Recombinant microorganisms and uses therefor

Abstract

Terpenes are valuable commercial products used in a diverse number of industries. Terpenes may be produced from petrochemical sources and from terpene feed-stocks, such as turpentine. However, these production methods are expensive, unsustainable and often cause environmental problems including contributing to climate change. Microbial fermentation provides an alternative option for the production of terpenes. One or more terpenes and/or precursors can be produced by microbial fermentation of a substrate comprising CO. Recombinant microorganisms may be used in such methods. Carboxydotrophic, acetogenic, recombinant microorganisms can be used in such methods. The recombinant microorgnsims may contain exogenous mevalonate (MVA) pathway enzymes and/or DXS pathway enzymes, for example.

We claim: 
     
         1 . Isolated, genetically engineered, carboxydotrophic, acetogenic bacteria which comprise an exogenous nucleic acid encoding an enzyme in a mevalonate pathway or in a DXS pathway or in a terpene biosynthesis pathway, whereby the bacteria express the enzyme, said enzyme selected from the group consisting of:
 a) thiolase (EC 2.3.1.9);   b) HMG-CoA synthase (EC 2.3.3.10);   c) HMG-CoA reductase (EC 1.1.1.88);   d) Mevalonate kinase (EC 2.7.1.36);   e) Phosphomevalonate kinase (EC 2.7.4.2);   f) Mevalonate Diphosphate decarboxylase (EC 4.1.1.33); 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC:2.2.1.7);   g) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC:1.1.1.267);   h) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC:2.7.7.60);   i) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC:2.7.1.148);   j) 2-C-methyl-D-erythritol 2;4-cyclodiphosphate synthase IspF (EC:4.6.1.12);   k) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC:1.17.7.1);   l) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC:1.17.1.2); geranyltranstransferase Fps (EC:2.5.1.10);   m) heptaprenyl diphosphate synthase (EC:2.5.1.10);   n) octaprenyl-diphosphate synthase (EC:2.5.1.90);   o) isoprene synthase (EC 4.2.3.27);   p) isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2); and   q) farnesene synthase (EC 4.2.3.46/EC 4.2.3.47).   
     
     
         2 . The bacteria of  claim 1  wherein in the absence of said nucleic acid, the bacteria do not express the enzyme. 
     
     
         3 . The bacteria of  claim 1  which express the enzyme under anaerobic conditions. 
     
     
         4 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 1  where the bacteria express the isoprene synthase enzyme and the bacteria are able to convert dimethylallyldiphosphate to isoprene. 
     
     
         5 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 4  wherein the isoprene synthase is a  Populus tremuloides  enzyme. 
     
     
         6 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 4  wherein the nucleic acid is codon optimized. 
     
     
         7 . The isolated, genetically engineered, carboxydrotrophic, acetogenic bacteria of  claim 4  wherein expression of the isoprene synthase is under the transcriptional control of a promoter for a pyruvate: ferredoxin oxidoreductase gene from  Clostridium autoethanogenum.    
     
     
         8 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 1  where the bacteria express the isopentyldiphosphate delta isomerase enzyme and the bacteria are able to convert dimethylallyldiphosphate to isopentyldiphosphate. 
     
     
         9 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 8  wherein the nucleic acid encodes a  Clostridium beijerinckii  isopentyldiphosphate delta isomerase. 
     
     
         10 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 8  wherein the nucleic acid is under the transcriptional control of a promoter for a pyruvate: ferredoxin oxidoreductase gene from  Clostridium autoethanogenum.    
     
     
         11 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 8  wherein the nucleic acid is under the transcriptional control of a promoter for a pyruvate: ferredoxin oxidoreductase gene from  Clostridium autoethanogenum  and downstream of a second nucleic acid encoding an isoprene synthase. 
     
     
         12 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 1  where the bacteria has a copy number of greater than 1 per genome of a nucleic acid encoding a1-deoxy-D-xylulose 5-phosphate synthase (DXS) enzyme. 
     
     
         13 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 12  further comprising a nucleic acid encoding an isoprene synthase. 
     
     
         14 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 12  further comprising a nucleic acid encoding an isopentyldiphosphate delta isomerase. 
     
     
         15 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 12  further comprising a nucleic acid encoding an isopentyldiphosphate delta isomerase and a nucleic acid encoding an isoprene synthase. 
     
     
         16 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 1  where the bacteria encodes a phosphomevalonate kinase (PMK enzyme, wherein the enzyme is not native to the bacteria. 
     
     
         17 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 16  wherein the enzymes are  Staphylococcus aureus  enzymes. 
     
     
         18 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 16  wherein the enzyme is expressed under the control of one or more  C. autoethanogenum  promoters. 
     
     
         19 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 16  further comprising a nucleic acid encoding thiolase (thlA/vraB), a nucleic acid encoding a HMG-CoA synthase (HMGS), and a nucleic acid encoding an HMG-CoA reductase (HMGR). 
     
     
         20 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 19  wherein the thiolase is  Clostridium acetobutylicum  thiolase. 
     
     
         21 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 16  further comprising a nucleic acid encoding a mevalonate disphosphate decarboxylase (PMD). 
     
     
         22 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 1  where the bacteria encodes alpha-farnesene synthase. 
     
     
         23 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 22  wherein the nucleic acid is codon optimized for expression in  C. autoethanogenum.    
     
     
         24 . The bacteria of  claim 22  wherein the alpha-farnesene synthase is a  Malus×domestica  alpha-farnesene synthase. 
     
     
         25 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 22  further comprising a nucleic acid segment encoding geranyltranstransferase. 
     
     
         26 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 25  wherein the gernayltranstransferase is an  E. coli  geranyltranstransferase. 
     
     
         27 . The isolated, genetically engineered, carboxydotrophic, acetogenic bacteria of  claim 1 , which is selected from the group consisting of  Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatologenes, Clostridium aceticum, Clostridium formicoaceticum, Clostridium magnum, Butyribacterium methylotrophicum, Acetobacterium woodii, Alkalibaculum bacchii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Sporomusa ovata, Sporomusa silvacetica, Sporomusa sphaeroides, Oxobacter pfennigii , and  Thermoanaerobacter kiuvi.    
     
     
         28 . A plasmid which can replicate in a carboxydotrophic, acetogenic bacteria which comprises a nucleic acid encoding an enzyme in a mevalonate pathway or in a DXS pathway or in a terpene biosynthesis pathway, whereby when the plasmid is in said bacteria the enzyme is expressed by said bacteria, said enzyme selected from the group consisting of:
 thiolase (EC 2.3.1.9);   a) HMG-CoA synthase (EC 2.3.3.10);   b) HMG-CoA reductase (EC 1.1.1.88);   c) Mevalonate kinase (EC 2.7.1.36);   d) Phosphomevalonate kinase (EC 2.7.4.2);   e) Mevalonate Diphosphate decarboxylase (EC 4.1.1.33); 1-deoxy-D-xylulose-5-phosphate synthase DXS (EC:2.2.1.7);   f) 1-deoxy-D-xylulose 5-phosphate reductoisomerase DXR (EC:1.1.1.267);   g) 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase IspD (EC:2.7.7.60);   h) 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase IspE (EC:2.7.1.148);   i) 2-C-methyl-D-erythritol 2;4-cyclodiphosphate synthase IspF (EC:4.6.1.12);   j) 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase IspG (EC:1.17.7.1);   k) 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC:1.17.1.2); geranyltranstransferase Fps (EC:2.5.1.10);   l) heptaprenyl diphosphate synthase (EC:2.5.1.10);   m) octaprenyl-diphosphate synthase (EC:2.5.1.90);   n) isoprene synthase (EC 4.2.3.27);   o) isopentenyl-diphosphate delta-isomerase (EC 5.3.3.2); and   p) farnesene synthase (EC 4.2.3.46/EC 4.2.3.47).