Microorganisms for producing organic acids
Organic acid-producing microorganisms and methods of using same. The organic acid-producing microorganisms comprise modifications that reduce or ablate AcsA activity or AcsA homolog activity. The modifications increase tolerance of the microorganisms to such organic acids as 3-hydroxypropionic acid, acrylic acid, propionic acid, lactic acid, and others. Further modifications to the microorganisms increase production of such organic acids as 3-hydroxypropionic acid, lactate, and others. Methods of producing such organic acids as 3-hydroxypropionic acid, lactate, and others with the modified microorganisms are provided. Methods of using acsA or homologs thereof as counter-selectable markers are also provided.
1. A microorganism comprising:
a modification that reduces or ablates AcsA activity or AcsA homolog activity in the microorganism; and
one or more recombinant nucleic acids configured to express an enzyme selected from the group consisting of a lactate dehydrogenase and a transhydrogenase, wherein the microorganism produces an increased amount of lactic acid compared to a corresponding microorganism not comprising the one or more recombinant nucleic acids.
2. The microorganism of claim 1 wherein the microorganism is a bacterium.
3. The microorganism of claim 1 wherein the microorganism is a cyanobacterium.
4. The microorganism of claim 1 wherein the one or more recombinant nucleic acids is configured to express a lactate dehydrogenase and a pyridine nucleotide transhydrogenase.
5. The microorganism of claim 1 wherein the one or more recombinant nucleic acids is configured to express a lactate dehydrogenase selected from the group consisting of a lactate dehydrogenase from Bacillus subtilis or a homolog thereof and a lactate dehydrogenase from Lactococcus lactis or a homolog thereof.
6. The microorganism of claim 1 wherein the lactate dehydrogenase is selected from the group consisting of a lactate dehydrogenase from Bacillus subtilis or a homolog thereof comprising a sequence at least about 80% identical to SEQ ID NO:18 and a lactate dehydrogenase from Lactococcus lactis or a homolog thereof comprising a sequence at least about 80% identical to SEQ ID NO:22.
7. The microorganism of claim 1 wherein the lactate dehydrogenase is selected from the group consisting of a lactate dehydrogenase from Bacillus subtilis or a homolog thereof comprising a sequence at least about 90% identical to SEQ ID NO:18 and a lactate dehydrogenase from Lactococcus lactis or a homolog thereof comprising a sequence at least about 90% identical to SEQ ID NO:22.
8. The microorganism of claim 1 wherein the lactate dehydrogenase is selected from the group consisting of a lactate dehydrogenase from Bacillus subtilis or a homolog thereof comprising a sequence at least about 90% identical to SEQ ID NO:18 and a lactate dehydrogenase from Lactococcus lactis or a homolog thereof comprising a sequence at least about 90% identical to SEQ ID NO:22.
9. The microorganism of claim 1 wherein the one or more recombinant nucleic acids is configured to express a soluble pyridine nucleotide transhydrogenase from Escherichia coli or a homolog thereof.
10. The microorganism of claim 9 wherein the soluble pyridine nucleotide transhydrogenase from Escherichia coli or the homolog thereof comprises a sequence at least about 80% identical to SEQ ID NO:20.
11. The microorganism of claim 9 wherein the soluble pyridine nucleotide transhydrogenase from Escherichia coli or the homolog thereof comprises a sequence at least about 90% identical to SEQ ID NO:20.
12. The microorganism of claim 9 wherein the soluble pyridine nucleotide transhydrogenase from Escherichia coli or the homolog thereof comprises a sequence at least about 95% identical to SEQ ID NO:20.
13. The microorganism of claim 1 wherein the microorganism is a bacterium, the one or more recombinant nucleic acids is configured to express a soluble pyridine nucleotide transhydrogenase from Escherichia coli or a homolog thereof comprising a sequence at least about 95% identical to SEQ ID NO:20 and a lactate dehydrogenase selected from the group consisting of a lactate dehydrogenase from Bacillus subtilis or a homolog thereof comprising a sequence at least about 95% identical to SEQ ID NO:18 and a lactate dehydrogenase from Lactococcus lactis or a homolog thereof comprising a sequence at least about 95% identical to SEQ ID NO:22.
14. The microorganism of claim 13 wherein the microorganism is a cyanobacterium.
15. The microorganism of claim 14 wherein the lactase dehydrogenase comprises the lactate dehydrogenase from Bacillus subtilis or the homolog thereof comprising the sequence at least about 95% identical to SEQ ID NO:18.
16. The microorganism of claim 14 wherein the lactase dehydrogenase comprises the lactate dehydrogenase from Lactococcus lactis or the homolog thereof comprising the sequence at least about 95% identical to SEQ ID NO:18.
17. A method of producing lactic acid comprising culturing a microorganism as recited in claim 1 .
18. The method of claim 17 wherein the culturing produces lactic acid to a concentration of at least about 5 mM.
19. The method of claim 17 wherein the culturing produces lactic acid at a rate of at least about 200 mg/L/Day.
20. The method of claim 17 wherein the culturing converts at least about 15% of carbon consumed by the microorganism to lactic acid.