Microbial production of 3,4-dihydroxybutyrate (3,4-DHBA), 2,3-dihydroxybutyrate (2,3-DHBA) and 3-hydroxybutyrolactone (3-HBL)
The invention relates to recombinant cells and their use in the production of 3,4-dihydroxybutyrate, 2,3-dihydroxybutyrate and 3-hydroxybutyrolactone.
1. An isolated cell that overexpresses a ycdW gene encoding a glyoxylate reductase enzyme, an aceA gene encoding an isocitrate lyase enzyme, and an aceK gene encoding an isocitrate dehydrogenase kinase/phosphatase enzyme, and that recombinantly expresses a pct gene encoding a propionyl-CoA transferase enzyme, at least one of a phaA, thil, atoB or bktB gene encoding a thiolase enzyme, at least one of a phaB or hbd gene encoding a reductase enzyme, and a tesB gene encoding a thioesterase B enzyme, wherein the isolated cell can produce a cell culture that contains at least 0.1 g L −1 2,3-dihydroxybutyrate (2,3-DHBA).
2. The isolated cell of claim 1 , wherein the isolated cell has reduced or eliminated expression of an iclR gene, an aceB gene and a gcl gene relative to a wild type cell.
3. The isolated cell of claim 1 , wherein overexpression of ycdW, aceA and aceK, and recombinant expression of pct, at least one of phaA, thil, atoB or bktB, at least one of phaB or hbd, and tesB, is induced through at least two independently inducible expression systems.
4. The isolated cell of claim 1 wherein the isolated cell is a bacterial cell, a fungal cell, a plant cell, an insect cell or an animal cell.
5. The isolated cell of claim 1 , wherein the isolated cell endogenously expresses one or more of ycdW, aceA, and aceK, and wherein endogenous expression of one or more of ycdW, aceA, and aceK is increased through modification of one or more of the gene(s), their promoter(s) and their ribosome binding sites (RBSs).
6. The isolated cell of claim 1 , wherein one or more of ycdW, aceA, aceK, pct, phaA, thil, atoB, bktB, phaB, hbd and tesB is expressed from a plasmid, or wherein one or more copies of ycdW, aceA, aceK, pct, phaA, thil, atoB, bktB, phaB, hbd and tesB is integrated into the genome of the isolated cell.
7. The isolated cell of claim 1 , wherein one or more of the ycdW, aceA aceK and tesB gene(s) is an Escherichia coli gene.
8. The isolated cell of claim 1 wherein one or more of the phaA, bktB and phaB gene(s) is a Ralstonia eutropha gene.
9. The isolated cell of claim 1 wherein the pct gene is a Megasphaera elsdenii gene.
10. A method for producing 2,3-DHBA comprising culturing the isolated cell of claim 1 to produce 2,3-DHBA, optionally wherein the method further comprises recovering the 2,3-DHBA from the cell culture.
11. A cell culture produced by culturing the isolated cell of claim 1 , wherein the cell culture contains at least 0.1 g L −1 2,3-DHBA.
12. A supernatant of a cell culture produced by culturing the isolated cell of claim 1 , optionally wherein the supernatant is subjected to lactonization and optionally wherein lactonization is achieved through acidification to reduce the pH of the supernatant.
13. The method of claim 10 , wherein the isolated cell is a bacterial cell, a fungal cell, a plant cell, an insect cell or an animal cell.
14. The method of claim 10 , wherein the isolated cell endogenously expresses one or more of ycdW, aceA, and aceK, and wherein endogenous expression of one or more of ycdW, aceA, and aceK is increased through modification of the gene(s) and/or their promoter(s) and/or their ribosome binding sites (RBSs).
15. The method of claim 10 , wherein one or more of ycdW, aceA, aceK, pct, phaA, thil, atoB, bktB, phaB, hbd and tesB are expressed from a plasmid, or wherein one or more copies of ycdW, aceA, aceK, pct, phaA, thil, atoB, bktB, phaB, hbd and tesB is integrated into the genome of the cell.
16. The method of claim 10 , wherein one or more of the ycdW, aceA, aceK, and tesB gene(s) is an Escherichia coli gene.
17. The method of claim 10 , wherein one or more of the phaA, bktB and phaB gene(s) is a Ralstonia eutropha gene.
18. The method of claim 10 , wherein the pct gene is a Megasphaera elsdenii gene.
19. A cell culture produced by the method of claim 10 , wherein the cell culture contains at least 0.1 g L −1 2,3-DHBA.
20. A supernatant of a cell culture produced by the method of claim 10 , optionally wherein the supernatant is subjected to lactonization and optionally wherein lactonization is achieved through acidification to reduce the pH of the supernatant.
21. The isolated cell of claim 4 wherein the cell is a yeast cell.
22. The method of claim 13 , wherein the isolated cell is a yeast cell.