COMPOSITIONS AND METHODS FOR BIOLOGICAL PRODUCTION OF BUTANE-BASED COMPOUNDS FROM A C1 SUBSTRATE
The present disclosure relates to biosynthetic methods for producing propylene from C 1 substrates (e.g., methane, methanol, carbon monoxide, syngas) and to genetically engineered organisms having propylene biosynthesis capability, as well as engineered organisms having a butyrate/butanol-producing pathway.
1 . A modified methanotrophic bacterium, wherein the modified methanotrophic bacterium comprises:
(a) a heterologous nucleic acid molecule encoding a crotonase, wherein the modified methanotrophic bacterium produces crotonyl-CoA from a C 1 substrate comprising methane; and
(b) a parent methanotrophic bacterium selected from Methylosinus trichosporium OB3b, Methylococcus capsulatus Bath, Methylomonas methanica 16a, Methylosinus trichosporium, Methylosinus sporium, Methylocystis parvus, Methylomonas methanica, Methylomonas albus, Methylobacter capsulatus, Methylobacterium organophilum, Methylomonas sp AJ-3670, Methylocella silvestris, Methylomicrobium alcaliphilum, or a growth variant thereof; and
wherein the modified methanotrophic bacterium produces n-butanol, butyraldehyde, or 1,4-butanediol.
2 . The modified methanotrophic bacterium of claim 1 , wherein the methanotrophic bacterium is an obligate methanotrophic bacterium.
3 . The modified methanotrophic bacterium of claim 1 , wherein the heterologous nucleic acid molecule further encodes a butyryl-CoA dehydrogenase, whereby a butyryl-CoA intermediate is produced and the modified methanotrophic bacterium produces n-butanol, butyraldehyde, or both.
4 . The modified methanotrophic bacterium of claim 3 , wherein the heterologous nucleic acid molecule further encodes an aldehyde dehydrogenase, and the modified methanotrophic bacterium produces n-butanol, butyraldehyde, or both.
5 . The modified methanotrophic bacterium of claim 1 , wherein the heterologous nucleic acid molecule further encodes a crotonyl-CoA hydratase, whereby a hydroxybutyryl-CoA intermediate is produced and the modified methanotrophic bacterium produces 1,4-butanediol.
6 . The modified methanotrophic bacterium of claim 4 , wherein the modified methanotrophic bacterium does not have a functional PHB synthase or a substantial amount of functional PHB synthase.
7 . The modified methanotrophic bacterium of claim 5 , wherein the modified methanotrophic bacterium does not have a functional PHB synthase or a substantial amount of functional PHB synthase.
8 . The modified methanotrophic bacterium of claim 6 , wherein the PHB synthase is encoded by phaC or phbC.
9 . The modified methanotrophic bacterium of claim 7 , wherein the PHB synthase is encoded by phaC or phbC.
10 . The modified methanotrophic bacterium of claim 4 , wherein the modified methanotrophic bacterium has a functional β-ketothiolase activity and a functional acetoacetyl coenzyme A reductase activity.
11 . The modified methanotrophic bacterium of claim 5 , wherein the modified methanotrophic bacterium has a functional β-ketothiolase activity and a functional acetoacetyl coenzyme A reductase activity.
12 . The modified methanotrophic bacterium of claim 10 , wherein the β-ketothiolase is encoded by phaA or phbA.
13 . The modified methanotrophic bacterium of claim 10 , wherein the acetoacetyl coenzyme A reductase is encoded by phaB or phbB.
14 . The modified methanotrophic bacterium of claim 11 , wherein the β-ketothiolase is encoded by phaA or phbA.
15 . The modified methanotrophic bacterium of claim 11 , wherein the acetoacetyl coenzyme A reductase is encoded by phaB or phbB.
16 . The modified methanotrophic bacterium of claim 6 , wherein the modified methanotrophic bacterium does not produce a substantial amount of polyhydroxybutyrate.
17 . The modified methanotrophic bacterium of claim 7 , wherein the modified methanotrophic bacterium does not produce a substantial amount of polyhydroxybutyrate.
18 . The modified methanotrophic bacterium of claim 1 , wherein the C 1 substrate comprising methane comprises natural gas.
19 . A method for producing n-butanol and/or butyraldehyde, comprising culturing the modified methanotrophic bacterium of claim 1 on a C1 substrate comprising methane under conditions sufficient to produce n-butanol and/or butyraldehyde.
20 . A method for producing 1,4-butanediol, comprising culturing the modified methanotrophic bacterium of claim 1 on a C1 substrate comprising methane under conditions sufficient to produce 1,4-butanediol.