MICROBIAL FERMENTATION FOR THE PRODUCTION OF TERPENES
The invention provides a method for producing a terpene or a precursor thereof by microbial fermentation. Typically, the method involves culturing a recombinant bacterium in the presence of a gaseous substrate whereby the bacterium produces a terpene or a precursor thereof, such as mevalonic acid, isopentenyl pyrophosphate, dimethylallyl pyrophosphate, isoprene, geranyl pyrophosphate, farnesyl pyrophosphate, and/or farnesene. The bacterium may comprise one or more exogenous enzymes, such as enzymes in mevalonate, DXS, or terpene biosynthesis pathways.
1 . A method for producing at least one terpene or a precursor thereof by microbial fermentation comprising providing a gaseous substrate comprising CO to a culture of a recombinant Clostridium bacterium and fermenting the culture to produce at least one terpene or a precursor thereof from the gaseous substrate.
2 . The method of claim 1 , wherein the terpene or precursor thereof is selected from the group consisting of mevalonic acid, isopentenyl pyrophosphate (IPP), dimethylallyl pyrophosphate (DMAPP), isoprene, geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), and farnesene.
3 . The method of claim 1 , wherein the bacterium comprises at least one exogenous nucleic acid encoding one or more of:
an enzyme in a mevalonate pathway selected from the group consisting of thiolase (EC 2.3.1.9), HMG-CoA synthase (EC 2.3.3.10), HMG-CoA reductase (EC 1.1.1.88), mevalonate kinase (EC 2.7.1.36), phosphomevalonate kinase (EC 2.7.4.2), and mevalonate diphosphate decarboxylase (EC 4.1.1.33);
an enzyme in a DXS pathway selected from the group consisting of 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC 2.2.1.7), 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC 1.1.1.267), 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC 2.7.7.60), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC 2.7.1.148), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase IspF (EC 4.6.1.12), 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC 1.17.7.1), and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC 1.17.1.2); or
an enzyme in a terpene biosynthesis pathway selected from the group consisting of geranyltranstransferase Fps (EC 2.5.1.10), heptaprenyl diphosphate synthase (EC 2.5.1.10), octaprenyl-diphosphate synthase (EC 2.5.1.90), isoprene synthase (EC 4.2.3.27), isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2), and farnesene synthase (EC 4.2.3.46/EC 4.2.3.47).
4 . The method of claim 3 , wherein the bacterium comprises exogenous nucleic acids encoding thiolase (EC 2.3.1.9), HMG-CoA synthase (EC 2.3.3.10), and HMG-CoA reductase (EC 1.1.1.88).
5 . The method of claim 3 , wherein the bacterium comprises exogenous nucleic acids encoding thiolase (EC 2.3.1.9), HMG-CoA synthase (EC 2.3.3.10), HMG-CoA reductase (EC 1.1.1.88), mevalonate kinase (EC 2.7.1.36), phosphomevalonate kinase (EC 2.7.4.2), and mevalonate diphosphate decarboxylase (EC 4.1.1.33).
6 . The method of claim 3 , wherein the bacterium comprises exogenous nucleic acids encoding isoprene synthase (EC 4.2.3.27), isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2), and 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC 2.2.1.7).
7 . The method of claim 3 , wherein the bacterium comprises an exogenous nucleic acid encoding isoprene synthase (EC 4.2.3.27) and produces isoprene.
8 . The method of claim 3 , wherein the bacterium comprises an exogenous nucleic acid encoding isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2) and produces isopentenyl pyrophosphate (IPP).
9 . The method of claim 3 , wherein the exogenous nucleic acid is codon optimized for expression in the bacterium.
10 . The method of claim 1 , wherein the bacterium is derived from a parental bacterium selected from the group consisting of Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium aceticum, Clostridium formicoaceticum, and Clostridium magnum.
11 . The method of claim 10 , wherein the parental bacterium is Clostridium autoethanogenum deposited under DSMZ accession number DSM23693.
12 . The method of claim 1 , comprising fermenting the culture under anaerobic conditions.
13 . The method of claim 1 , wherein the gaseous substrate comprises at least 20% CO.
14 . The method of claim 13 , wherein the gaseous substrate comprises at least 40% CO.
15 . The method of claim 3 , wherein the gaseous substrate comprises at least 20% CO.
16 . The method of claim 15 , wherein the gaseous substrate comprises at least 40% CO.
17 . The method of claim 1 , wherein the gaseous substrate further comprises at least one of CO 2 or H 2 .
18 . The method of claim 1 , wherein the gaseous substrate is derived from an industrial process selected from the group consisting of ferrous metal products manufacturing, non-ferrous products manufacturing, petroleum refining, coal gasification, electric power production, carbon black production, ammonia production, methanol production, and coke manufacturing.
19 . The method of claim 1 , wherein the gaseous substrate is syngas.