, L-iduronic acid, idaric acid, glucaric acid. Any of the products can be produced using glucose as a substrate or using any intermediate in any of the methods or pathways of the invention.
1. A method for producing a product of an enzymatic or chemical pathway from a starting substrate, the pathway comprising one or more conversion steps selected from the group consisting of:
contacting guluronic acid with a uronate dehydrogenase to perform an enzymatic conversion of guluronic acid into D-glucarate (Step 7);
contacting 5-ketogluconate (5-KGA) with an isomerase to perform an enzymatic conversion of 5-ketogluconate (5-KGA) into L-Iduronic acid (Step 15);
contacting L-Iduronic acid with a uronate dehydrogenase to perform an enzymatic conversion of L-Iduronic acid into Idaric acid (Step 7b); and
contacting 5-ketogluconate with a gluconate dehydratase to perform an enzymatic conversion of 5-ketogluconate into 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 16); and
contacting 1,5-gluconolactone with an alditol oxidase to perform an enzymatic conversion of 1,5-gluconolactone to gulurono-lactone (Step 19).
2. The method of claim 1 wherein the one or more conversion steps comprises contacting guluronic acid with a uronate dehydrogenase to perform the enzymatic conversion of guluronic acid into D-glucarate (Step 7).
3. The method of claim 1 wherein the one or more conversion steps comprises contacting 5-ketogluconate (5-KGA) with an isomerase to perform the enzymatic conversion of 5-ketogluconate (5-KGA) into L-Iduronic acid (Step 15).
4. The method of claim 1 wherein the one or more conversion steps comprises contacting L-Iduronic acid with a uronate dehydrogenase to perform the enzymatic conversion of L-Iduronic acid into Idaric acid Step 7b).
5. The method of claim 1 wherein the one or more conversion steps comprises contacting 5-ketogluconate with a gluconate dehydratase to perform the enzymatic conversion of 5-ketogluconate into 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 16).
6. The method of claim 1 wherein the one or more conversion steps comprises contacting 1,5-gluconolactone with an alditol oxidase to perform the enzymatic conversion of 1,5-gluconolactone to gulurono-lactone (Step 19).
7. The method of claim 1 wherein the pathway is an enzymatic pathway and the product is 5-dehydro-4-deoxy-glucarate (DDG).
8. The method of claim 1 wherein the substrate is glucose and the product is 5-dehydro-4-deoxy-glucarate (DDG), comprising the steps of:
contacting D-glucose with an oxygen dependent glucose oxidase or an NAD(P)-dependent glucose dehydrogenase to perform an enzymatic conversion of D-glucose to 1,5-gluconolactone (Step 1);
contacting 1,5-gluconolactone with an alditol oxidase to perform the enzymatic conversion of 1,5-gluconolactone to gulurono-lactone (Step 19);
contacting gulurono-lactone with a lactonase to perform an enzymatic conversion of gulurono-lactone to guluronic acid (Step 1B), or converting gulurono-lactone to guluronic acid by spontaneous reaction in an aqueous solution;
contacting guluronic acid with a uronate dehydrogenase to perform the enzymatic conversion of guluronic acid to D-glucarate (Step 7);
contacting D-glucarate with a glucarate dehydratase to perform an enzymatic conversion of D-glucarate to 5-dehydro-4-deoxy-glucarate (DDG) (Step 8).
9. A method of claim 1 wherein the substrate is glucose and the product is DDG, comprising the steps of:
contacting D-glucose with an oxygen dependent glucose oxidase to produce 1,5-gluconolactone (Step 1);
hydrolyzing 1,5-gluconolactone to gluconic acid (Step 1a);
contacting gluconic acid with an NAD(P)-dependent dehydrogenase to produce 5-ketogluconate (5-KGA) (Step 14);
contacting 5-ketogluconate (5-KGA) with an isomerase to produce L-Iduronic acid (Step 15);
contacting L-Iduronic acid with a uronate dehydrogenase to produce Idaric acid (Step 7b); and
contacting Idaric acid with a glucarate dehydratase to produce DDG (Step 8a).
10. A method of claim 1 wherein the substrate is glucose and the product is DDG, comprising the steps of:
contacting D-glucose with an oxygen dependent glucose oxidase and an NAD(P)-dependent glucose dehydrogenase to produce 1,5-gluconolactone (Step 1);
hydrolyzing 1,5-gluconolactone to gluconic acid (Step 1a);
contacting gluconic acid with an NAD(P)-dependent dehydrogenase to produce 5-ketogluconate (5-KGA) (Step 14);
contacting 5-ketogluconate (5-KGA) with a gluconate dehydratase to produce 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 16);
contacting 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) with a glucoronate isomerase to produce 4-deoxy-5-threo-hexosulose uronate (DTHU) (Step 4); and
oxidizing 4-deoxy-5-threo-hexosulose uronate (DTHU) to DDG (Step 5).
11. A method of claim 1 wherein the substrate is glucose and the product is DDG, comprising the steps of:
contacting D-glucose with an oxygen dependent glucose oxidase to produce 1,5-gluconolactone (Step 1);
hydrolyzing 1,5-gluconolactone to gluconic acid (Step 1a);
contacting gluconic acid with an NAD(P)-dependent dehydrogenase to produce 5-ketogluconate (5-KGA) (Step 14);
contacting 5-ketogluconate (5-KGA) with an isomerase to produce L-Iduronic acid (Step 15);
contacting L-Iduronic acid with a uronate dehydrogenase to produce 4-deoxy-5-threo-hexosulose uronate (DTHU) (Step 7b); and
oxidizing 4-deoxy-5-threo-hexosulose uronate (DTHU) to produce DDG (Step 5).
12. The method of claim 8 further comprising the step of converting the DDG to 2,5-furan-dicarboxylic acid (FDCA) by contacting the DDG with an inorganic acid.
13. The method of claim 9 further comprising the step of converting the DDG to 2,5-furan-dicarboxylic acid (FDCA) by contacting the DDG with an inorganic acid.
14. The method of claim 10 further comprising the step of converting the DDG to 2,5-furan-dicarboxylic acid (FDCA) by contacting the DDG with an inorganic acid.
15. The method of claim 11 further comprising the step of converting the DDG to 2,5-furan-dicarboxylic acid (FDCA) by contacting the DDG with an inorganic acid.
16. The method of claim 2 wherein the uronate dehydrogenase comprises SEQ ID NO: 1-3 or a homolog having at least 70% sequence identity to SEQ ID NOs: 1-3; or is encoded by a nucleic acid of SEQ ID NOs: 4-6 or a homolog having at least 70% sequence identity to a nucleic acid of SEQ ID NOs: 4-6.
17. The method of claim 3 wherein the isomerase comprises SEQ ID NOs: 7-19 or a homolog having at least 70% sequence identity to an isomerase of SEQ ID NOs: 7-19; or is encoded by a nucleic acid of SEQ ID NOs: 20-32 or a homolog having at least 70% sequence identity to a nucleic acid of SEQ ID NOs: 20-32.
18. The method of claim 5 wherein the gluconate dehydratase comprises SEQ ID NOs: 33-35 or a homolog having at least 70% sequence identity to a gluconate dehydratase of SEQ ID NOs: 33-35; or is encoded by a nucleic acid of SEQ ID NOs: 36-38 or a homolog having at least 70% sequence identity to a nucleic acid of SEQ ID NOs: 36-38.
19. The method of claim 6 wherein the alditol oxidase comprises SEQ ID NOs: 39-46 or a homolog having at least 70% sequence identity to an alditol oxidase of SEQ ID NOs: 39-46; or is encoded by a nucleic acid of SEQ ID NOs: 47-54 or a homolog having at least 70% sequence identity to a nucleic acid of SEQ ID NOs: 47-54.
20. The method of claim 4 wherein the uronate dehydrogenase comprises SEQ ID NO: 1-3 or a homolog having at least 70% sequence identity to SEQ ID NOs: 1-3; or is encoded by a nucleic acid of SEQ ID NOs: 4-6 or a homolog having at least 70% sequence identity to a nucleic acid of SEQ ID NOs: 4-6.