Metabolic engineering of clostridium tyrobutyricum for butanol production
View Patent ↗This invention relates to compositions, systems, and methods for producing biofuels, such as butanol, and related compounds. More specifically, provided are methods of making recombinant microorganizms having non-naturally occurring metabolic pathways for the production of biofuels, and methods of producing biofuels using such organizms. Also provided are metabolically engineered microorganizms capable of producing butanol from a substrate.
1. A recombinant microorganism capable of producing butanol, which is constructed by transferring a heterologous DNA comprising the base sequences of SEQ ID No: 9 and SEQ ID No: 10 into a non-solventogenic host organism.
2. The recombinant microorganism of claim 1 , wherein the host organism is Clostridium tyrobutyricum.
3. The recombinant microorganism of claim 2 , wherein the host organism has been transformed with vector pMAD72.
4. The recombinant microorganism of claim 2 , wherein the host organism has been transformed with vector pMAD22.
5. The recombinant microorganism of claim 1 , wherein the recombinant microorganism is capable of producing butanol from glucose.
6. The recombinant microorganism of claim 4 , wherein the recombinant microorganism is capable of producing butanol at a titer greater than 10 g/L and a yield greater than 0.3 g/g substrate.
7. The recombinant microorganism of claim 1 , wherein the recombinant microorganism is capable of producing butanol from a monosaccharide, oligosaccharide, or polysaccharide.
8. The recombinant microorganism of claim 1 , which is further constructed by transferring a foreign gene for inactivating acetate kinase to the host organism.
9. The recombinant microorganism of claim 1 , which is further constructed by transferring a foreign gene or a portion thereof for inactivating phosphotransbutyrylase to the host organism.
10. The recombinant microorganism of claim 1 , which is further constructed by transferring a foreign gene for inactivating phosphotransacetylase to the host organism.
11. The recombinant microorganism of claim 1 , wherein the transfer of SEQ ID NO: 9 is accomplished using a vector that includes a thiolase promoter sequence.
12. A method for producing butanol, comprising the steps of culturing a recombinant microorganism of claim 1 in a culture medium containing glucose, and collecting butanol from a culture thereof.
13. A method for producing butanol, comprising the steps of culturing a recombinant microorganism of claim 8 in a culture medium containing glucose, and collecting butanol from a culture thereof.
14. A vector containing the DNA sequences of SEQ. ID No: 9 and SEQ. ID No: 10.
15. A method of metabolically engineering the recombinant microorganism of claim 1 capable of producing butanol from a substrate, comprising the steps of:
obtaining a microbial host capable of naturally producing butyric acid, and
transferring a foreign gene that encodes an enzyme having both alcohol/aldehyde dehydrogenase and butanol hydrogenase activity into the microbial host.
16. The method of claim 15 , further comprising the step of transferring to the microbial host a foreign gene for inactivating acetate kinase.
17. The method of claim 15 , further comprising the step of transferring to the microbial host a foreign gene for inactivating phosphotransbutyrylase.
18. The method of claim 15 , further comprising the step of transferring to the microbial host a foreign gene for inactivating phosphotransacetylase.
19. The method of claim 15 , wherein the microbial host is C. tyrobutyricum.
20. The method of claim 15 , wherein the microbial host is selected from the group consisting of Clostridium butyricum, Clostridium thermobutyricum, Clostridium cellulovorans, Clostridium carboxidivorans, Butyribacterium methylotrophicum, Clostridium polysaccharolyticum, Clostridium populeti , and Clostridium kluyveri.
21. An isolated and biologically pure culture of a bacterial mutant strain having the ATCC deposit No. PTA-12145.
22. The recombinant microorganism of claim 8 , where the foreign gene for inactivating acetate kinase is DNA comprising the base sequence of SEQ. ID NO: 11.
23. The recombinant microorganism of claim 10 , wherein the foreign gene for inactivating phosphotransacetylase is DNA comprising the base sequence of SEQ. ID NO: 12.
24. The recombinant organism of claim 11 , wherein the thiolase promoter sequence is a DNA comprising the base sequence of SEQ. ID NO: 10.
25. The recombinant microorganism of claim 1 , wherein the transfer of SEQ ID NO: 9 is accomplished using a vector that includes pCB102 replicon from Clostridium butyricum.
26. The recombinant microorganism of claim 1 , wherein the transfer of SEQ ID NO: 9 is accomplished using a vector that includes pBP1 replicon from Clostridium botulinum.