Recombinant microorganisms and uses therefor
Carboxydotrophic acetogenic microorganisms do not produce MEK and/or 2-butanol. They lack the biosynthesis pathways to make these products. In addition, they produce the intermediate (R,R)-2,3-butanediol whereas the production of MEK and 2-butanol requires production of the intermediate (R,S)-2,3-butanediol. Nonetheless, the production of MEK and/or 2-butanol can be accomplished using recombinant microorganisms adapted to express or overexpress key enzymes in the MEK and/or 2-butanol biosynthesis pathways. Such microorganisms, such as the carboxydotrophic acetogen Clostridium autoethanogenum , can ferment substrates comprising CO. The overall scheme involves the production of 2-butanol from (R,S)-2,3-butanediol and the conversion of (R)-acetoin to (S)-2,3-butanediol. These steps are involved in the production of both MEK and 2-butanol. Such fermentation methods offer a means of using carbon monoxide from industrial processes which would otherwise be released into the atmosphere and pollute the environment.
1. Genetically engineered, carboxydotrophic, acetogenic bacteria comprising an exogenous nucleic acid encoding a meso-2,3-butanediol dehydrogenase enzyme and an exogenous nucleic acid encoding a diol/glycerol dehydratase enzyme, wherein the bacteria are Clostridium autoethanogenum or Clostridium ljungdahlii.
2. The bacteria of claim 1 , wherein the bacteria comprise a knock-out mutation in one or both of a D-(−)2,3-butanediol dehydrogenase gene and an alcohol dehydrogenase gene.
3. The bacteria of claim 1 , wherein the bacteria comprise an exogenous nucleic acid encoding a reactivation protein of the diol/glycerol dehydratase.
4. A nucleic acid encoding a meso-2,3-butanediol dehydrogenase codon-optimized for Clostridium autoethanogenum , wherein the nucleic acid comprises SEQ ID NO: 5.
5. A nucleic acid encoding a diol/glycerol dehydratase codon-optimized for Clostridium autoethanogenum , wherein the nucleic acid comprises SEQ ID NO: 4.
6. The bacteria of claim 1 , wherein the meso-2,3-butanediol dehydrogenase is Klebsiella pneumoniae meso-2,3-butanediol dehydrogenase.
7. The bacteria of claim 1 , wherein the diol/glycerol dehydratase is Klebsiella oxytoca diol/glycerol dehydratase or Clostridium butyricum diol/glycerol dehydratase.
8. The bacteria of claim 1 , wherein the bacteria are derived from parental bacterium Clostridium autoethanogenum deposited under DSMZ Accession No. DSM23693.
9. The bacteria of claim 6 , wherein the bacteria comprise a knock-out mutation in one or both of a D-(−)2,3-butanediol dehydrogenase gene and an alcohol dehydrogenase gene.
10. The bacteria of claim 7 , wherein the bacteria comprise a knock-out mutation in one or both of a D-(−)2,3-butanediol dehydrogenase gene and an alcohol dehydrogenase gene.
11. The bacteria of claim 6 , wherein the bacteria comprise an exogenous nucleic acid encoding a reactivation protein of the diol/glycerol dehydratase.
12. The bacteria of claim 7 , wherein the bacteria comprise an exogenous nucleic acid encoding a reactivation protein of the diol/glycerol dehydratase.
13. The bacteria of claim 1 , wherein the nucleic acid encoding the meso-2,3-butanediol dehydrogenase comprises SEQ ID NO: 5.
14. The bacteria of claim 1 , wherein the nucleic acid encoding the diol/glycerol dehydratase comprises SEQ ID NO: 4.
15. The bacteria of claim 1 , wherein the bacteria produce one or both of methyl ethyl ketone and 2-butanol.