Fermentative production of alcohols
View Patent ↗The invention relates to the fields of industrial microbiology and alcohol production. The invention also relates to the development of a microorganism capable of producing fermentation products via an engineered pathway, and uses of the microorganism. The invention also relates to the methods to improve cell viability and productivity and the use of recycling and acid washing to increase the yield of fermentation products.
1. A process for producing isobutanol comprising:
(a) providing a recombinant yeast host cell comprising an engineered isobutanol biosynthetic pathway, wherein the engineered isobutanol biosynthetic pathway comprises polynucleotides encoding polypeptides that catalyze the substrate to product conversion of (i) pyruvate to acetolactate, (ii) acetolactate to 2,3-dihydroxyisovalerate, (iii) 2,3-dihydroxyisovalerate to 2-ketoisovalerate, (iv) 2-ketoisovalerate to isobutyraldehyde, and (v) isobutyraldehyde to isobutanol,
(b) contacting the recombinant yeast host cell with a fermentation medium comprising one or more carbon substrates under conditions wherein isobutanol is produced;
(c) collecting the recombinant yeast host cell;
(d) recovering isobutanol from the fermentation medium;
(e) contacting the collected recombinant yeast host cell of (c) with one or more carbon substrates under conditions wherein isobutanol is produced;
(f) repeating steps (c)-(e); and, optionally exposing the collected recombinant yeast host cell of (c) to conditions of pH less than or equal to about 3.0 in the presence of at least about 0.3% isobutanol.
2. The process of claim 1 , wherein steps c)-e) are repeated at least ten times.
3. The process of claim 1 , wherein the recombinant yeast host cell does not express or has reduced expression of pyruvate decarboxylase.
4. The process of claim 1 , wherein the recombinant yeast host cell does not express or has reduced expression of glyceraldehyde-3-phosphate dehydrogenase.
5. The process of claim 1 , wherein the recombinant yeast host cell does not express or has reduced expression of phosphodiesterase.
6. The process of claim 5 , wherein the phosphodiesterase is PDE1.
7. The process of claim 1 , wherein the recombinant yeast host cell does not express or has reduced expression of butanediol dehydrogenase (BDH1).
8. The process of claim 1 , wherein the recombinant yeast host cell is present at a cell density of at least about 2 gdcw/L during the contacting of (b).
9. The process of claim 1 , wherein the recombinant yeast host cell of (b) maintains its specific productivity for at least ten cycles of repeating steps (c)-(e).
10. The process of claim 1 , wherein isobutanol is produced in (b) at an effective rate of at least about 0.1 g/gdcw/h.
11. The process of claim 1 , wherein the contacting of (b) occurs in the presence of an extractant.
12. The process of claim 1 , wherein the contacting of (b) and (e) occur under anaerobic or microaerobic conditions.
13. The process of claim 3 wherein the recombinant yeast host cell does not express or has reduced expression of a gene encoding acetolactate reductase.
14. The process of claim 13 wherein the reduction in expression is the result of an insertion, mutation, substitution, and/or deletion of a gene encoding YMR226C.
15. The process of claim 1 , wherein the substrate to product conversion of pyruvate to acetolactate is catalyzed by acetolactate synthase, the substrate to product conversion of acetolactate to 2,3-dihydroxyisovalerate is catalyzed by acetohydroxy acid reductoisomerase, the substrate to product conversion of 2,3-dihydroxyisovalerate to 2-ketoisovalerate is catalyzed by acetohydroxy acid dehydratase, the substrate to product conversion of 2-ketoisovalerate to isobutyraldehyde is catalyzed by a branched-chain α-keto acid decarboxylase, and the substrate to product conversion of isobutyraldehyde to isobutanol is catalyzed by a branched-chain alcohol dehydrogenase.
16. The process of claim 1 , wherein the recombinant yeast host cell comprises at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide affecting Fe—S cluster biosynthesis, wherein the polypeptide affecting Fe—S cluster biosynthesis is selected from AFT1, AFT2, FRA2, GRX3, or CCC1.
17. The process of claim 1 , wherein the recombinant yeast host cell comprises a deletion, mutation, and/or substitution in an endogenous gene encoding one or more polypeptides selected from pyruvate decarboxylase, glyceraldehyde-3-phosphate dehydrogenase, AFT1, AFT2, FRA2, GRX3, and CCC1.
18. The process of claim 1 , wherein the isobutanol is recovered by distillation, liquid-liquid extraction, decantation, adsorption, gas stripping, membrane evaporation, pervaporation, or combinations thereof.
19. The process of claim 1 , wherein solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or combinations thereof.
20. The process of claim 11 , wherein the extractant is selected from the group consisting of C 12 to C 22 fatty alcohols, C 12 to C 22 fatty acids, esters of C 12 to C 22 fatty acids, C 12 to C 22 fatty aldehydes, and mixtures thereof.
21. The process of claim 11 , wherein the extractant is selected from the group consisting of oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, alkyl alkanols, 1-undecanol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, methyl oleate, undecanal, lauric aldehyde, 20-methylundecanal, trioctyl phosphine oxide, and mixtures thereof.