ENGINEERED MICROORGANISMS FOR PRODUCING ISOPROPANOL
In an embodiment, there is disclosed a recombinant microbial host cell having each of the DNA molecules encoding a polypeptide or group of polypeptides that catalyze the conversion: (i) Acetyl-CoA to Acetate and CoA (conversion 1) (ii) Acetyl-CoA to Acetoacetyl-CoA and CoA (conversion 2) (iii) Acetoacetyl-CoA and Acetate to Acetoacetate and Acetyl-CoA (conversion 3.1) (iv) Acetoacetate to Acetone and CO2 (conversion 4) (v) Acetone and NAD(P)H and H+ to Isopropanol and NAD(P)+ (conversion 5) wherein at least one DNA molecule is heterologous to the microbial host cell and wherein the microbial host cell produces isopropanol. In another embodiment, a method is disclosed for the production of isopropanol including providing a recombinant microbial host cell, the host cell of (i) with a fermentable carbon substrate in a fermentation medium under conditions whereby isopropanol is produced, and recovering the isopropanol.
1 . A recombinant microbial host cell comprising each of the DNA molecules encoding a polypeptide or group of polypeptides that catalyze the conversion:
(i) Acetyl-CoA to Acetate and CoA (conversion 1)
(ii) Acetyl-CoA to Acetoacetyl-CoA and CoA (conversion 2)
(iii) Acetoacetyl-CoA and Acetate to Acetoacetate and Acetyl-CoA (conversion 3.1)
(iv) Acetoacetate to Acetone and CO2 (conversion 4)
(v) Acetone and NAD(P)H and H+ to Isopropanol and NAD(P)+ (conversion 5)
wherein the at least one DNA molecule is heterologous to said microbial host cell and wherein said microbial host cell produces isopropanol.
2 . A host cell according to claim 1 , wherein the host cell produces isopropanol at a yield of greater than 25% of theoretical.
3 . A host cell according to claim 1 , wherein the host cell produces isopropanol at a yield of greater than 40% of theoretical.
4 . A host cell according to claim 1 , wherein the host cell produces isopropanol at a yield of greater than 50% of theoretical.
5 . A host cell according to claim 1 , wherein the host cell produces isopropanol at a yield of greater than 75% of theoretical.
6 . A host cell according to claim 1 , wherein the group of polypeptides that catalyzes conversion 1 consists of phosphate acetyltransferase and acetate kinase.
7 . A host cell according to claim 6 , wherein the phosphate acetyltransferase is encoded by the E. coli gene pta and wherein the acetate kinase is encoded by the E. coli gene ackAB.
8 . A host cell according to claim 1 , wherein the polypeptide that catalyzes conversion 2 is acetyl-CoA-acetyltransferase.
9 . A host cell according to claim 8 , wherein the acetyl-CoA acetyltransferase has an amino acid sequence of SEQ ID NO:4.
10 . A host cell according to claim 1 , wherein the polypeptide that catalyzes conversion 3.1 is acetoacetyl-CoA:acetate/butyrate coenzyme-A transferase.
11 . A host cell according to claim 10 , wherein the acetoacetyl-CoA:acetate/butyrate coenzyme-A transferase is encoded by the C. acetobutyrlicum genes ctfA and ctfB which have corresponding amino acid sequence of SEQ ID NO:5 and 6.
12 . A host cell according to claim 1 , wherein the polypeptide that catalyzes conversion 4 is acetoacetate decarboxylase.
13 . A host cell according to claim 12 , wherein the acetoacetate decarboxylase has an amino acid sequence of SEQ ID NO:7.
14 . A host cell according to claim 1 , wherein the polypeptide that catalyzes conversion 5 is a secondary alcohol dehydrogenase.
15 . A host cell according to claim 14 , wherein said secondary alcohol dehydrogenase is heterologous to said microorganism.
16 . A host cell according to claim 14 , wherein said secondary alcohol dehydrogenase is not heterologous to said microorganism.
17 . A host cell according to claim 14 , wherein said secondary alcohol dehydrogenase is from Clostridium beijerinckii , from Burkholderia spp., or from Thermoanaerobacter brockii.
18 . A host cell according to claim 17 , wherein said Clostridium beijerinckii is strain NRRL B593 or strain NESTE 225.
19 . A host cell according to claim 14 , wherein said alcohol dehydrogenase has an amino acid sequence of SEQ ID NO:8.
20 . A host cell according to claim 1 , wherein said microorganism comprises deletion or inactivation of competing acetyl-CoA consuming genes.
21 . A host cell according to claim 1 , wherein said microorganism is an E. coli strain which comprises deletion or inactivation of a gene or genes selected from the group consisting of poxB, adhE, ldhA, frdABCD, succinate dehydrogenase, malate dehydrogenase, alpha-ketoglutarate dehydrogenase and combinations thereof.
22 . A host cell according to claim 1 , wherein said microorganism is an E. coli strain which comprises deletion or inactivation of a gene or genes selected from the group consisting of poxB, ldhA, frdABCD and combinations thereof.
23 . A host cell according to claim 1 , wherein the cell is selected from the group consisting of: a bacterium, a cyanobacterium, a filamentous fungus and a yeast.
24 . A host cell according to claim 1 , wherein said microorganism is an E. coli.
25 . A host cell according to claim 1 , wherein said microorganism is a Saccharomyces cerevisiae.
26 . A host cell according to claim 1 , wherein said microorganism is a member of the genus Salmonella.
27 . A host cell according to claim 1 , wherein said microorganism is a member of the genus Bacillus.
28 . A host cell according to claim 1 , wherein said microorganism is a member of the genus Clostridium.
29 . A host cell according to claim 1 , wherein said microorganism is a member of a genus selected from the group consisting of Pichia, Hansenula, Yarrowia, Aspergillus, Kluyveromyces, Pachysolen, Rhodotorula, Zygosaccharomyces, Galactomyces, Schizosaccharomyces, Torulaspora, Debaryomyces, Williopsis, Dekkera, Kloeckera, Metschnikowia or Candida.
30 . A host cell according to claim 1 , wherein said microorganism is a member of a genus selected from the group consisting of Arthrobacter, Bacillus, Brevibacterium, Clostridium, Corynebacterium, Gluconobacter, Nocardia, Pseudomonas, Rhodococcus, Streptomyces , or Xanthomonas.
31 . A host cell according to claim 1 , wherein all of said polypeptides are heterologous to said microorganism.
32 . A recombinant microbial host cell comprising each of the DNA molecules encoding a polypeptide that catalyzes the conversion:
(i) Acetyl-CoA to Acetoacetyl-CoA and CoA (conversion 2)
(ii) Acetoacetyl-CoA+H2O→Acetoacetate+CoA (conversion 3.2)
(iii) Acetoacetate to Acetone and CO2 (conversion 4)
(iv) Acetone and NAD(P)H and H+ to Isopropanol and NAD(P)+ (conversion 5)
wherein the at least one DNA molecule is heterologous to said microbial host cell and wherein said microbial host cell produces isopropanol.
33 - 59 . (canceled)
60 . A method for the production of isopropanol comprising:
(a) providing a recombinant microbial host cell comprising each of the DNA molecules encoding a polypeptide or group of polypeptides that catalyze the conversion:
(i) Acetyl-CoA to Acetate and CoA (conversion 1)
(ii) Acetyl-CoA to Acetoacetyl-CoA and CoA (conversion 2)
(iii) Acetoacetyl-CoA and Acetate to Acetoacetate and Acetyl-CoA (conversion 3.1)
(iv) Acetoacetate to Acetone and CO2 (conversion 4)
(v) Acetone and NAD(P)H and H+ to Isopropanol and NAD(P)+ (conversion 5)
wherein the at least one DNA molecule is heterologous to said microbial host cell;
(b) contacting the host cell of (i) with a fermentable carbon substrate in a fermentation medium under conditions whereby isopropanol is produced; and
(c) recovering said isopropanol.
61 - 84 . (canceled)
85 . An isopropanol containing fermentation medium produced by a method comprising:
(a) providing recombinant microbial host cell comprising each of the DNA molecules encoding a polypeptide or group of polypeptides that catalyze the conversion:
(i) Acetyl-CoA to Acetate and CoA (conversion 1)
(ii) Acetyl-CoA to Acetoacetyl-CoA and CoA (conversion 2)
(iii) Acetoacetyl-CoA and Acetate to Acetoacetate and Acetyl-CoA (conversion 3.1)
(iv) Acetoacetate to Acetone and CO2 (conversion 4)
(v) Acetone and NAD(P)H and H+ to Isopropanol and NAD(P)+ (conversion 5)
wherein the at least one DNA molecule is heterologous to said microbial host cell;
(b) contacting the host cell of (i) with a fermentable carbon substrate in a fermentation medium under conditions whereby isopropanol is produced; and
(c) recovering said isopropanol.
86 . Isopropanol produced by a method comprising:
(a) providing recombinant microbial host cell comprising each of the DNA molecules encoding a polypeptide or group of polypeptides that catalyze the conversion:
(i) Acetyl-CoA to Acetate and CoA (conversion 1)
(ii) Acetyl-CoA to Acetoacetyl-CoA and CoA (conversion 2)
(iii) Acetoacetyl-CoA and Acetate to Acetoacetate and Acetyl-CoA (conversion 3.1)
(iv) Acetoacetate to Acetone and CO2 (conversion 4)
(v) Acetone and NAD(P)H and H+ to Isopropanol and NAD(P)+ (conversion 5)
wherein the at least one DNA molecule is heterologous to said microbial host cell;
(b) contacting the host cell of (i) with a fermentable carbon substrate in a fermentation medium under conditions whereby isopropanol is produced; and
(c) recovering said isopropanol.
87 . A method for the production of isopropanol comprising:
(a) providing a recombinant microbial host cell comprising each of the DNA molecules encoding a polypeptide that catalyzes the conversion:
(i) Acetyl-CoA to Acetoacetyl-CoA and CoA (conversion 2)
(ii) Acetoacetyl-CoA+H2O→Acetoacetate+CoA (conversion 3.2)
(iii) Acetoacetate to Acetone and CO2 (conversion 4)
(iv) Acetone and NAD(P)H and H+ to Isopropanol and NAD(P)+ (conversion 5)
wherein the at least one DNA molecule is heterologous to said microbial host cell;
(b) contacting the host cell of (i) with a fermentable carbon substrate in a fermentation medium under conditions whereby isopropanol is produced; and
(c) recovering said isopropanol.
88 - 108 . (canceled)
109 . An isopropanol containing fermentation medium produced by a method comprising:
(a) providing a recombinant microbial host cell comprising each of the DNA molecules encoding a polypeptide that catalyzes the conversion:
(i) Acetyl-CoA to Acetoacetyl-CoA and CoA (conversion 2)
(ii) Acetoacetyl-CoA+H2O→Acetoacetate+CoA (conversion 3.2)
(iii) Acetoacetate to Acetone and CO2 (conversion 4)
(iv) Acetone and NAD(P)H and H+ to Isopropanol and NAD(P)+ (conversion 5)
wherein the at least one DNA molecule is heterologous to said microbial host cell;
(b) contacting the host cell of (i) with a fermentable carbon substrate in a fermentation medium under conditions whereby isopropanol is produced; and
(c) recovering said isopropanol.
110 . Isopropanol produced by a method comprising:
(a) providing a recombinant microbial host cell comprising each of the DNA molecules encoding a polypeptide that catalyzes the conversion:
(i) Acetyl-CoA to Acetoacetyl-CoA and CoA (conversion 2)
(ii) Acetoacetyl-CoA+H2O→Acetoacetate+CoA (conversion 3.2)
(iii) Acetoacetate to Acetone and CO2 (conversion 4)
(iv) Acetone and NAD(P)H and H+ to Isopropanol and NAD(P)+ (conversion 5)
wherein the at least one DNA molecule is heterologous to said microbial host cell;
(b) contacting the host cell of (i) with a fermentable carbon substrate in a fermentation medium under conditions whereby isopropanol is produced; and
(c) recovering said isopropanol.