# US 10,308,910 B2 · Partial adaption for butanol production

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- Number: US 10,308,910 B2
- Document ID: US10308910B2
- Status: Granted
- Kind: B2
- Application number: 15/492,568
- Filed: 2017-04-20
- Published: 2017-10-05
- Granted: 2019-06-04
- Earliest claimed: 2013-07-03
- Assignees: Butamax Advanced Biofuels LLC
- Applicants: Butamax Advanced Biofuels LLC
- Inventors: Ritu Bhalla; Steven D. Doig; Kakasaheb Suresh Konde; Ranjan Patnaik
- Web page: https://patentgrep.com/patent/US10308910B2

## CPC

- C12N 1/36: Adaptation or attenuation of cells
- C12N 15/52: Genes encoding for enzymes or proenzymes
- C12P 7/16: Butanols
- Y02E 50/10: Biofuels, e.g. bio-diesel
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- Y02E: REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION

## 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

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 1)

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).

### Claim 4 (depends on 1)

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.

### Claim 5 (depends on 4)

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

### Claim 6 (depends on 5)

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

### Claim 7 (depends on 6)

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

### Claim 8 (depends on 7)

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

### Claim 9 (depends on 8)

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.

### Claim 10 (depends on 4)

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.

### Claim 11 (depends on 10)

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.

### Claim 12 (depends on 10)

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

### Claim 13 (depends on 12)

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.

### Claim 14 (depends on 4)

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

### Claim 15 (depends on 14)

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.

### Claim 16 (depends on 4)

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.

### Claim 17 (depends on 16)

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.

### Claim 18 (depends on 16)

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.

### Claim 19 (depends on 16)

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

### Claim 20 (depends on 1)

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.

### Claim 21 (depends on 20)

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.

### Claim 22 (depends on 21)

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

### Claim 23 (depends on 22)

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

### Claim 24 (depends on 23)

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.

### Claim 25 (depends on 20)

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

### Claim 26 (depends on 20)

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