Microbial fermentation for the production of terpenes

Abstract

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.