Recombinant microorganism and methods of production thereof

Abstract

A novel genetically modified microorganisms capable of using CO to produce 1-butanol and/or a precursor thereof, novel methyltransferases and nucleic acids encoding same, methods for producing genetically modified microorganisms using said novel methyltransferases, and methods of producing 1-butanol and/or a precursor thereof by microbial fermentation.

1 .- 65 . (canceled) 
     
     
         66 . An acetogenic recombinant microorganism which one or a combination of produces 1-butanol and a precursor thereof as the main fermentation product. 
     
     
         67 . An acetogenic recombinant microorganism as claimed in  claim 66 , wherein the microorganism is capable of producing one or both 1-butanol and a precursor thereof by fermentation from a substrate comprising CO at a concentration of greater than approximately 1 mM or 0.075 grams per liter of fermentation broth. 
     
     
         68 . An acetogenic recombinant microorganism as claimed in  claim 66 , wherein the microorganism further comprises exogenous nucleic acids adapted to express one or more enzymes in the butanol biosynthesis pathway. 
     
     
         69 . An acetogenic recombinant microorganism as claimed in  claim 68 , wherein the microorganism further comprises one or more exogenous nucleic acids adapted to express one or more of enzymes chosen from the group consisting of:
 Thiolase   3-hydroxybutyryl-CoA dehydrogenase   Crotonase/crotonyl-CoA hydratase   Butyryl-CoA dehydrogenase   Electron transport flavoprotein   Phosphotransbutyrylase;   butyrate kinase;   ferredoxin dependent aldehyde oxidoreductase;   butyraldehyde dehydrogenase;   butanol dehydrogenase; and,   a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase.   
     
     
         70 . An acetogenic recombinant microorganism as claimed in  claim 69 , wherein the microorganism comprises one or more exogenous nucleic acids encoding each of Thiolase, 3-hydroxybutyryl-CoA dehydrogenase, Crotonase, and Butyryl-CoA dehydrogenase. 
     
     
         71 . An acetogenic recombinant microorganism as claimed in  claim 70 , wherein the microorganism further comprises one or more exogenous nucleic acids encoding at least one electron transport flavoprotein chosen from the group consisting of Electron Transfer Flavoprotein A and Electron Transfer Flavoprotein B. 
     
     
         72 . An acetogenic recombinant microorganism as claimed in  claim 70 , wherein the microorganism further comprises one or more exogenous nucleic acids encoding one or more of butyraldehyde dehydrogenase, butanol dehydrogenase and a bifunctional butyraldehyde dehydrogenase/butanol dehydrogenase. 
     
     
         73 . An acetogenic recombinant microorganism as claimed in  claim 70 , wherein the microorganism further comprises one or more exogenous nucleic acids encoding one or more of Phosphotransbutyrylase, butyrate kinase, ferredoxin dependent aldehyde oxidoreductase and butanol dehydrogenase. 
     
     
         74 . An acetogenic recombinant microorganism as claimed in  claim 66 , wherein the microorganism is selected from the group comprising  Clostridium autoethanogenum, Clostridium ljungdahlii Clostridium ragsdalei, Clostridium carboxidivorans, Clostridium drakei, Clostridium scatalogenes, Clostridium aceticum, Clostridium formicoaceticum, Butyribacterium limosum, Acetobacterium woodii, Blautia producta, Eubacterium limosum, Moorella thermoacetica, Moorella thermautotrophica, Oxobacter pfennigii , and  Thermoanaerobacter kiuvi.    
     
     
         75 . An acetogenic recombinant microorganism as claimed in  claim 74 , wherein the microorganism is  Clostridium autoethanogenum  DSM23693. 
     
     
         76 . A method for the production of one or both of 1-butanol and a precursor thereof by microbial fermentation comprising fermenting a substrate using a recombinant microorganism as claimed in  claim 66 . 
     
     
         77 . A method as claimed in  claim 76 , wherein one or both of 1-butanol and a precursor thereof is the main fermentation product. 
     
     
         78 . A method as claimed in  claim 76 , wherein at least one or both 1-butanol and a precursor thereof is produced in a yield of from approximately 0.075 grams per liter of fermentation broth to approximately 20 grams per liter. 
     
     
         79 . A method as claimed in  claim 78 , wherein the yield is from approximately 0.15 grams per liter to approximately 1.54 grams per liter. 
     
     
         80 . A method as claimed in  claim 78 , wherein the yield is approximately 10 grams per liter, approximately 5 grams per liter, or approximately 2 grams per liter. 
     
     
         81 . A method as claimed in  claim 78 , wherein the yield of 1-butanol is up to the limit at which butanol becomes toxic. 
     
     
         82 . A method as claimed in  claim 76 , wherein the substrate comprises CO. 
     
     
         83 . A method as claimed in  claim 82 , wherein the substrate comprises at least about 20% to about 100% CO by volume. 
     
     
         84 . A recombinant microorganism having the defining characteristics of the microorganism deposited at the DSMZ under the accession number DSM24138. 
     
     
         85 . (canceled) 
     
     
         86 . The method of  claim 76 , wherein the substrate comprises an industrial waste gas.