Compositions and methods for recombinant biosynthesis of propane
Provided are genetically engineered microorganism that catalyze the synthesis of propane and/or butanol from a suitable substrate such as glucose. Also provided are methods of engineering said genetically engineered microorganism and methods of producing propane and/or butanol using the genetically engineered microorganism.
1. A genetically engineered bacterium, which expresses both an enzyme for butyraldehyde synthesis and a heterologous polypeptide having aldehyde deformylating oxygenase activity and produces propane from butyraldehyde as a precursor independent of fatty acid synthesis pathways.
2. The genetically engineered bacterium of claim 1 , which further produces butanol.
3. The genetically engineered bacterium of claim 1 , wherein propane is produced independent of aldehyde-alcohol dehydrogenase (AdhE2).
4. The genetically engineered bacterium of claim 1 , comprising a deletion of aldehyde reductase (Δahr) enzyme, alcohol dehydrogenase (ΔyqhD) enzyme or a combination thereof.
5. The genetically engineered bacterium of claim 4 , wherein the bacterium has been transformed with a first plasmid vector comprising a first nucleotide sequence encoding a polypeptide having acetyl-CoA acetyltransferase (AtoB) activity, a second nucleotide sequence encoding a polypeptide having 3-hydroxybutyrl-CoA dehydrogenase (Hbd) activity, a third nucleotide sequence encoding a polypeptide having 3-hydroxybutyryl-CoA dehydratase (Crt) activity and a fourth nucleotide sequence encoding a polypeptide having trans-2-enoyl-CoA reductase (Ter) activity.
6. The genetically engineered bacterium of claim 5 , wherein the bacterium has been co-transformed with a second plasmid vector selected from the group consisting of:
a second plasmid vector comprising a first nucleotide sequence encoding a polypeptide having acyl-CoA thioester hydrolase (YciA) activity, a second nucleotide sequence encoding a polypeptide having the activity of a maturation factor for phosphopantetheinyl transferase (Sfp) and a third nucleotide sequence encoding a polypeptide having carboxylic acid reductase (CAR) activity; and
a second plasmid vector comprising a nucleotide sequence encoding a polypeptide having aldehyde-alcohol dehydrogenase (AdhE2) activity.
7. The genetically engineered bacterium of claim 1 , wherein the bacterium has been transformed with a first plasmid vector comprising a first nucleotide sequence encoding a polypeptide having acetyl-CoA acetyltransferase (AtoB) activity, a second nucleotide sequence encoding a polypeptide having 3-hydroxybutyrl-CoA dehydrogenase (Hbd) activity, a third nucleotide sequence encoding a polypeptide having 3-hydroxybutyryl-CoA dehydratase (Crt) activity and a fourth nucleotide sequence encoding a polypeptide having trans-2-enoyl-CoA reductase (Ter) activity.
8. The genetically engineered bacterium of claim 7 , wherein the bacterium has been co-transformed with a second plasmid vector selected from the group consisting of:
a second plasmid vector comprising a nucleotide sequence encoding a polypeptide having aldehyde-alcohol dehydrogenase (AdhE2) activity; and
a second plasmid vector comprising a nucleotide sequence encoding a polypeptide having acyl-CoA thioester hydrolase (YciA) activity, a second nucleotide sequence encoding a polypeptide having the activity of a maturation factor for phosphopantetheinyl transferase (Sfp) and a third nucleotide sequence encoding a polypeptide having carboxylic acid reductase (CAR) activity.
9. The genetically engineered bacterium of claim 1 , wherein the bacterium has been transformed with a first plasmid vector comprising a first nucleotide sequence encoding a polypeptide having acetoacetyl-CoA synthase (NphT7) activity, a second nucleotide sequence encoding a polypeptide having 3-hydroxybutyrl-CoA dehydrogenase (Hbd) activity, a third nucleotide sequence encoding a polypeptide having 3-hydroxybutyryl-CoA dehydratase (Crt) activity and a fourth nucleotide sequence encoding a polypeptide having trans-2-enoyl-CoA reductase (Ter) activity.
10. The genetically engineered bacterium of claim 9 , wherein the bacterium has been transformed with a second plasmid vector selected from the group consisting of:
a second plasmid vector comprising a nucleotide sequence encoding a polypeptide having aldehyde-alcohol dehydrogenase (AdhE2) activity; and
a second plasmid vector comprising a nucleotide sequence encoding a polypeptide having acyl-CoA thioester hydrolase (YciA) activity, a second nucleotide sequence encoding a polypeptide having the activity of a maturation factor for phosphopantetheinyl transferase (Sfp) and a third nucleotide sequence encoding a polypeptide having carboxylic acid reductase (CAR) activity.
11. The genetically engineered bacterium of claim 9 , wherein the bacterium has been co-transformed with a second plasmid vector selected from the group consisting of:
a second plasmid vector comprising a first nucleotide sequence encoding a polypeptide having acyl-CoA thioester hydrolase (YciA) activity, a second nucleotide sequence encoding a polypeptide having activity of a maturation factor for phosphopantetheinyl transferase (Sfp) and a third nucleotide sequence encoding a polypeptide having carboxylic acid reductase (Car) activity; and
a second plasmid vector comprising a nucleotide sequence encoding a polypeptide having aldehyde-alcohol dehydrogenase (AdhE2) activity.
12. The genetically engineered bacterium of claim 11 wherein the bacterium has been co-transformed with a third plasmid vector comprising a nucleotide sequence encoding a polypeptide having aldehyde deformylating oxygenase (ADO) activity.
13. The genetically engineered bacterium of claim 11 , wherein the bacterium has been co-transformed with a third plasmid vector comprising a nucleotide sequence encoding a polypeptide having aldehyde deformylating oxygenase activity wherein alanine at position 134 is substituted with phenylalanine (ADOA 134 F).
14. The genetically engineered bacterium of claim 12 , wherein the bacterium further comprises a polynucleotide encoding one or more ferredoxins so as to increase supply of electrons.
15. The genetically engineered bacterium of claim 14 , the ferredoxin is PetF (ssl0020).
16. The genetically engineered bacterium of claim 1 which further expresses:
(i) a heterologous polypeptide having acetyl-CoA acetyltransferase activity;
(ii) a heterologous polypeptide having 3-hydroxybutyrl-CoA dehydrogenase activity;
(iii) a heterologous polypeptide having 3-hydroxybutyryl-CoA dehydratase activity;
(iv) a heterologous polypeptide having trans-2-enoyl-CoA reductase activity; and
(v) a heterologous polypeptide having aldehyde-alcohol dehydrogenase activity.
17. The genetically engineered bacterium of claim 1 which further expresses:
(i) a heterologous polypeptide having acetyl-CoA acetyltransferase activity;
(ii) a heterologous polypeptide having 3-hydroxybutyrl-CoA dehydrogenase activity;
(iii) a heterologous polypeptide having 3-hydroxybutyryl-CoA dehydratase activity;
(iv) a heterologous polypeptide having trans-2-enoyl-CoA reductase activity;
(v) a heterologous polypeptide having acyl-CoA thioester hydrolase activity;
(vi) a heterologous polypeptide having the activity of a maturation factor for phosphopantetheinyl transferase; and
(vii) a heterologous polypeptide having carboxylic acid reductase activity.
18. The genetically engineered bacterium of claim 1 , wherein the enzyme for butyraldehyde synthesis is endogenously expressed.
19. A method for producing propane comprising:
providing the genetically engineered bacterium of claim 1 ; and
culturing the bacterium, so as to produce propane.
20. A method for producing butanol comprising:
providing the genetically engineered bacterium of claim 2 ; and
culturing the bacterium, so as to produce butanol.