Partial adaption for butanol production

• US 10,308,910 B2
• Filed: 04/20/2017
• Issued: 06/04/2019
• Est. Priority Date: 07/03/2013
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: Bhalla, Ritu, Doig, Steven D., Konde, Kakasaheb Suresh, Patnaik, Ranjan
• Primary Examiner(s): Noakes, Suzanne M
• Assistant Examiner(s): Lee, Jae W
• Application Number:
  US15/492,568
• Publication Number:
  US 20170283765A1
• Time in Patent Office: 775 Days
• Field of Search: None
• CPC Class Codes: C12N 1/36 Adaptation or attenuation of cells
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Provided herein are processes for producing an improved culture of cells comprising an engineered butanol biosynthetic pathway. The processes comprise (a) providing a cell culture of recombinant microorganisms comprising an engineered butanol biosynthetic pathway, wherein the engineered butanol biosynthetic pathway is minimal or not activated; and (b) growing the culture of recombinant microorganisms under adaptive conditions whereby pathway activation is increased to produce an improved cell culture and whereby the improved cell culture is capable of continuing to grow in fermentation.

Claims

1. A process for producing an improved culture of cells comprising an engineered butanol biosynthetic pathway:

a) providing a cell culture of recombinant microorganisms comprising an engineered butanol biosynthetic pathway, wherein the recombinant microorganisms produce butanol;

b) growing the culture of recombinant microorganisms in a propagation phase wherein the propagation phase is characterized by aerobic conditions and a butanol concentration of about 0 g/L to about 10 g/L; and

c) growing the culture of step b) in a production phase wherein the production phase is characterized by anaerobic conditions and an effective titer of butanol is at least 10 g/L,

wherein the recombinant microorganism is selected from a genus from the group consisting of Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Lactococcus, Enterococcus, Alcaligenes, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluveromyces, Yarrowia, Pichia, Zygosaccharomyces, Debaryomyces, Candida, Brettanomyces, Pachysolen, Hansenula, Issatchenkia, Trichosporon, Yamadazyma, and Saccharomyces.

2. The process of claim 1, wherein the engineered butanol biosynthetic pathway is selected from the group consisting of:

(a) a 1-butanol biosynthetic pathway;

(b) a 2-butanol biosynthetic pathway; and

(c) an isobutanol biosynthetic pathway.

3. The process of claim 1, wherein the rate of butanol production of the provided cell culture of recombinant microorganisms is less than 0.20 grams of butanol per gram of dry cell weight of cells per hour (g/g dcw/hr).

4. The process of claim 1, wherein the cell culture of recombinant microorganisms is grown under adaptive conditions selected from at least one of a source of carbon substrate, a dissolved oxygen concentration, a temperature, a pH, a substrate concentration, a butanol concentration, a 2-butanone concentration, or a component of the fermentation medium.

5. The process of claim 4, wherein the adaptive condition comprises a substrate concentration and a dissolved oxygen concentration.

6. The process of claim 5, wherein the substrate is glucose.

7. The process of claim 6, wherein the glucose concentration is about 1 g/L to about 50 g/L.

8. The process of claim 7, wherein the dissolved oxygen concentration is greater than 5%.

9. The process of claim 8, wherein specific oxygen uptake rate (Sp. OUR) of the recombinant microorganism is about 0.5 millimoles per gram cells per hour (mM/g cells/hr) to about 5 mM/g cells/hr.

10. The process of claim 4, wherein the cell culture is characterized by at least one of an increase in biomass production, a reduction in the amount of time for the fermentation, or a reduction or elimination of the production of an inhibitory product.

11. The process of claim 10, wherein growing the culture under adaptive conditions increases the biomass production of the culture to a cell density of about 5 g/L to about 15 g/L.

12. The process of claim 10, wherein the inhibitory product is isobutyric acid.

13. The process of claim 12, wherein the growing the culture under adaptive conditions reduces the isobutyric acid concentration to about 0.1 g/L to about 2.5 g/L.

14. The process of claim 4, wherein the dissolved oxygen concentration is less than 5%.

15. The process of claim 14, wherein specific oxygen uptake rate (Sp. OUR) of the recombinant microorganism is about 0 millimoles per gram cells per hour (mM/g cells/hour) to about 2.5 mM/g cells/hr.

16. The process of claim 4, wherein the cell culture is characterized by at least one of an increase in biomass production, a reduction in the amount of time for the fermentation, an increase in butanol yield, an increase in butanol productivity, an increase in biomass yield, or a delay in the production of an inhibitory product.

17. The process of claim 16, wherein growing the culture under adaptive conditions increases the biomass production of the culture such that the culture is capable of growing for at least one generation in the fermentation.

18. The process of claim 16, wherein growing the culture under adaptive conditions increases the biomass production of the culture such that the culture is capable of growing for at least two generations in the fermentation.

19. The process of claim 16, wherein the butanol yield is increased to at least 30 g/L.

20. The process of claim 1, further comprising contacting the recombinant microorganisms with a fermentable carbon substrate in a fermentation medium under conditions whereby the butanol is produced and recovering the butanol.

21. The process of claim 20, wherein the butanol is recovered by distillation, decantation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, pervaporation, or combinations thereof.

22. The process of claim 21, wherein the butanol is recovered by liquid-liquid extraction.

23. The process of claim 22, wherein the fermentation medium is contacted with a water immiscible extractant.

24. The process of claim 23, wherein the extractant is selected from the group consisting of C₁₂ to C₂₂ fatty alcohols, C₁₂ to C₂₂ fatty acids, esters of C₁₂ to C₂₂ fatty acids, C₁₂ to C₂₂ fatty aldehydes, and mixtures thereof.

25. The process of claim 20, wherein solids are removed from the fermentation medium by centrifugation, filtration, decantation, or combinations thereof.

26. The process of claim 20, wherein the butanol is isobutanol.