METABOLIC ENGINEERING OF YEASTS FOR THE PRODUCTION OF 1-BUTANOL
The present invention provides genetically modified yeast cells, and methods of using those yeast cells, to produce 1-butanol. The yeast cell can be selected from the genera Saccharomyces, Candida, Pichia, Kluyveromyces, Issatchenkia, Yarrowia, Rhodotorula, Hansenula, Schizochytrium , or Thraustochytrium . The yeast cell of the invention overexpresses at least one enzyme that catalyzes one or more butanoate pathways selected from the group consisting of pyruvate to acetyl-CoA, acetyl-CoA to acetoacetyl-CoA, acetoacetyl-CoA to 3-hydroxybutanoyl-CoA, 3-hydroxybutanoyl-CoA to crotonoyl-CoA, crotonoyl-CoA to butyryl-CoA to butyraldehyde, and butyraldehyde to 1-butanol. Some embodiments overexpress enzymes that are endogenous to the bacterium Clostridium acetobutylicum . The genetically modified yeast cell can further be subjected to other desired genetic changes, such as deletion or disruption of one or more native PDC genes.
1 . A genetically modified yeast cell having an active metabolic pathway from pyruvate to 1-butanol, wherein the active metabolic pathway from pyruvate to 1-butanol includes the reactions a) pyruvate to acetyl-CoA, b) acetyl-CoA to acetoacetyl-CoA, c) acetoacetyl-CoA to 3-hydroxybutanoyl-CoA, d) 3-hydroxybutanoyl-CoA to crotonoyl-CoA, d) crotonoyl-CoA to butyryl-CoA, f) butyryl-CoA to butyraldehyde, and g) butyraldehyde to 1-butanol.
2 - 4 . (canceled)
5 . The genetically modified yeast cell of claim 1 , which contains at least one active, exogenous gene that produces an enzyme that catalyzes the reaction of pyruvate to acetyl-CoA.
6 . The genetically modified yeast cell of claim 5 , wherein the exogenous gene is a pyruvate formate lyase (pfl) gene or a pyruvate dehydrogenase (pdh) gene.
7 - 9 . (canceled)
10 . The genetically modified yeast cell of claim 5 , which contains at least one active, exogenous gene that produces an enzyme that catalyzes the reaction of acetyl-CoA to acetoacetyl-CoA.
11 . The genetically modified yeast cell of claim 10 , wherein the exogenous gene that produces an enzyme that catalyzes the reaction of acetyl-CoA to acetoacetyl-CoA is an acetyl-CoA C-acetyltransferase gene.
12 . (canceled)
13 . The genetically modified yeast cell of claim 5 , which contains at least one active, exogenous gene that produces an enzyme that catalyzes the reaction of acetoacetyl-CoA to 3-hydroxybutanoyl-CoA.
14 . The genetically modified yeast cell of claim 13 , wherein the exogenous gene that produces an enzyme that catalyzes the reaction of acetoacetyl-CoA to 3-hydroxybutanoyl-CoA is an acetyl-3-hydroxylbutyryl-COA dehydrogenase (hbd) gene.
15 . (canceled)
16 . The genetically modified yeast cell claim 5 , which contains at least one active, exogenous gene that produces an enzyme that catalyzes the reaction of 3-hydroxybutanoyl-CoA to crotonoyl-CoA.
17 . The genetically modified yeast cell of claim 16 , wherein the exogenous gene that produces an enzyme that catalyzes the reaction of 3-hydroxybutanoyl-CoA to crotonoyl-CoA is a 3-hydroxybutyryl-CoA dehydratase gene.
18 . (canceled)
19 . The genetically modified yeast cell of claim 5 , which contains at least one active, exogenous gene that produces an enzyme that catalyzes the reaction of crotonoyl-CoA to butyryl-CoA.
20 . The genetically modified yeast cell of claim 19 , wherein the exogenous gene that produces an enzyme that catalyzes the reaction of crotonoyl-CoA to butyryl-CoA is a 3-butyryl-CoA dehydrogenase (bcd) gene.
21 . (canceled)
22 . The genetically modified yeast cell of claim 5 , which contains at least one active, exogenous gene that produces an enzyme that catalyzes the reaction of butyryl-CoA to butyraldehyde.
23 . The genetically modified yeast cell of claim 22 , wherein the exogenous gene that produces an enzyme that catalyzes the reaction of butyryl-CoA to butyraldehyde is a butyryl-CoA dehydrogenase gene.
24 . (canceled)
25 . The genetically modified yeast cell of claim 5 , which contains at least one active, exogenous gene that produces an enzyme that catalyzes the reaction of butyraldehyde to 1-butanol.
26 . The genetically modified yeast cell of claim 25 , wherein the exogenous gene that produces an enzyme that catalyzes the reaction of butyraldehyde to 1-butanol is a 1-butanol dehydrogenase gene.
27 . (canceled)
28 . The genetically modified yeast cell of claim 5 , which overexpresses at least one native gene that catalyzes at least one of reactions a)-g).
29 . The yeast cell of claim 3 , which further has deletion or disruption of a native pathway from pyruvate to ethanol.
30 . (canceled)
31 . The yeast cell of claim 29 , which further has deletion or disruption of one or more native alcohol dehydrogenase genes.
32 . The yeast cell of claim 3 , wherein the yeast is of the genera Saccharomyces, Candida, Pichia, Kluyveromyces, Issatchenkia, Yarrowia, Rhodotorula, Hansenula, Schizochytrium , or Thraustochytrium.
33 . The yeast cell of claim 32 , wherein the yeast species is selected from the group consisting of Issatchenkia orientalis, Hansenula ofunaensis, H. polymorphs, H. anomala, Schizochytrium limacinum, Thraustochytrium striatum, T. roseum , and T. aureum.
34 . The yeast cell of claim 33 , wherein the yeast species is Issatchenkia orientalis.
35 . A process for making 1-butanol, comprising culturing a yeast cell of claim 3 in the presence of a fermentable carbon source under conditions such that the yeast cells metabolize the carbon source and convert at least a portion of the carbon source to 1-butanol.
36 . The process of claim 35 , wherein ethanol is produced in a yield of 10% or less.
37 . (canceled)
38 . The process of claim 36 , wherein ethanol is not detectably produced.
39 . The process of any claim 35 , wherein at least one carbon source is selected from the group consisting of glucose, xylose, arabinose, sucrose, fructose, cellulose, glucose oligomers, and glycerol.
40 . (canceled)
41 . The process of claim 35 , wherein the final yield of 1-butanol on the carbon source is at least 50% of theoretical.
42 . (canceled)
43 . The process of claim 41 , wherein the broth titer of 1-butanol reaches at least 50 g/L.
44 . (canceled)