# Patent

## Title
Method for producing butanol using two-phase extractive fermentation

## Abstract

A method of making butanol from at least one fermentable carbon source that overcomes the issues of toxicity resulting in an increase in the effective titer, the effective rate, and the effective yield of butanol production by fermentation utilizing a recombinant microbial host wherein the butanol is extracted into specific organic extractants during fermentation.

## Background
This application is a continuation-in-part of U.S. application Ser. No. 12/478,389, filed Jun. 4, 2009, herein incorporated by reference, and which claims the benefit of priority from U.S. Provisional Application No. 61/058,567, filed Jun. 4, 2008, herein incorporated by reference.


FIELD OF THE INVENTION
The invention relates to the field of biofuels. More specifically, the invention relates to a method for producing butanol through microbial fermentation, in which the butanol product is removed by extraction into a water immiscible organic extractant during the fermentation.
BACKGROUND OF THE INVENTION
Butanol is an important industrial chemical, with a variety of applications, such as use as a fuel additive, as a feedstock chemical in the plastics industry, and as a foodgrade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this chemical will likely increase.
Several chemical synthetic methods are known; however, these methods of producing butanol use starting materials derived from petrochemicals and are generally expensive and are not environmentally friendly. Several methods of producing butanol by fermentation are also known, for example the ABE process which is the fermentive process producing a mixture of acetone, 1-butanol and ethanol. Acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum is one of the oldest known industrial fermentations; as is also the pathways and genes responsible for the production of these solvents. Production of 1-butanol by the ABE process is limited by the toxic effect of the 1-butanol on Clostridium acetobutylicum. In situ extractive fermentation methods using specific organic extractants which are nontoxic to the bacterium have been reported to enhance the production of 1-butanol by fermentation using Clostridium acetobutylicum (Roffler et al., Biotechnol. Bioeng. 31:135-143, 1988; Roffler et al., Bioprocess Engineering 2:1-12, 1987; and Evans et al., Appl. Environ. Microbiol. 54:1662-1667, 1988).
In contrast to the native Clostridium acetobutylicum described above, recombinant microbial production hosts expressing 1-butanol, 2-butanol, and isobutanol biosynthetic pathways have also been described. These recombinant hosts have the potential of producing butanol in higher yields compared to the ABE process because they do not produce byproducts such as acetone and ethanol. However, the problem with these recombinant hosts is that biological production of butanol appears to be limited by butanol toxicity thresholds to the host microorganism used in the fermentation. Extractive fermentation methods have not been applied to the production of butanols using recombinant microbial strains.
The present invention satisfies the above need and provides a method of making butanol from at least one fermentable carbon source that overcome the issues of toxicity resulting in an increase in the effective titer, the effective rate, and the effective yield of butanol production by fermentation utilizing a recombinant microbial host wherein the butanol is extracted into specific organic extractants during fermentation.
SUMMARY OF THE INVENTION
The invention provides a method for recovering butanol from a fermentation medium, the method comprising:

    
    
        a) providing a fermentation medium comprising butanol, water, and a genetically modified microorganism that produces butanol from at least one fermentable carbon source;
        b) contacting the fermentation medium with i) at least one first water immiscible organic extractant 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, C12 to C22 fatty amides and mixtures thereof, and optionally (ii) a second water immiscible organic extractant 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, C12 to C22 fatty amides and mixtures thereof to form a two-phase mixture comprising an aqueous phase and a butanol-containing organic phase;
        c) separating the butanol-containing organic phase from the aqueous phase; and
        d) recovering the butanol from the butanol-containing organic phase to produce recovered butanol.
    
    


The invention provides a method for the production of butanol comprising the steps of:

    
    
        a) providing a genetically modified microorganism that produces butanol from at least one fermentable carbon source;
        b) growing the microorganism in a biphasic fermentation medium comprising an aqueous phase and a water immiscible organic extractant 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, wherein said biphasic fermentation medium comprises from about 3% to about 60% by volume of said water immiscible organic extractant, for a time sufficient to allow extraction of the butanol into the organic extractant to form a butanol-containing organic phase;
        c) separating the butanol-containing organic phase from the aqueous phase; and
        d) recovering the butanol from the butanol-containing organic phase to produce recovered butanol.
    
    


An embodiment of the invention provides a method for the production of butanol comprising the steps of:

    
    
        a) providing a genetically modified microorganism that produces butanol from at least one fermentable carbon source;
        b) growing the microorganism in a fermentation medium wherein the microorganism produces said butanol into the fermentation medium to produce a butanol-containing fermentation medium;
        c) contacting the butanol-containing fermentation medium with i) at least one first water immiscible organic extractant 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, C12 to C22 fatty amides and mixtures thereof, and optionally (ii) a second water immiscible organic extractant 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, C12 to C22 fatty amides and mixtures thereof to form a two-phase mixture comprising an aqueous phase and a butanol-containing organic phase;
        d) separating the butanol-containing organic phase from the aqueous phase; and
        e) recovering the butanol from the butanol-containing organic phase.
    
    


An embodiment of the invention provides a method for the production of butanol comprising the steps of:

    
    
        a) providing a genetically modified microorganism that produces butanol from at least one fermentable carbon source;
        b) growing the microorganism in a fermentation medium under aerobic conditions for a time sufficient to reach a preselected growth level;
        c) switching to microaerobic or anaerobic conditions to stimulate butanol production into the fermentation medium to form a butanol-containing fermentation medium;
        d) contacting the butanol-containing fermentation medium with i) at least one first water immiscible organic extractant 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, C12 to C22 fatty amides and mixtures thereof, and optionally (ii) a second water immiscible organic extractant 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, C12 to C22 fatty amides and mixtures thereof to form a two-phase mixture comprising an aqueous phase and a butanol-containing organic phase;
        e) separating the butanol-containing organic phase from the aqueous phase; and
        f) recovering the butanol from the butanol-containing organic phase.
    
    


Another embodiment of the invention provides a method for the production of butanol comprising the steps of:

    
    
        a) providing a fermentation medium comprising butanol, water, and a genetically modified microorganism that produces butanol from a fermentation medium comprising at least one fermentable carbon source;
        b) contacting the fermentation medium via a co-current or counter-current extractant stream with i) at least one first water immiscible organic extractant 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, and optionally (ii) a second water immiscible organic extractant 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, C12 to C22 fatty amides and mixtures thereof to form a two-phase mixture comprising an aqueous phase and a butanol-containing organic phase;
        c) separating the butanol-containing organic phase from the aqueous phase; and
        d) recovering the butanol from the butanol-containing organic phase to produce recovered butanol.
    
    


The present extractive fermentation methods provide butanol, including all butanol isomers, which is known to have an energy content similar to that of gasoline and which can be blended with any fossil fuel. Butanol is favored as a fuel or fuel additive as it yields only CO2 and little or no SOX or NOX when burned in the standard internal combustion engine. Additionally butanol is less corrosive than ethanol, the most preferred fuel additive to date.
In addition to its utility as a biofuel or fuel additive, the butanol produced from the present methods has the potential of impacting hydrogen distribution problems in the emerging fuel cell industry. Fuel cells today are plagued by safety concerns associated with hydrogen transport and distribution. Butanol can be easily reformed for its hydrogen content and can be distributed through existing gas stations in the purity required for either fuel cells or vehicles.
Finally, the present methods produce butanol from plant derived carbon sources, avoiding the negative environmental impact associated with standard petrochemical processes for butanol production.



BRIEF DESCRIPTION OF THE FIGURE AND SEQUENCE DESCRIPTIONS
FIG. 1 is a graph showing the concentration of isobutanol in the fermentation medium (i.e., aqueous phase) during a fermentation using oleyl alcohol as the organic extractant with gas stripping (▪) as described in Example 6, and during a fermentation with gas stripping alone (●), as described in Example 7. FIG. 1 represents data generated using a recombinant Escherichia coli producing isobutanol.
FIG. 2 is a graph showing the concentration of isobutanol in the fermentation medium (i.e., aqueous phase) during a fermentation using oleyl alcohol as the organic extractant with gas stripping (▪) as described in Example 8, and during a fermentation with gas stripping alone (●), as described in Example 9. FIG. 2 represents data generated using a recombinant Saccharomyces cerevisiae producing isobutanol.
FIG. 3 schematically illustrates one embodiment of the methods of the invention, in which the first water immiscible extractant and the optional second water immiscible extractant are combined in a vessel prior to contacting the fermentation medium with the extractant in a fermentation vessel.
FIG. 4 schematically illustrates one embodiment of the methods of the invention, in which the first water immiscible extractant and the optional second water immiscible extractant are added separately to a fermentation vessel in which the fermentation medium is contacted with the extractant.
FIG. 5 schematically illustrates one embodiment of the methods of the invention, in which the first water immiscible extractant and the optional second water immiscible extractant are added separately to different fermentation vessels for contacting of the fermentation medium with the extractant.
FIG. 6 schematically illustrates one embodiment of the methods of the invention, in which extraction of the product occurs downstream of the fermentor and the first water immiscible extractant and the optional second water immiscible extractant are combined in a vessel prior to contacting the fermentation medium with the extractant in a different vessel.
FIG. 7 schematically illustrates one embodiment of the methods of the invention, in which extraction of the product occurs downstream of the fermentor and the first water immiscible extractant and the optional second water immiscible extractant are added separately to a vessel in which the fermentation medium is contacted with the extractant.
FIG. 8 schematically illustrates one embodiment of the methods of the invention, in which extraction of the product occurs downstream of the fermentor and the first water immiscible extractant and the optional second water immiscible extractant are added separately to different vessels for contacting of the fermentation medium with the extractant.
FIG. 9 schematically illustrates one embodiment of the methods of the invention, in which extraction of the product occurs in at least one batch fermentor via co-current flow of an extractant at or near the bottom of a fermentation mash to fill the fermentor with extractant which flows out of the fermentor at a point at or near the top of the fermentor.



The following sequences conform with 37 C.F.R. 1.821 1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPO and PCT (Rules 5.2 and 49.5 (a bis), and Section 208 and Annex C of the Administrative Instructions).







TABLE 1







Summary of Gene and Protein SEQ ID Numbers












SEQ ID NO:
SEQ ID NO:



Description
Nucleic acid
Peptide















Klebsiella pneumoniae budB
1
2



(acetolactate synthase)



E. coli ilvC (acetohydroxy acid
3
4



reductoisomerase)



E. coli ilvD (acetohydroxy acid
5
6



dehydratase)



Lactococcus lactis kivD
7
8



(branched-chain α-keto acid



decarboxylase), codon



optimized



Achromobacter xylosoxidans.
9
10



butanol dehydrogenase



(sadB) gene



Bacillus subtilis alsS
32
33



(acetolactate synthase)



Pf5.llvC-Z4B8 (KARI)
36
37



S. cerevisiae ILV5
40
41



(acetohydroxy acid



reductoisomerase; KARI)



B. subtilis ketoisovalerate
43
44



decarboxylase (KivD) codon



optimized



Horse liver alcohol
45
46



dehydrogenase (HADH)



codon optimized



Streptococcus mutans ilvD
58
59



acetohydroxy acid



dehydratase










SEQ ID NOs:11-22 are the nucleotide sequences of the primers used to construct the recombinant Escherichia coli strain described in the General Methods section of the Examples herein below.
SEQ ID NO:23 is the nucleotide sequence of the pflB gene from Escherichia coli strain K-12 MG1655.
SEQ ID NO:24 is the nucleotide sequence of the IdhA gene from Escherichia coli strain K-12 MG1655.
SEQ ID NO:25 is the nucleotide sequence of the adhE gene from Escherichia coli strain K-12 MG1655.
SEQ ID NO:26 is the nucleotide sequence of the frdA gene from Escherichia coli strain K-12 MG1655.
SEQ ID NO:27 is the nucleotide sequence of the frdB gene from Escherichia coli strain K-12 MG1655.
SEQ ID NO:28 is the nucleotide sequence of the frdC gene from Escherichia coli strain K-12 MG1655.
SEQ ID NO:29 is the nucleotide sequence of the frdD gene from Escherichia coli strain K-12 MG1655.
SEQ ID NO; 30 is the nucleotide sequence of pLH475-Z4B8.
SEQ ID NO; 31 is the nucleotide sequence of the CUP1 promoter.
SEQ ID NO; 34 is the nucleotide sequence of the CYC1 terminator.
SEQ ID NO; 35 is the nucleotide sequence of the ILV5 promoter.
SEQ ID NO; 38 is the nucleotide sequence of the ILV5 terminator.
SEQ ID NO; 39 is the nucleotide sequence of the FBA1 promoter.
SEQ ID NO; 42 is the nucleotide sequence of pLH468.
SEQ ID NO; 47 is the nucleotide sequence of pNY8.
SEQ ID NO; 48 is the nucleotide sequence of the GPD1 promoter.
SEQ ID NOs:49, 50, 54, 55, 62-71, 73-83 and 85-86 are the nucleotide sequences of primers used in the examples.
SEQ ID NO; 51 is the nucleotide sequence of pRS425::GPM-sadB.
SEQ ID NO; 52 is the nucleotide sequence of the GPM1 promoter.
SEQ ID NO:53 is the nucleotide sequence of the ADH1 terminator.
SEQ ID NO:56 is the nucleotide sequence of pRS423 FBA ilvD(Strep).
SEQ ID NO:57 is the nucleotide sequence of the FBA terminator.
SEQ ID NO:60 is the nucleotide sequence of the GPM-sadB-ADHt segment.
SEQ ID NO:61 is the nucleotide sequence of pUC19-URA3r.
SEQ ID NO:72 is the nucleotide sequence of the ilvD-FBA1t segment.
SEQ ID NO:84 is the nucleotide sequence of the URA3r2 template DNA.
DETAILED DESCRIPTION
As used above and throughout the description of the invention, the following terms, unless otherwise indicated, shall be defined as follows:
The term “butanol” as used herein, refers to 1-butanol, 2-butanol, isobutanol, or mixtures thereof.
The term “aerobic conditions” as used herein, means growth conditions in the presence of oxygen.
The term “microaerobic conditions” as used herein, means growth conditions with low levels of oxygen (i.e., below normal atmospheric oxygen levels).
The term “anaerobic conditions” as used herein, means growth conditions in the absence of oxygen.
The term “fermentable carbon source” as used herein, refers to a carbon source capable of being metabolized by the microorganisms disclosed herein. Suitable fermentable carbon sources include, but are not limited to, monosaccharides, such as glucose or fructose; disaccharides, such as lactose or sucrose; oligosaccharides; polysaccharides, such as starch or cellulose; one carbon substrates; and mixtures thereof.
The term “extractant” as used herein refers to organic solvent used to extract any butanol isomer.
The term “biphasic fermentation medium” as used herein, refers to a two-phase growth medium comprising a fermentation medium (i.e., the aqueous phase) and a suitable amount of a water immiscible organic extractant.
The term “butanol biosynthetic pathway” as used herein, refers to an enzyme pathway to produce 1-butanol, 2-butanol, or isobutanol.
The term “1-butanol biosynthetic pathway” as used herein, refers to an enzyme pathway to produce 1-butanol from acetyl-coenzyme A (acetyl-CoA).
The term “2-butanol biosynthetic pathway” as used herein, refers to an enzyme pathway to produce 2-butanol from pyruvate.
The term “isobutanol biosynthetic pathway” as used herein, refers to an enzyme pathway to produce isobutanol from pyruvate.
The term “fatty acid” as used herein, refers to a carboxylic acid having a long, aliphatic chain (i.e., C11 to C22), which is either saturated or unsaturated.
The term “fatty alcohol” as used herein, refers to an alcohol having a long, aliphatic chain (i.e., C11 to C22), which is either saturated or unsaturated.
The term “fatty aldehyde” as used herein, refers to an aldehyde having a long, aliphatic chain (i.e., C11 to C22), which is either saturated or unsaturated.
The term “effective titer” as used herein, refers to the total amount of butanol produced by fermentation per liter of fermentation medium. The total amount of butanol includes the amount of butanol in the fermentation medium, and the amount of butanol recovered from the organic extractant and from the gas phase, if gas stripping is used.
The term “minimal media” as used herein, refers to growth media that contain the minimum nutrients possible for growth, generally without the presence of amino acids. A minimal medium typically contains a fermentable carbon source and various salts, which may vary among microorganisms and growing conditions; these salts generally provide essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow the microorganism to synthesize proteins and nucleic acids.
The term “defined media” as used herein, refers to growth media that have known quantities of all ingredients. e.g., a defined carbon source and nitrogen source, and trace elements and vitamins required by the microorganism.
The term “OD” as used herein, refers to optical density.
The term “OD600” as used herein, refers to the optical density at a wavelength of 600 nm.
The term “id” as used herein, refers to internal diameter.
The term “Aq” as used herein, refers to aqueous phase.
The term “Org” as used herein, refers to organic phase.
The term “IPTG” as used herein, refers to isopropyl β-D-thiogalactopyranoside.
The term “vvm” as used herein, refers to volume to volume per minute.
The term “ATCC” as used herein, refers to the American Type Culture Collection, Manassas, Va.
The term “vol” means volume.
The term “rpm” means revolutions per minute.
The term “sec” means second(s).
The term “min” means minute(s).
The term “h” means hour(s).
The term “μL” means microliter.
The term “mL” means milliliter(s).
The term “L” means liter(s).
The term “mL/min” means milliliters per minute.
The term “mmol” means millimole(s).
The term “mM” means millimolar.
The term “M” means molar.
The term “μm” means micrometer.
The term “g” means grams.
The term “μg” means microgram.
The term “g/g” means gram per gram.
The term “g/L” means grams per liter.
The term “μg/mL” means microgram per liter.
The term “mg/L” means micgram per liter.
The term “mmol/min/mg” means millimole per minute per milligram.
The term “g/L/h” means grams per liter per hour.
The term “HPLC” means high pressure liquid chromatography.
The term “GC” means gas chromatography.
Genetically Modified Microorganisms
Microbial hosts for butanol production may be selected from bacteria, cyanobacteria, filamentous fungi and yeasts. The microbial host used should be tolerant to the butanol product produced, so that the yield is not limited by toxicity of the product to the host. The selection of a microbial host for butanol production is described in detail below.
Microbes that are metabolically active at high titer levels of butanol are not well known in the art. Although butanol-tolerant mutants have been isolated from solventogenic Clostridia, little information is available concerning the butanol tolerance of other potentially useful bacterial strains. Most of the studies on the comparison of alcohol tolerance in bacteria suggest that butanol is more toxic than ethanol (de Cavalho et al., Microsc. Res. Tech. 64:215-22 (2004) and Kabelitz et al., FEMS Microbiol. Lett. 220:223-227 (2003)). Tomas et al. (J. Bacteriol. 186:2006-2018 (2004)) report that the yield of 1-butanol during fermentation in Clostridium acetobutylicum may be limited by butanol toxicity. The primary effect of 1-butanol on Clostridium acetobutylicum is disruption of membrane functions (Hermann et al., Appl. Environ. Microbiol. 50:1238-1243 (1985)).
The microbial hosts selected for the production of butanol should be tolerant to butanol and should be able to convert carbohydrates to butanol using the introduced biosynthetic pathway as described below. The criteria for selection of suitable microbial hosts include the following: intrinsic tolerance to butanol, high rate of carbohydrate utilization, availability of genetic tools for gene manipulation, and the ability to generate stable chromosomal alterations.
Suitable host strains with a tolerance for butanol may be identified by screening based on the intrinsic tolerance of the strain. The intrinsic tolerance of microbes to butanol may be measured by determining the concentration of butanol that is responsible for 50% inhibition of the growth rate (IC50) when grown in a minimal medium. The IC50 values may be determined using methods known in the art. For example, the microbes of interest may be grown in the presence of various amounts of butanol and the growth rate monitored by measuring the optical density at 600 nanometers. The doubling time may be calculated from the logarithmic part of the growth curve and used as a measure of the growth rate. The concentration of butanol that produces 50% inhibition of growth may be determined from a graph of the percent inhibition of growth versus the butanol concentration. Preferably, the host strain should have an IC50 for butanol of greater than about 0.5%. More suitable is a host strain with an IC50 for butanol that is greater than about 1.5%. Particularly suitable is a host strain with an IC50 for butanol that is greater than about 2.5%.
The microbial host for butanol production should also utilize glucose and/or other carbohydrates at a high rate. Most microbes are capable of utilizing carbohydrates. However, certain environmental microbes cannot efficiently use carbohydrates, and therefore would not be suitable hosts.
The ability to genetically modify the host is essential for the production of any recombinant microorganism. Modes of gene transfer technology that may be used include by electroporation, conjugation, transduction or natural transformation. A broad range of host conjugative plasmids and drug resistance markers are available. The cloning vectors used with an organism are tailored to the host organism based on the nature of antibiotic resistance markers that can function in that host.
The microbial host also may be manipulated in order to inactivate competing pathways for carbon flow by inactivating various genes. This requires the availability of either transposons or chromosomal integration vectors to direct inactivation. Additionally, production hosts that are amenable to chemical mutagenesis may undergo improvements in intrinsic butanol tolerance through chemical mutagenesis and mutant screening.
Based on the criteria described above, suitable microbial hosts for the production of butanol include, but are not limited to, members of the genera, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Pediococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces. Preferred hosts include: Escherichia coli, Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, Pediococcus pentosaceus, Pediococcus acidilactici, Bacillus subtilis and Saccharomyces cerevisiae. 
Microorganisms mentioned above may be genetically modified to convert fermentable carbon sources into butanol, specifically 1-butanol, 2-butanol, or isobutanol, using methods known in the art. Particularly suitable microorganisms include Escherichia Lactobacillus, and Saccharomyces, where E. coli, L. plantarum and S. cerevisiae are particularly preferred. Additionally, the microorganism may be a butanol-tolerant strain of one of the microorganisms listed above that is isolated using the method described by Bramucci et al. (copending and commonly owned U.S. patent application Ser. No. 11/761,497; and WO 2007/146377). An example of one such strain is Lactobacillus plantarum strain PN0512 (ATCC: PTA-7727, biological deposit made Jul. 12, 2006 for U.S. patent application Ser. No. 11/761,497).
These microorganisms can be genetically modified to contain a 1-butanol biosynthetic pathway to produce 1-butanol, as described by Donaldson et al. in WO 2007/041269, incorporated herein by reference. For example, the microorganism may be genetically modified to express a 1-butanol biosynthetic pathway comprising the following enzyme-catalyzed substrate to product conversions:
a) acetyl-CoA to acetoacetyl-CoA;
b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA;
c) 3-hydroxybutyryl-CoA to crotonyl-CoA;
d) crotonyl-CoA to butyryl-CoA;
e) butyryl-CoA to butyraldehyde; and
f) butyraldehyde to 1-butanol.
The microorganisms may also be genetically modified to express a 2-butanol biosynthetic pathway to produce 2-butanol, as described by Donaldson et al. in U.S. Patent Application Publication Nos. 2007/0259410 and 2007/0292927, WO 2007/130518 and WO 2007/130521, all of which are incorporated herein by reference. For example, in one embodiment the microorganism may be genetically modified to express a 2-butanol biosynthetic pathway comprising the following enzyme-catalyzed substrate to product conversions:
a) pyruvate to alpha-acetolactate;
b) alpha-acetolactate to acetoin;
c) acetoin to 2,3-butanediol;
d) 2,3-butanediol to 2-butanone; and
e) 2-butanone to 2-butanol.
The microorganisms may also be genetically modified to express an isobutanol biosynthetic pathway to produce isobutanol, as described by Donaldson et al. in U.S. Patent Application Publication No. 2007/0092957 and WO 2007/050671, both of which are incorporated herein by reference. For example, the microorganism may be genetically modified to contain an isobutanol biosynthetic pathway comprising the following enzyme-catalyzed substrate to product conversions:
a) pyruvate to acetolactate;
b) acetolactate to 2,3-dihydroxyisovalerate;
c) 2,3-dihydroxyisovalerate to α-ketoisovalerate;
d) α-ketoisovalerate to isobutyraldehyde; and
e) isobutyraldehyde to isobutanol.
The microorganism genetically modified to be capable of converting fermentable carbon sources into butanol may be a recombinant Escherichia coli strain that comprises an isobutanol biosynthetic pathway, as described above, and deletions of the following genes to eliminate competing pathways that limit isobutanol production, pflB, given as SEQ ID NO:23, (encoding for pyruvate formate lyase), IdhA, given as SEQ ID NO:24, (encoding for lactate dehydrogenase), adhE, given as SEQ ID NO:25, (encoding for alcohol dehydrogenase), and at least one gene comprising the frdABCD operon (encoding for fumarate reductase), specifically, frdA, given as SEQ ID NO:26, frdB, given as SEQ ID NO:27, frdC, given as SEQ ID NO:28, and frdD, given as SEQ ID NO:29,
The Escherichia coli strain may comprise: (a) an isobutanol biosynthetic pathway encoded by the following genes: budB (given as SEQ ID NO:1) from Klebsiella pneumoniae encoding acetolactate synthase (given as SEQ ID NO:2), ilvC (given as SEQ ID NO:3) from E. coli encoding acetohydroxy acid reductoisomerase (given as SEQ ID NO:4), ilvD (given as SEQ ID NO:5) from E. coli encoding acetohydroxy acid dehydratase (given as SEQ ID NO:6), kivD (given as SEQ ID NO:7) from Lactococcus lactis encoding the branched-chain keto acid decarboxylase (given as SEQ ID NO:8), and sadB (given as SEQ ID NO:9) from Achromobacter xylosoxidans encoding a butanol dehydrogenase (given as SEQ ID NO:10); and (b) deletions of the following genes: pflB (SEQ ID NO:23), IdhA (SEQ ID NO:24) adhE (SEQ ID NO:25), and frdB (SEQ ID NO:27). The enzymes encoded by the genes of the isobutanol biosynthetic pathway catalyze the substrate to product conversions for converting pyruvate to isobutanol, as described above. Specifically, acetolactate synthase catalyzes the conversion of pyruvate to acetolactate, acetohydroxy acid reductoisomerase catalyzes the conversion of acetolactate to 2,3-dihydroxyisovalerate, acetohydroxy acid dehydratase catalyzes the conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate, branched-chain keto acid decarboxylase catalyzes the conversion of α-ketoisovalerate to isobutyraldehyde, and butanol dehydrogenase catalyzes the conversion of isobutyraldehyde to isobutanol. This recombinant Escherichia coli strain can be constructed using methods known in the art, as exemplified in the General Methods Section of the Examples herein below.
The Saccharomyces cerevisiae strain may comprise: an isobutanol biosynthetic pathway encoded by the following genes: alsS coding region from Bacillus subtilis (SEQ ID NO:32) encoding acetolactate synthase (SEQ ID NO:33), ILV5 from S. cerevisiae (SEQ ID NO:40) encoding acetohydroxy acid reductoisomerase (KAR1; SEQ ID NO:41) and/or a mutant KAR1 such as encoded by Pf5.IlvC-Z4B8 (SEQ ID NO:36; protein SEQ ID NO:37), ilvD from Streptococcus mutans (SEQ ID NO:58) encoding acetohydroxy acid dehydratase (SEQ ID NO:59), kivD from Bacillus subtilis (SEQ ID NO:43) encoding the branched-chain keto acid decarboxylase (SEQ ID NO:44), and sadB from Achromobacter xylosoxidans (SEQ ID NO:9) encoding a butanol dehydrogenase (SEQ ID NO:10). The enzymes encoded by the genes of the isobutanol biosynthetic pathway catalyze the substrate to product conversions for converting pyruvate to isobutanol, as described herein.
A preferred yeast strain expressing an isobutanol pathway has acetolactate synthase (ALS) activity in the cytosol and has deletions of the endogenous pyruvate decarboxylase (PDC) genes as described in commonly owned and co-pending U.S. Patent Application No. 61/058,970, which is herein incorporated by reference. This combination of cytosolic ALS and reduced PDC expression was found to greatly increase flux from pyruvate to acetolactate, which then flows to the pathway for production of isobutanol.
This recombinant Saccharomyces cerevisiae strain can be constructed using methods known in the art, as exemplified in the General Methods section of the Examples herein below.
Organic Extractants
Extractants useful in the Methods described herein are water immiscible organic solvents. Suitable organic extractants should meet the criteria for an ideal solvent for a commercial two-phase extractive fermentation for the production or recovery of butanol. Specifically, the extractant should (i) be nontoxic to the butanol-producing microorganisms such as, for example, genetically modified Escherichia coli, Lactobacillus plantarum, and Saccharomyces cerevisiae, (ii) be substantially immiscible with the fermentation medium, (iii) have a high partition coefficient for the extraction of butanol, (iv) have a low partition coefficient for the extraction of nutrients, (v) have a low tendency to form emulsions with the fermentation medium, and (vi) be low cost and nonhazardous. Suitable organic extractants for use in the Methods disclosed herein are 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, C12 to C22 fatty amides and mixtures thereof. As used herein, the term “mixtures thereof” encompasses both mixtures within and mixtures between these group members, for example mixtures within C12 to C22 fatty alcohols, and also mixtures between C12 to C22 fatty alcohols and C12 to C22 fatty acids, for example.
In some instances extractants having less than 12-carbon chain lengths can be harmful to the microorganism and therefore harmful to the process of providing butanol via a biosynthetic path. In the case of an 11-carbon extractant, the effect on a microorganism can be dependent on the conditions, but can be harmful. In the case where a C11 fatty alcohol, C11 fatty acid, an ester of a C12 fatty acid, a C11 aldehyde, and mixtures thereof can be deleterious to the process, for example in the case where a microorganism is adversely affected by the C11 compound under the conditions used, such use is to be avoided. Suitable organic extractants are further selected from the group consisting of oleyl alcohol (CAS No. 143-28-2), behenyl alcohol (CAS No. 661-19-8), cetyl alcohol (CAS No. 36653-82-4), lauryl alcohol, also referred to as 1-dodecanol (CAS No. 112-53-8), myristyl alcohol (112-72-1), stearyl alcohol (CAS No. 112-92-5), 1-undecanol (CAS No. 112-42-5), oleic acid (CAS No. 112-80-1), lauric acid (CAS No. 143-07-7), myristic acid (CAS No. 544-63-8), stearic acid (CAS No. 57-11-4), methyl myristate CAS No. 124-10-7), methyl oleate (CAS No. 112-62-9), undecanal (CAS No. 112-44-7), lauric aldehyde (CAS No. 112-54-9), 2-methylundecanal (CAS No. 110-41-8), oleamide (CAS No. 301-02-0), linoleamide (CAS No. 3999-01-7), palmitamide (CAS No. 629-54-9) and stearylamide (CAS No. 124-26-5) and mixtures thereof. These organic extractants are available commercially from various sources, such as Sigma-Aldrich (St. Louis, Mo.), in various grades, many of which may be suitable for use in extractive fermentation to produce or recover butanol. Technical grades contain a mixture of compounds, including the desired component and higher and lower fatty components. For example, one commercially available technical grade oleyl alcohol contains about 65% oleyl alcohol and a mixture of higher and lower fatty alcohols.
One of reasonable skill in the art can appreciate that it may be advantageous to use a mixture of the organic extractants. For example, solvent mixtures may be used to increase the partition coefficient of the product. Additionally, solvent mixtures may be used to adjust and optimize physical characteristics of the solvent, such as the density, boiling point, and viscosity.
Methods for Producing Butanol Using Two-Phase Extractive Fermentation
The microorganism may be cultured in a suitable fermentation medium in a suitable fermentor to produce butanol. Any suitable fermentor may be used including a stirred tank fermentor, an airlift fermentor, a bubble fermentor, or any combination thereof. Materials and methods for the maintenance and growth of microbial cultures are well known to those skilled in the art of microbiology or fermentation science (See for example, Bailey et al., Biochemical Engineering Fundamentals, second edition, McGraw Hill, New York, 1986). Consideration must be given to appropriate fermentation medium, pH, temperature, and requirements for aerobic, microaerobic, or anaerobic conditions, depending on the specific requirements of the microorganism, the fermentation, and the process. The fermentation medium used is not critical, but it must support growth of the microorganism used and promote the biosynthetic pathway necessary to produce the desired butanol product. A conventional fermentation medium may be used, including, but not limited to, complex media containing organic nitrogen sources such as yeast extract or peptone and at least one fermentable carbon source; minimal media; and defined media. Suitable fermentable carbon sources include, but are not limited to, monosaccharides, such as glucose or fructose; disaccharides, such as lactose or sucrose; oligosaccharides; polysaccharides, such as starch or cellulose; one carbon substrates; and mixtures thereof. In addition to the appropriate carbon source, the fermentation medium may contain a suitable nitrogen source, such as an ammonium salt, yeast extract or peptone, minerals, salts, cofactors, buffers and other components, known to those skilled in the art (Bailey et al. supra). Suitable conditions for the extractive fermentation depend on the particular microorganism used and may be readily determined by one skilled in the art using routine experimentation.
Methods for Recovering Butanol Using Two-Phase Extractive Fermentation
Butanol may be recovered from a fermentation medium containing butanol, water, at least one fermentable carbon source, and a microorganism that has been genetically modified (that is, genetically engineered) to produce butanol via a biosynthetic pathway from at least one carbon source. Such genetically modified microorganisms can be selected from the group consisting of Escherichia coli, Lactobacillus plantarum, and Saccharomyces cerevisiae. The first step in the process is contacting the fermentation medium with a water immiscible organic extractant, described above, to form a two-phase mixture comprising an aqueous phase and a butanol-containing organic phase. “Contacting” means the fermentation medium and the organic extractant are brought into physical contact at any time during the fermentation process. In one embodiment, the fermentation medium further comprises ethanol, and the butanol-containing organic phase can contain ethanol.
The organic extractant may contact the fermentation medium at the start of the fermentation forming a biphasic fermentation medium. Alternatively, the organic extractant may contact the fermentation medium after the microorganism has achieved a desired amount of growth, which can be determined by measuring the optical density of the culture.
Further, the organic extractant may contact the fermentation medium at a time at which the butanol level in the fermentation medium reaches a preselected level, for example, before the butanol concentration reaches a toxic level. The butanol concentration may be monitored during the fermentation using methods known in the art, such as gas chromatography or high performance liquid chromatography.
Fermentation may be run under aerobic conditions for a time sufficient for the culture to achieve a preselected level of growth, as determined by optical density measurement. An inducer may then be added to induce the expression of the butanol biosynthetic pathway in the modified microorganism, and fermentation conditions are switched to microaerobic or anaerobic conditions to stimulate butanol production, as described in detail in Example 6 herein below. The extractant is added after the switch to microaerobic or anaerobic conditions.
After contacting the fermentation medium with the organic extractant, the butanol product partitions into the organic extractant, decreasing the concentration in the aqueous phase containing the microorganism, thereby limiting the exposure of the production microorganism to the inhibitory butanol product. The volume of the organic extractant to be used depends on a number of factors, including the volume of the fermentation medium, the size of the fermentor, the partition coefficient of the extractant for the butanol product, and the fermentation mode chosen, as described below. The volume of the organic extractant is about 3% to about 60% of the fermentor working volume.
The next step is separating the butanol-containing organic phase from the aqueous phase using methods known in the art, including but not limited to, siphoning, decantation, centrifugation, using a gravity settler, membrane-assisted phase splitting, and the like. Recovery of the butanol from the butanol-containing organic phase can be done using methods known in the art, including but not limited to, distillation, adsorption by resins, separation by molecular sieves, pervaporation, and the like. Specifically, distillation may be used to recover the butanol from the butanol-containing organic phase.
Gas stripping may be used concurrently with the organic extractant to remove the butanol product from the fermentation medium. Gas stripping may be done by passing a gas such as air, nitrogen, or carbon dioxide through the fermentation medium, thereby forming a butanol-containing gas phase. The butanol product may be recovered from the butanol-containing gas phase using methods known in the art, such as using a chilled water trap to condense the butanol, or scrubbing the gas phase with a solvent.
Any butanol remaining in the fermentation medium after the fermentation run is completed may be recovered by continued extraction using fresh or recycled organic extractant. Alternatively, the butanol can be recovered from the fermentation medium using methods known in the art, such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, pervaporation, and the like.
The two-phase extractive fermentation method may be carried out in a continuous mode in a stirred tank fermentor. In this mode, the mixture of the fermentation medium and the butanol-containing organic extractant is removed from the fermentor. The two phases are separated by means known in the art including, but not limited to, siphoning, decantation, centrifugation, using a gravity settler, membrane-assisted phase splitting, and the like, as described above. After separation, the fermentation medium may be recycled to the fermentor or may be replaced with fresh medium. Then, the extractant is treated to recover the butanol product as described above. The extractant may then be recycled back into the fermentor for further extraction of the product. Alternatively, fresh extractant may be continuously added to the fermentor to replace the removed extractant. This continuous mode of operation offers several advantages. Because the product is continually removed from the reactor, a smaller volume of organic extractant is required enabling a larger volume of the fermentation medium to be used. This results in higher production yields. The volume of the organic extractant may be about 3% to about 50% of the fermentor working volume; 3% to about 20% of the fermentor working volume; or 3% to about 10% of the fermentor working volume. It is beneficial to use the smallest amount of extractant in the fermentor as possible to maximize the volume of the aqueous phase, and therefore, the amount of cells in the fermentor. The process may be operated in an entirely continuous mode in which the extractant is continuously recycled between the fermentor and a separation apparatus and the fermentation medium is continuously removed from the fermentor and replenished with fresh medium. In this entirely continuous mode, the butanol product is not allowed to reach the critical toxic concentration and fresh nutrients are continuously provided so that the fermentation may be carried out for long periods of time. The apparatus that may be used to carryout these modes of two-phase extractive fermentations are well known in the art. Examples are described, for example, by Kollerup et al. in U.S. Pat. No. 4,865,973.
Batchwise fermentation mode may also be used. Batch fermentation, which is well known in the art, is a closed system in which the composition of the fermentation medium is set at the beginning of the fermentation and is not subjected to artificial alterations during the process. In this mode, a volume of organic extractant is added to the fermentor and the extractant is not removed during the process. Although this mode is simpler than the continuous or the entirely continuous modes described above, it requires a larger volume of organic extractant to minimize the concentration of the inhibitory butanol product in the fermentation medium. Consequently, the volume of the fermentation medium is less and the amount of product produced is less than that obtained using the continuous mode. The volume of the organic solvent in the batchwise mode may be 20% to about 60% of the fermentor working volume; or 30% to about 60% of the fermentor working volume. It is beneficial to use the smallest volume of extractant in the fermentor as possible, for the reason described above.
Fed-batch fermentation mode may also be used. Fed-batch fermentation is a variation of the standard batch system, in which the nutrients, for example glucose, are added in increments during the fermentation. The amount and the rate of addition of the nutrient may be determined by routine experimentation. For example, the concentration of critical nutrients in the fermentation medium may be monitored during the fermentation. Alternatively, more easily measured factors such as pH, dissolved oxygen, and the partial pressure of waste gases, such as carbon dioxide, may be monitored. From these measured parameters, the rate of nutrient addition may be determined. The amount of organic solvent used in this mode is the same as that used in the batch-wise mode, described above.
Extraction of the product may be done downstream of the fermentor, rather than in situ. In this external mode, the extraction of the butanol product into the organic extractant is carried out on the fermentation medium removed from the fermentor. The amount of organic solvent used is about 20% to about 60% of the fermentor working volume; or 30% to about 60% of the fermentor working volume. The fermentation medium may be removed from the fermentor continuously or periodically, and the extraction of the butanol product by the organic extractant may be done with or without the removal of the cells from the fermentation medium. The cells may be removed from the fermentation medium by means known in the art including, but not limited to, filtration or centrifugation. After separation of the fermentation medium from the extractant by means described above, the fermentation medium may be recycled into the fermentor, discarded, or treated for the removal of any remaining butanol product. Similarly, the isolated cells may also be recycled into the fermentor. After treatment to recover the butanol product, the extractant may be recycled for use in the extraction process. Alternatively, fresh extractant may be used. In this mode the solvent is not present in the fermentor, so the toxicity of the solvent is much less of a problem. If the cells are separated from the fermentation medium before contacting with the solvent, the problem of solvent toxicity is further reduced. Furthermore, using this external mode there is less chance of forming an emulsion and evaporation of the solvent is minimized, alleviating environmental concerns.
A method for the production of butanol is provided, wherein a microorganism that has been genetically modified of being capable of converting at least one fermentable carbon source into butanol, is grown in a biphasic fermentation medium. The biphasic fermentation medium comprises an aqueous phase and a water immiscible organic extractant, as described above, wherein the biphasic fermentation medium comprises from about 3% to about 60% by volume of the organic extractant. The microorganism may be grown in the biphasic fermentation medium for a time sufficient to extract butanol into the extractant to form a butanol-containing organic phase. In the case where the fermentation medium further comprises ethanol, the butanol-containing organic phase may contain ethanol. The butanol-containing organic phase is then separated from the aqueous phase, as described above. Subsequently, the butanol is recovered from the butanol-containing organic phase, as described above.
Isobutanol may be produced by extractive fermentation with the use of a modified Escherichia coli or Saccharomyces cerevisiae strain in combination with oleyl alcohol as the organic extractant. Using the method described herein provides a high effective titer for isobutanol. Atsumi et al. (Nature 451(3):86-90, 2008) report isobutanol titers up to 22 g/L using fermentation with an Escherichia coli that was genetically modified to contain an isobutanol biosynthetic pathway. Butanol produced by the method disclosed herein has an effective titer of greater than 22 g per liter of the fermentation medium. Alternatively, the butanol produced by methods disclosed has an effective titer of at least 25 g per liter of the fermentation medium. Alternatively, the butanol produced by methods described herein has an effective titer of at least 30 g per liter of the fermentation medium. Alternatively, the butanol produced by methods described herein has an effective titer of at least 37 g per liter of the fermentation medium.
Without being held to theory, it is believed that the higher butanol titer obtained with the extractive fermentation method disclosed herein results, in part, from the removal of the toxic butanol product from the fermentation medium, thereby keeping the level below that which is toxic to the microorganism.
The use of the organic extractant oleyl alcohol has an additional beneficial effect that is surprising and not well understood at the time of presenting this invention. Specifically, the use of oleyl alcohol as the extractant in combination with gas stripping provides significantly higher titers than gas stripping alone, even though gas stripping alone is effective in keeping the butanol below toxic levels. Organic extractants comprising or consisting essentially of oleyl alcohol can provide improved titers in the processes described herein.
Referring now to FIG. 3, there is shown a schematic representation of one embodiment of processes for producing and recovering butanol using in situ extractive fermentation. An aqueous stream 10 of at least one fermentable carbon source is introduced into a fermentor 20, which contains at least one microorganism (not shown) genetically modified to convert the at least one fermentable carbon source into butanol. A stream of the first water immiscible extractant 12 and a stream of the optional second water immiscible extractant 14 are introduced to a vessel 16, in which the extractants are combined to form the extractant 18. A stream of the extractant 18 is introduced into the fermentor 20, whereby contact between the fermentation medium and the extractant to form a two-phase mixture comprising an aqueous phase and a butanol-containing organic phase occurs. A stream 26 comprising both the aqueous and organic phases is introduced into a vessel 38, in which separation of the aqueous and organic phases is performed to produce a butanol-containing organic phase 40 and an aqueous phase 42.
Referring now to FIG. 4, there is shown a schematic representation of one embodiment of processes for producing and recovering butanol using in situ extractive fermentation. An aqueous stream 10 of at least one fermentable carbon source is introduced into a fermentor 20, which contains at least one microorganism (not shown) genetically modified to convert the at least one fermentable carbon source into butanol. A stream of the first water immiscible extractant 12 and a stream of the optional second water immiscible extractant 14 are introduced separately to the fermentor 20, whereby contact between the fermentation medium and the extractant to form a two-phase mixture comprising an aqueous phase and a butanol-containing organic phase occurs. A stream 26 comprising both the aqueous and organic phases is introduced into a vessel 38, in which separation of the aqueous and organic phases is performed to produce a butanol-containing organic phase 40 and an aqueous phase 42.
Referring now to FIG. 5, there is shown a schematic representation of one embodiment of processes for producing and recovering butanol using in situ extractive fermentation. An aqueous stream 10 of at least one fermentable carbon source is introduced into a first fermentor 20, which contains at least one microorganism (not shown) genetically modified to convert the at least one fermentable carbon source into butanol. A stream of the first water immiscible extractant 12 is introduced to the fermentor 20, and a stream 22 comprising a mixture of the first solvent and the contents of fermentor 20 is introduced into a second fermentor 24. A stream of the optional second water immiscible extractant 14 is introduced into the second fermentor 24, whereby contact between the fermentation medium and the extractant to form a two-phase mixture comprising an aqueous phase and a butanol-containing organic phase occurs. A stream 26 comprising both the aqueous and organic phases is introduced into a vessel 38, in which separation of the aqueous and organic phases is performed to produce a butanol-containing organic phase 40 and an aqueous phase 42.
Referring now to FIG. 6, there is shown a schematic representation of one embodiment of processes for producing and recovering butanol in which extraction of the product is performed downstream of the fermentor, rather than in situ. An aqueous stream 110 of at least one fermentable carbon source is introduced into a fermentor 120, which contains at least one microorganism (not shown) genetically modified to convert the at least one fermentable carbon source into butanol. A stream of the first water immiscible extractant 112 and a stream of the optional second water immiscible extractant 114 are introduced to a vessel 116, in which the water immiscible extractants are combined to form the extractant 118. At least a portion, shown as stream 122, of the fermentation medium in fermentor 120 is introduced into vessel 124. A stream of the extractant 118 is also introduced into vessel 124, whereby contact between the fermentation medium and the extractant to form a two-phase mixture comprising an aqueous phase and a butanol-containing organic phase occurs. A stream 126 comprising both the aqueous and organic phases is introduced into a vessel 138, in which separation of the aqueous and organic phases is performed to produce a butanol-containing organic phase 140 and an aqueous phase 142.
Referring now to FIG. 7, there is shown a schematic representation of one embodiment of processes for producing and recovering butanol in which extraction of the product is performed downstream of the fermentor, rather than in situ. An aqueous stream 110 of at least one fermentable carbon source is introduced into a fermentor 120, which contains at least one microorganism (not shown) genetically modified to convert the at least one fermentable carbon source into butanol. A stream of the first water immiscible extractant 112 and a stream of the optional second water immiscible extractant 114 are introduced separately to a vessel 124, in which the water immiscible extractants are combined to form the extractant 118. At least a portion, shown as stream 122, of the fermentation medium in fermentor 120 is also introduced into vessel 124, whereby contact between the fermentation medium and the extractant to form a two-phase mixture comprising an aqueous phase and a butanol-containing organic phase occurs. A stream 126 comprising both the aqueous and organic phases is introduced into a vessel 138, in which separation of the aqueous and organic phases is performed to produce a butanol-containing organic phase 140 and an aqueous phase 142.
Referring now to FIG. 8, there is shown a schematic representation of one embodiment of processes for producing and recovering butanol in which extraction of the product is performed downstream of the fermentor, rather than in situ. An aqueous stream 110 of at least one fermentable carbon source is introduced into a fermentor 120, which contains at least one microorganism (not shown) genetically modified to convert the at least one fermentable carbon source into butanol. A stream of the first water immiscible extractant 112 is introduced to a vessel 128, and at least a portion, shown as stream 122, of the fermentation medium in fermentor 120 is also introduced into vessel 128. A stream 130 comprising a mixture of the first water immiscible extractant and the contents of fermentor 120 is introduced into a second vessel 132. A stream of the optional second water immiscible extractant 114 is introduced into the second vessel 132, whereby contact between the fermentation medium and the extractant to form a two-phase mixture comprising an aqueous phase and a butanol-containing organic phase occurs. A stream 134 comprising both the aqueous and organic phases is introduced into a vessel 138, in which separation of the aqueous and organic phases is performed to produce a butanol-containing organic phase 140 and an aqueous phase 142.
The extractive processes described herein can be run as batch processes or can be run in a continuous mode where fresh extractant is added and used extractant is pumped out such that the amount of extractant in the fermentor remains constant during the entire fermentation process. Such continuous extraction of products and byproducts from the fermentation can increase effective rate, titer and yield.
In yet another embodiment, it is also possible to operate the liquid-liquid extraction in a flexible co-current or, alternatively, counter-current way that accounts for the difference in batch operating profiles when a series of batch fermentors are used. In this scenario the fermentors are filled with fermentable mash which provides at least one fermentable carbon source and microorganism in a continuous fashion one after another for as long as the plant is operating. Referring to FIG. 9, once Fermentor F100 fills with mash and microorganism, the mash and microorganism feeds advance to Fermentor F101 and then to Fermentor F102 and then back to Fermentor F100 in a continuous loop. The fermentation in any one fermentor begins once mash and microorganism are present together and continues until the fermentation is complete. The mash and microorganism fill time equals the number of fermentors divided by the total cycle time (fill, ferment, empty and clean). If the total cycle time is 60 hours and there are 3 fermentors then the fill time is 20 hours. If the total cycle time is 60 hours and there are 4 fermentors then the fill time is 15 hours.
Adaptive co-current extraction follows the fermentation profile assuming the fermentor operating at the higher broth phase titer can utilize the extracting solvent stream richest in butanol concentration and the fermentor operating at the lowest broth phase titer will benefit from the extracting solvent stream leanest in butanol concentration. For example, referring again to FIG. 9, consider the case where Fermentor F100 is at the start of a fermentation and operating at relatively low butanol broth phase (B) titer, Fermentor F101 is in the middle of a fermentation operating at relatively moderate butanol broth phase titer and Fermentor F102 is near the end of a fermentation operating at relatively high butanol broth phase titer. In this case, lean extracting solvent (S), with minimal or no extracted butanol, can be fed to Fermentor F100, the “solvent out” stream (S′) from Fermentor F100 having an extracted butanol component can then be fed to Fermentor F101 as its “solvent in” stream and the solvent out stream from F101 can then e fed to Fermentor F102 as its solvent in stream. The solvent out stream from F102 can then be sent to be processed to recover the butanol present in the stream. The processed solvent stream from which most of the butanol is removed can be returned to the system as lean extracting solvent and would be the solvent in feed to Fermentor F100 above.
As the fermentations proceed in an orderly fashion the valves in the extracting solvent manifold can be repositioned to feed the leanest extracting solvent to the fermentor operating at the lowest butanol broth phase titer. For example, assume (a) Fermentor F102 completes its fermentation and has been reloaded and fermentation begins anew, (b) Fermentor F100 is in the middle of its fermentation operating at moderate butanol broth phase titer and (c) Fermentor F101 is near the end of its fermentation operating at relatively higher butanol broth phase titer. In this scenario the leanest extracting solvent would feed F102, the extracting solvent leaving F102 would feed Fermentor F100 and the extracting solvent leaving Fermentor F100 would feed Fermentor F101.
The advantage of operating this way can be to maintain the broth phase butanol titer as low as possible for as long as possible to realize improvements in productivity. Additionally, it can be possible to drop the temperature in the other fermentors that have progressed further into fermentation that are operating at higher butanol broth phase titers. The drop in temperature can allow for improved tolerance to the higher butanol broth phase titers.
EXAMPLES
The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.
All solvents (that is, extractants) were obtained from Sigma-Aldrich (St. Louis, Mo.) and were used without further purification. The oleyl alcohol used was technical grade, which contained a mixture of oleyl alcohol (65%) and higher and lower fatty alcohols. The purity of the other solvents used was as follows: oleic acid, 65 to 88%; octanoic acid, 98%; nonanol, 98%; 1-dodecanol, 98%; 1-nonanal, 95%, and 1-decanol, 98%. Isobutanol was obtained from Sigma-Aldrich and was used without further purification.
General Methods
Construction of Recombinant Escherichia coli Strain NGCI-031
A recombinant Escherichia coli strain comprising an isobutanol biosynthetic pathway and deletions of the following genes, pflB (SEQ ID NO:23, encoding for pyruvate formate lyase), IdhA (SEQ ID NO:24, encoding for lactate dehydrogenase), adhE (SEQ ID NO:25, encoding for alcohol dehydrogenase), and frdB (SEQ ID NO:27, encoding a subunit of fumarate reductase), was constructed as described below. The genes in the isobutanol biosynthetic pathway were budB from Klebsiella pneumoniae (given as SEQ ID NO:1), ilvC from Escherichia coli (given as SEQ ID NO:3), ilvD from Escherichia coli (given as SEQ ID NO:5), kivD from Lactococcus lactis (given as SEQ ID NO:7), and sadB from Achromobacter xylosoxidans (given as SEQ ID NO:9). The construction of the recombinant strain was done in two steps. First, an Escherichia coli strain having the aforementioned gene deletions was constructed. Then, the genes encoding the isobutanol biosynthetic pathway were introduced into the strain.
Construction of Recombinant Escherichia coli Strain Having Deletions of pflB, IdhA, adhE and frdB Genes
The Keio collection of E. coli strains (Baba et al., Mol. Syst. Biol., 2:1-11, 2006) was used for the production of the E. coli strain having the intended gene deletions, which is referred to herein as the four-knock out E. coli strain. The Keio collection is a library of single gene knockouts created in strain E. coli BW25113 by the method of Datsenko and Wanner (Datsenko, K. A. & Wanner, B. L., Proc. Natl. Acad. Sci., U.S.A. 97 6640-6645, 2000). In the collection, each deleted gene was replaced with a FRT-flanked kanamycin marker that was removable by Flp recombinase. The four-knock out E. coli strain was constructed by moving the knockout-kanamycin marker from the Keio donor strain by P1 transduction to a recipient strain. After each P1 transduction to produce a knockout, the kanamycin marker was removed by Flp recombinase. This markerless strain acted as the new donor strain for the next P1 transduction.
The four-knock out E. coli strain was constructed in Keio strain JW0886 by P1vir transductions with P1 phage lysates prepared from three Keio strains in addition to JW0886. The Keio strains used are listed below:

    
    
        JW0886: the kan marker is inserted in the pflB gene
        JW4114: the kan marker is inserted in the frdB gene
        JW1375: the kan marker is inserted in the IdhA gene
        JW1228: the kan marker is inserted in the adhE gene
    
    


P1vir transductions were carried out as described by Miller with some modifications (Miller, J. H. 1992. A Short Course in Bacterial Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Briefly, to prepare a transducing lysate, cells of the donor strain were grown overnight in Luria-Bertani (LB) medium at 37° C. while shaking. An overnight growth of these cells was sub-cultured into LB medium containing 0.005 M CaCl2 and placed in a 37° C. water bath with no aeration. One hour prior to adding phage, the cells were incubated at 37° C. with shaking. After final growth of the cells, a 1.0 mL aliquot of the culture was dispensed into 14-mL tubes and approximately 107 P1vir phage was added. The tubes were incubated in a 37° C. water bath for 20 min, after which 2.5 mL of 0.8% LB top agar was added to each tube. The contents of the tubes were spread on an LB agar plate and were incubated at 37° C. The following day the soft agar layer was scraped into a centrifuge tube. The surface of the plate was washed with LB medium and added to the centrifuge tube, followed by a few drops of CHCl3 and then the tube was vigorously agitated using a vortex mixer. After centrifugation at 4,000 rpm for 10 min, the supernatant containing the P1vir lysate was collected.
For transduction, the recipient strain was grown overnight in 1-2 mL of LB medium at 37° C. with shaking. Cultures were pelleted by centrifugation in a microcentrifuge (Eppendorf) at 10,000 rpm for 1 min at room temperature. The cell pellet was resuspended in an equal volume of MC buffer (0.1 M MgSO4, 0.005 M CaCl2), dispensed into tubes in 0.1 mL aliquots and 0.1 mL and 0.01 mL of P1vir lysate was added. A control tube containing no P1vir lysate was also included. The tubes were incubated for 20 min at 37° C. after which time, 0.2 mL of 0.1 M sodium citrate was added to stop the P1 infection. One mL of LB medium was added to each tube before the tubes were incubated at 37° C. for 1 h. After incubation the cells were pelleted as described above, resuspended in 50-200 μL of LB prior to spreading on the LB plates containing 25 μg/mL of kanamycin and were incubated overnight at 37° C. Transductants were screened by colony PCR with chromosome specific primers flanking the region upstream and downstream of the kanamycin marker insertion.
Removal of the kanamycin marker from the chromosome was obtained by transforming the kanamycin-resistant strain with plasmid pCP20 (Cherepanov, P. P. and Wackernagel, W., Gene, 158: 9-14, 1995) followed by spreading onto LB ampicillin (100 μg/mL) plates and incubating at 30° C. The pCP20 plasmid carries the yeast FLP recombinase under the control of the λPR promoter. Expression from this promoter is controlled by the cI857 temperature-sensitive repressor residing on the plasmid. The origin of replication of pCP20 is also temperature sensitive. Ampicillin resistant colonies were streaked onto LB agar plates and incubated at 42° C. The higher incubation temperature simultaneously induced expression of the FLP recombinase and cured the pCP20 plasmid from the cell. Isolated colonies were patched to grids onto the LB plates containing kanamycin (25 μg/mL), and LB ampicillin (100 μg/mL) plates and LB plates. The resulting kanamycin-sensitive, ampicillin-sensitive colonies were screened by colony PCR to confirm removal of the kanamycin marker from the chromosome.
For colony PCR amplifications the HotStarTaq Master Mix (Qiagen, Valencia, Calif.; catalog no. 71805-3) was used according to the manufacturer's protocol. Into a 25 μL Master Mix reaction containing 0.2 μM of each chromosome specific PCR primer, a small amount of a colony was added. Amplification was carried out in a DNA Thermocycler GeneAmp 9700 (PE Applied Biosystems, Foster City, Calif.). Typical colony PCR conditions were as follows: 15 min at 95° C.; 30 cycles of 95° C. for 30 sec, annealing temperature ranging from 50-58° C. for 30 sec, primers extended at 72° C. with an extension time of approximately 1 min/kb of DNA; then 10 min at 72° C. followed by a hold at 4° C. PCR product sizes were determined by gel electrophoresis by comparison with known molecular weight standards.
For transformations, electrocompetent cells of E. coli were prepared as described by Ausubel, F. M., et al., (Current Protocols in Molecular Biology, 1987, Wiley-Interscience,). Cells were grown in 25-50 mL of LB medium at 30-37° C. and harvested at an OD600 of 0.5-0.7 by centrifugation at 10,000 rpm for 10 min. These cells are washed twice in sterile ice-cold water in a volume equal to the original starting volume of the culture. After the final wash cells were resuspended in sterile water and the DNA to be transformed was added. The cells and DNA were transferred to chilled cuvettes and electroporated in a Bio-Rad Gene Pulser II according to manufacturer's instructions (Bio-Rad Laboratories, Inc Hercules, Calif.).
Strain JW0886 (ΔpflB::kan) was transformed with plasmid pCP20 and spread on LB plates containing 100 μg/mL of ampicillin at 30° C. Ampicillin resistant transformants were then selected, streaked on LB plates and grown at 42° C. Isolated colonies were patched onto the ampicillin and kanamycin selective medium plates and LB plates. Kanamycin-sensitive and ampicillin-sensitive colonies were screened by colony PCR with primers pflB CkUp (SEQ ID NO:11) and pflB CkDn (SEQ ID NO:12). A 10 μL aliquot of the PCR reaction mix was analyzed by gel electrophoresis. The expected approximate 0.4 kb PCR product was observed confirming removal of the marker and creating the “JW0886 markerless” strain. This strain had a deletion of the pflB gene.
The “JW0886 markerless” strain was transduced with a P1vir lysate from JW4114 (frdB::kan) and streaked onto the LB plates containing 25 μg/mL of kanamycin. The kanamycin-resistant transductants were screened by colony PCR with primers frdB CkUp (SEQ ID NO:13) and frdB CkDn (SEQ ID NO: 14). Colonies that produced the expected approximate 1.6 kb PCR product were made electrocompetent, as described above, and transformed with pCP20 for marker removal as described above. Transformants were first spread onto LB plates containing 100 μg/mL of ampicillin at 30° C. and ampicillin resistant transformants were then selected and streaked on LB plates and grown at 42° C. Isolated colonies were patched onto ampicillin and the kanamycin selective medium plates and LB plates. Kanamycin-sensitive, ampicillin-sensitive colonies were screened by PCR with primers frdB CkUp (SEQ ID NO:13) and frdB CkDn (SEQ ID NO: 14). The expected approximate 0.4 kb PCR product was observed confirming marker removal and creating the double knockout strain, “ΔpflB frdB”.
The double knockout strain was transduced with a P1vir lysate from JW1375 (ΔldhA::kan) and spread onto the LB plates containing 25 μg/mL of kanamycin. The kanamycin-resistant transductants were screened by colony PCR with primers IdhA CkUp (SEQ ID NO:15) and IdhA CkDn (SEQ ID NO:16). Clones producing the expected 1.1 kb PCR product were made electrocompetent and transformed with pCP20 for marker removal as described above. Transformants were spread onto LB plates containing 100 μg/mL of ampicillin at 30° C. and ampicillin resistant transformants were streaked on LB plates and grown at 42° C. Isolated colonies were patched onto ampicillin and kanamycin selective medium plates and LB plates. Kanamycin-sensitive, ampicillin-sensitive colonies were screened by PCR with primers IdhA CkUp (SEQ ID NO:15) and IdhA CkDn (SEQ ID NO:16) for a 0.3 kb product. Clones that produced the expected approximate 0.3 kb PCR product confirmed marker removal and created the triple knockout strain designated the “three-knock out strain” (ΔpflB frdB IdhA).
The “three-knock out strain” was transduced with a P1vir lysate from JW1228 (ΔadhE::kan) and spread onto the LB plates containing 25 μg/mL kanamycin. The kanamycin-resistant transductants were screened by colony PCR with primers adhE CkUp (SEQ ID NO: 17) and adhE CkDn (SEQ ID NO:18). Clones that produced the expected 1.6 kb PCR product were made electrocompetent and transformed with pCP20 for marker removal. Transformants were spread onto LB plates containing 100 μg/mL of ampicillin at 30° C. Ampicillin resistant transformants were streaked on LB plates and grown at 42° C. Isolated colonies were patched onto ampicillin and kanamycin selective plates and LB plates. Kanamycin-sensitive, ampicillin-sensitive colonies were screened by PCR with the primers adhE CkUp (SEQ ID NO: 17) and adhE CkDn (SEQ ID NO:18). Clones that produced the expected approximate 0.4 kb PCR product were named the “four-knock out strain” (ΔpflB frdB IdhA adhE).
Introduction of the Set of Genes Encoding an Isobutanol Biosynthetic Pathway into the Four-Knock Out E. coli Strain.
The plasmid pTrc99A::budB-ilvC-ilvD-kivD was constructed as described in Examples 9-14 of copending and commonly owned U.S. Patent Application Publication No. 2007/0092957, which are incorporated herein by reference. This plasmid comprised the following genes, budB encoding acetolactate synthase from Klebsiella pneumoniae (SEQ ID NO:1), ilvC gene encoding acetohydroxy acid reductoisomerase from E. coli (SEQ ID NO:3), ilvD encoding acetohydroxy acid dehydratase from E. coli (SEQ ID NO:5), and kivD encoding the branched-chain keto acid decarboxylase from Lactococcus lactis (SEQ ID NO:7). The sadB gene from Achromobacter xylosoxidans encoding a butanol dehydrogenase (SEQ ID NO:9) was subcloned into the pTrc99A::budB-ilvC-ilvD-kivD plasmid as described below.
A DNA fragment encoding a butanol dehydrogenase (DNA: SEQ ID NO:9; protein: SEQ ID NO:10) from Achromobacter xylosoxidans (disclosed in copending and commonly owned U.S. Patent Application No. 61/048,291) was amplified from A. xylosoxidans genomic DNA using standard conditions. The DNA was prepared using a Gentra Puregene kit (Gentra Systems, Inc., Minneapolis, Minn.; catalog number D-5500A) following the recommended protocol for gram negative organisms. PCR amplification was done using forward and reverse primers N473 and N469 (SEQ ID NOs:19 and 20, respectively) with Phusion high Fidelity DNA Polymerase (New England Biolabs, Beverly, Mass.). The PCR product was TOPO-Blunt cloned into pCR4 BLUNT (Invitrogen) to produce pCR4Blunt::sadB, which was transformed into E. coli Mach-1 cells. Plasmid was subsequently isolated from four clones, and the sequence verified.
The sadB coding region was then cloned into the vector pTrc99a (Amann et al., Gene 69: 301-315, 1988). The pCR4Blunt::sadB was digested with EcoRI, releasing the sadB fragment, which was ligated with EcoRI-digested pTrc99a to generate pTrc99a::sadB. This plasmid was transformed into E. coli Mach 1 cells and the resulting transformant was named Mach1/pTrc99a::sadB. The activity of the enzyme expressed from the sadB gene in these cells was determined to be 3.5 mmol/min/mg protein in cell-free extracts when analyzed using isobutyraldehyde as the standard.
Then, the sadB gene was subcloned into pTrc99A::budB-ilvC-ilvD-kivD as follows. The sadB coding region was amplified from pTrc99a::sadB using primers N695A (SEQ ID NO:21) and N696A (SEQ ID NO:22) with Phusion High Fidelity DNA Polymerase (New England Biolabs, Beverly, Mass.). Amplification was carried out with an initial denaturation at 98° C. for 1 min, followed by 30 cycles of denaturation at 98° C. for 10 sec, annealing at 62° C. for 30 sec, elongation at 72° C. for 20 sec and a final elongation cycle at 72° C. for 5 min, followed by a 4° C. hold. Primer N695A contained an AvrII restriction site for cloning and a RBS (ribosomal binding site) upstream of the ATG start codon of the sadB coding region. The N696A primer included an XbaI site for cloning. The 1.1 kb PCR product was digested with AvrII and XbaI (New England Biolabs, Beverly, Mass.) and gel purified using a Qiaquick Gel Extraction Kit (Qiagen Inc., Valencia, Calif.)). The purified fragment was ligated with pTrc99A::budB-ilvC-ilvD-kivD, that had been cut with the same restriction enzymes, using T4 DNA ligase (New England Biolabs, Beverly, Mass.). The ligation mixture was incubated at 16° C. overnight and then transformed into E. coli Mach 1TM competent cells (Invitrogen) according to the manufacturer's protocol. Transformants were obtained following growth on LB agar with 100 μg/ml of ampicillin. Plasmid DNA from the transformants was prepared with QIAprep Spin Miniprep Kit (Qiagen Inc., Valencia, Calif.) according to manufacturer's protocols. The resulting plasmid was called pTrc99A::budB-ilvC-ilvD-kivD-sadB. Electrocompetent four-knock out E. coli cells, prepared as described above, were transformed with pTrc99A::budB-ilvC-ilvD-kivD-sadB. Transformants were streaked onto LB agar plates containing 100 μg/mL of ampicillin. The resulting recombinant E. coli strain comprised an isobutanol biosynthetic pathway, encoded by plasmid pTrc99A::budB-ilvC-ilvD-kivD-sadB, and deletions of pflB, frdB, IdhA, and adhE genes and was designated as strain NGCI-031.
Construction of the Yeast Strain NGI-049
NGI-049 is a Saccharomyces cerevisiae strain with insertion-inactivation of endogenous PDC1, PDC5, and PDC6 genes, and containing expression vectors pLH475-Z4B8 and pLH468. PDC1, PDC5, and PDC6 genes encode the three major isozymes of pyruvate decarboxylase. The strain expresses genes encoding enzymes for an isobutanol biosynthetic pathway that are integrated or on plasmids.
Expression Vector pLH475-Z4B8
The pLH475-Z4B8 plasmid (SEQ ID NO:30) was constructed for expression of ALS and KAR1 in yeast. pLH475-Z4B8 is a pHR81 vector (ATCC #87541) containing the following chimeric genes: 1) the CUP1 promoter (SEQ ID NO:31), acetolactate synthase coding region from Bacillus subtilis (AlsS; SEQ ID NO:32; protein SEQ ID NO:33) and CYC1 terminator (SEQ ID NO:34); 2) an ILV5 promoter (SEQ ID NO:35), Pf5.IlvC-Z4B8 coding region (SEQ ID NO:36; protein SEQ ID NO:37) and ILV5 terminator (SEQ ID NO:38); and 3) the FBA1 promoter (SEQ ID NO:39), S. cerevisiae KAR1 coding region (ILV5; SEQ ID NO:40; protein SEQ ID NO:41) and CYC1 terminator.
The Pf5.IlvC-Z4B8 coding region is a sequence encoding KAR1 derived from Pseudomonas fluorescens but containing mutations, that was described in commonly owned and co-pending U.S. patent application Ser. No. 12/337,736, which is herein incorporated by reference. The Pf5.IlvC-Z4B8 encoded KAR1 (SEQ ID NO:37;) has the following amino acid changes as compared to the natural Pseudomonas fluorescens KAR1:
C33L: cysteine at position 33 changed to leucine,
R47Y: arginine at position 47 changed to tyrosine,
S50A: serine at position 50 changed to alanine,
T52D: threonine at position 52 changed to asparagine,
V53A: valine at position 53 changed to alanine,
L61F: leucine at position 61 changed to phenylalanine,
T80I: threonine at position 80 changed to isoleucine,
A156V: alanine at position 156 changed to threonine, and
G170A: glycine at position 170 changed to alanine.
The Pf5.IlvC-Z4B8 coding region was synthesized by DNA 2.0 (Palo Alto, Calif.; SEQ ID NO:6) based on codons that were optimized for expression in Saccharomyces cerevisiae. 
Expression Vector pLH468
The pLH468 plasmid (SEQ ID NO:42) was constructed for expression of DHAD, KivD and HADH in yeast.
Coding regions for B. subtilis ketoisovalerate decarboxylase (KivD) and Horse liver alcohol dehydrogenase (HADH) were synthesized by DNA2.0 based on codons that were optimized for expression in Saccharomyces cerevisiae (SEQ ID NO:43 and 45, respectively) and provided in plasmids pKivDy-DNA2.0 and pHadhy-DNA2.0. The encoded proteins are SEQ ID NOs:44 and 46, respectively. Individual expression vectors for KivD and HADH were constructed. To assemble pLH467 (pRS426::PGRD1-kivDy-GPD1t), vector pNY8 (SEQ ID NO:47; also named pRS426.GPD-ald-GPDt, described in commonly owned and co-pending US Patent App. Pub. US2008/0182308, Example 17, which is herein incorporated by reference) was digested with AscI and SfiI enzymes, thus excising the GPD1 promoter and the ald coding region. A GPD1 promoter fragment (SEQ ID NO:48) from pNY8 was PCR amplified to add an AscI site at the 5′ end, and an SpeI site at the 3′ end, using 5′ primer OT1068 and 3′ primer OT1067 (SEQ ID NOs:49 and 50). The AscI/SfiI digested pNY8 vector fragment was ligated with the GPD1 promoter PCR product digested with AscI and SpeI, and the SpeI-SfiI fragment containing the codon optimized kivD coding region isolated from the vector pKivD-DNA2.0. The triple ligation generated vector pLH467 (pRS426::PGPD1-kivDy-GPD1t). pLH467 was verified by restriction mapping and sequencing.
pLH435 (pRS425::PGPM1-Hadhy-ADH1t) was derived from vector pRS425::GPM-sadB (SEQ ID NO:51) which is described in commonly owned and co-pending U.S. Patent App. No. 61/058,970, Example 3, which is herein incorporated by reference. pRS425::GPM-sadB is the pRS425 vector (ATCC #77106) with a chimeric gene containing the GPM1 promoter (SEQ ID NO:52), coding region from a butanol dehydrogenase of Achromobacter xylosoxidans (sadB; SEQ ID NO:9; protein SEQ ID NO:10: disclosed in commonly owned and co-pending U.S. Patent App. No. 61/048,291), and ADH1 terminator (SEQ ID NO:53). pRS425::GPMp-sadB contains BbvI and PacI sites at the 5′ and 3′ ends of the sadB coding region, respectively. A NheI site was added at the 5′ end of the sadB coding region by site-directed mutagenesis using primers OT1074 and OT1075 (SEQ ID NO:54 and 55) to generate vector pRS425-GPMp-sadB-NheI, which was verified by sequencing. pRS425::PGPM1-sadB-NheI was digested with NheI and PacI to drop out the sadB coding region, and ligated with the NheI-PacI fragment containing the codon optimized HADH coding region from vector pHadhy-DNA2.0 to create pLH435.
To combine KivD and HADH expression cassettes in a single vector, yeast vector pRS411 (ATCC #87474) was digested with SacI and NotI, and ligated with the SacI-SalI fragment from pLH467 that contains the PGPD1-kivDy-GPD1t cassette together with the SalI-NotI fragment from pLH435 that contains the PGPM1-Hadhy-ADH1t cassette in a triple ligation reaction. This yielded the vector pRS411::PGPD1-kivDy-PGPM1-Hadhy (pLH441), which was verified by restriction mapping.
In order to generate a co-expression vector for all three genes in the lower isobutanol pathway: ilvD, kivDy and Hadhy, we used pRS423 FBA ilvD(Strep) (SEQ ID NO:56), which is described in commonly owned and co-pending U.S. Patent Application No. 61/100,792, as the source of the IlvD gene. This shuttle vector contains an F1 origin of replication (nt 1423 to 1879) for maintenance in E. coli and a 2 micron origin (nt 8082 to 9426) for replication in yeast. The vector has an FBA promoter (nt 2111 to 3108; SEQ ID NO:39) and FBA terminator (nt 4861 to 5860; SEQ ID NO:57). In addition, it carries the H is marker (nt 504 to 1163) for selection in yeast and ampicillin resistance marker (nt 7092 to 7949) for selection in E. coli. The ilvD coding region (nt 3116 to 4828; SEQ ID NO:58; protein SEQ ID NO:59) from Streptococcus mutans UA159 (ATCC #700610) is between the FBA promoter and FBA terminator forming a chimeric gene for expression. In addition there is a lumio tag fused to the ilvD coding region (nt 4829-4849).
The first step was to linearize pRS423 FBA ilvD(Strep) (also called pRS423-FBA(SpeI)-ilvD(Streptococcus mutans)-Lumio) with SacI and SacII (with SacII site blunt ended using T4 DNA polymerase), to give a vector with total length of 9,482 bp. The second step was to isolate the kivDy-hADHy cassette from pLH441 with SacI and KpnI (with KpnI site blunt ended using T4 DNA polymerase), which gives a 6,063 by fragment. This fragment was ligated with the 9,482 by vector fragment from pRS423-FBA(SpeI)-IlvD (Streptococcus mutans)-Lumio. This generated vector pLH468 (pRS423::PFBA1-ilvD(Strep)Lumio-FBA1t-PGPD1-kivDy-GPD1t-PGPM1-hadhy-ADH1t), which was confirmed by restriction mapping and sequencing.
Construction of pdc6::GPMp1-sadB Integration Cassette and PDC6 Deletion:
A pdc6::GPM1p-sadB-ADH1t-URA3r integration cassette was made by joining the GPM-sadB-ADHt segment (SEQ ID NO:60) from pRS425::GPM-sadB (described above) to the URA3r gene from pUC19-URA3r. pUC19-URA3r (SEQ ID NO:61) contains the URA3 marker from pRS426 (ATCC #77107) flanked by 75 bp homologous repeat sequences to allow homologous recombination in vivo and removal of the URA3 marker. The two DNA segments were joined by SOE PCR (as described by Horton et al. (1989) Gene 77:61-68) using as template pRS425::GPM-sadB and pUC19-URA3r plasmid DNAs, with Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-5405) and primers 114117-11A through 114117-11D (SEQ ID NOs:62, 63, 64 and 65), and 114117-13A and 114117-13B (SEQ ID NOs:66 and 67).
The outer primers for the SOE PCR (114117-13A and 114117-13B) contained 5′ and 3′  ̃50 bp regions homologous to regions upstream and downstream of the PDC6 promoter and terminator, respectively. The completed cassette PCR fragment was transformed into BY4700 (ATCC #200866) and transformants were maintained on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformants were screened by PCR using primers 112590-34G and 112590-34H (SEQ ID NOs:68 and 69), and 112590-34F and 112590-49E (SEQ ID NOs:70 and 71) to verify integration at the PDC6 locus with deletion of the PDC6 coding region. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth. The resulting identified strain has the genotype: BY4700 pdc6::PGPM1-sadB-ADH1t.
Construction of pdc1:: PDC1-ilvD Integration Cassette and PDC1 Deletion:
A pdc1::PDC1p-ilvD-FBA1t-URA3r integration cassette was made by joining the ilvD-FBA1t segment (SEQ ID NO:72) from pLH468 (described above) to the URA3r gene from pUC19-URA3r by SOE PCR (as described by Horton et al. (1989) Gene 77:61-68) using as template pLH468 and pUC19-URA3r plasmid DNAs, with Phusion DNA polymerase (New England Biolabs Inc., Beverly, Mass.; catalog no. F-5405) and primers 114117-27A through 114117-27D (SEQ ID NOs:73, 74, 75 and 76).
The outer primers for the SOE PCR (114117-27A and 114117-27D) contained 5′ and 3′  ̃50 bp regions homologous to regions downstream of the PDC1 promoter and downstream of the PDC1 coding sequence. The completed cassette PCR fragment was transformed into BY4700 pdc6::PGPM1-sadB-ADH1t and transformants were maintained on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202). Transformants were screened by PCR using primers 114117-36D and 135 (SEQ ID NOs:77 and 78), and primers 112590-49E and 112590-30F (SEQ ID NOs:70 and 79) to verify integration at the PDC1 locus with deletion of the PDC1 coding sequence. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth. The resulting identified strain “NYLA67” has the genotype: BY4700 pdc6::GPM1p-sadB-ADH1t pdc1::PDC1p-ilvD-FBA1t.
HIS3 Deletion
To delete the endogenous HIS3 coding region, a his3::URA3r2 cassette was PCR-amplified from URA3r2 template DNA (SEQ ID NO:84). URA3r2 contains the URA3 marker from pRS426 (ATCC #77107) flanked by 500 bp homologous repeat sequences to allow homologous recombination in vivo and removal of the URA3 marker. PCR was done using Phusion DNA polymerase and primers 114117-45A and 114117-45B (SEQ ID NOs:85 and 86) which generated a  ̃2.3 kb PCR product. The HIS3 portion of each primer was derived from the 5′ region upstream of the HIS3 promoter and 3′ region downstream of the coding region such that integration of the URA3r2 marker results in replacement of the HIS3 coding region. The PCR product was transformed into NYLA67 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on synthetic complete media lacking uracil and supplemented with 2% glucose at 30° C. Transformants were screened to verify correct integration by replica plating of transformants onto synthetic complete media lacking histidine and supplemented with 2% glucose at 30° C. The URA3r marker was recycled by plating on synthetic complete media supplemented with 2% glucose and 5-FOA at 30° C. following standard protocols. Marker removal was confirmed by patching colonies from the 5-FOA plates onto SD-URA media to verify the absence of growth. The resulting identified strain “NYLA73” has the genotype: BY4700 pdc6::GPM1p-sadB-ADH1t pdc1::PDC1p-ilvD-FBA1t Δhis3.
Construction of pdc5::kanMX Integration Cassette and PDC5 Deletion:
A pdc5::kanMX4 cassette was PCR-amplified from strain YLR134W chromosomal DNA (ATCC No. 403-4091) using Phusion DNA polymerase and primers PDC5::KanMXF and PDC5::KanMXR (SEQ ID NOs:80 and 81) which generated a  ̃2.2 kb PCR product. The PDC5 portion of each primer was derived from the 5′ region upstream of the PDC5 promoter and 3′ region downstream of the coding region such that integration of the kanMX4 marker results in replacement of the PDC5 coding region. The PCR product was transformed into NYLA73 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp. 201-202) and transformants were selected on YP media supplemented with 1% ethanol and geneticin (200 μg/ml) at 30° C. Transformants were screened by PCR to verify correct integration at the PDC locus with replacement of the PDC5 coding region using primers PDC5kofor and N175 (SEQ ID NOs:82 and 83). The identified correct transformants have the genotype: BY4700 pdc6::GPM1p-sadB-ADH1t pdc1::PDC1p-ilvD-FBA1t Δhis3 pdc5::kanMX4.
Plasmid vectors pLH468 and pLH475-Z4B8 were simultaneously transformed into strain BY4700 pdc6::GPM1p-sadB-ADH1t pdc1::PDC1p-ilvD-FBA1t Δhis3 pdc5::kanMX4 using standard genetic techniques (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).and maintained on synthetic complete media lacking histidine and uracil, and supplemented with 1% ethanol at 30° C.
GC Method for Determination of Isobutanol
The following GC method was used to determine the amount of isobutanol in the aqueous phase and organic phase in Examples 1-7 described below. The GC method utilized an HP-InnoWax column (30 m×0.32 mm ID, 0.25 μm film) from Agilent Technologies (Santa Clara, Calif.). The carrier gas was helium at a flow rate of 1 mL/min measured at 150° C. with constant head pressure; injector split was 1:10 at 200° C.; oven temperature was 45° C. for 1 min, 45° C. to 230° C. at 10° C./min, and 230° C. for 30 sec. Flame ionization detection was used at 260° C. with 40 mL/min helium makeup gas. Culture broth samples were filtered through 0.2 μm spin filters before injection. Depending on the analytical sensitivity desired, either 0.1 μL or 0.5 μL injection volumes were used. Calibrated standard curves were generated for the following compounds: ethanol, isobutanol, acetoin, meso-2,3-butanediol, and (2S,3S)-2,3-butanediol. Analytical standards were also utilized to identify retention times for isobutyraldehyde, isobutyric acid, and isoamyl alcohol. Under these conditions, the isobutanol retention time was about 5.33 minutes.
HPLC Method for Determination of Glucose and Isobutanol in the Aqueous Phase
For Examples 7 and 8, isobutanol in the organic phase was determined using the GC method described above. For Examples 7 and 8, isobutanol and glucose concentrations in the aqueous phase were measured by HPLC (Waters Alliance Model, Milford, Mass. or Agilent 1100 Series, Santa Clara, Calif.) using a Shodex sugar SH1011 column, 8.0 mm×300 mm, (Showa Denko K.K., Kanagawa, Japan (through Thompson Instruments, Clear Brook, Va.)) using 0.01 N aqueous sulfuric acid, isocratic, as the eluant. The sample was passed through a 0.2 μm syringe filter (PALL GHP membrane) into an HPLC vial. The HPLC run conditions were as follows:
Injection volume: 10 μL
Flow rate: 0.80 mL/minute
Run time: 32 minutes
Column Temperature: 50° C.
Detector: refractive index
Detector temperature: 40° C.
UV detection: 210 nm, 4 nm bandwidth
After the run, concentrations in the sample were determined from standard curves for each of the compounds. The retention times were 27.0 and 8.7 minutes for isobutanol and glucose, respectively.
Example 1
Screening of Solvents
The purpose of this Example was to screen various organic solvents for use in the extractive fermentation of butanols. Solvent characteristics that were investigated were the partitioning of isobutanol between the solvent and an aqueous phase, the emulsion forming tendency of the solvent in the two-phase system, and the biocompatibility of the solvent with a wild-type Saccharomyces cerevisiae strain.
The partitioning of isobutanol between water and the following solvents: oleic acid (CAS No. 112-80-1), oleyl alcohol (CAS No. 143-28-2), octanoic acid (CAS No. 124-07-2), 1-nonanol (CAS No. 28473-21-4), 1-dodecanol (CAS No. 112-53-8), 1-nonanal (CAS No. 124-19-6), and 1-decanol (CAS No. 112-30-1) was investigated. Isobutanol was added to water to give aqueous solutions having a final isobutanol concentration of 10, 30, 50, and 70 g/L. These aqueous butanol solutions (12 mL) were added to test tubes and 4 mL of the solvent to be tested was added. Each solvent was tested in duplicate at each isobutanol concentration. The tubes were incubated for 3 hours with mixing at 30° C. After that time, the aqueous phase and the solvent phase from each tube were separated by centrifugation and analyzed for isobutanol using the GC Method described in the General Methods Section herein above. The partition coefficient for isobutanol between each solvent phase and the aqueous phase was calculated, i.e., Kp=[Isobutanol]Org/[isobutanol]Aq. The results are summarized in Table 2.







TABLE 2







Partition Coefficients for Isobutanol










Solvent
Average Partition Coefficient







oleic acid
2.7



oleyl alcohol
3.7



octanoic acid
6.7



nonanol
6.7



1-dodecanol
5.2



1-nonanal
5.7



1-decanol
4.7










As can be seen from the data in the table, all the solvents tested had a favorable partition coefficient for isobutanol.
The biocompatibility of each solvent was determined using shake flask studies with Saccharomyces cerevisiae BY4741 obtained from ATCC. Seed shake flasks containing 700 mL of yeast extract/peptone/dextrose (YPD) medium were inoculated with 200 μL of S. cerevisiae BY4741 inoculum and incubated overnight at 30° C. with shaking at 250 rpm until the OD600 reached about 0.5. Samples were withdrawn from the culture for measurement of OD600 using a spectrophotometer and glucose concentration using HPLC.
The resulting seed culture was divided into eight 125 mL flasks as follows. Into one flask, which served as a control, 100 mL of the seed culture was added. Into the remaining seven flasks, 75 mL of the seed culture and 25 mL of the solvent to be tested was added. The flasks were incubated at 30° C., with shaking at 250 rpm in a table top shaker (Innova 4230, New Brunswick Scientific, Edison, N.J.) and samples were removed from each flask at various times and the OD600 and glucose concentration were measured. The results are summarized in Tables 3 and 4.







TABLE 3







Optical Density Results













OD600
OD600
OD600
OD600
OD600


Solvent
t = 0
t = 2 h
t = 4
t = 6 h
t = 24 h





none (control)
0.5
1.0
2.6
3.4
4.7


oleic acid
0.5
0.8
1.7
2.8
4.4


oleyl alcohol
0.5
1.0
2.0
2.4
3.5


octanoic acid
0.5
0.9
0.8
0.7
1.0


nonanol
0.5
0.5
0.7
0.6
0.7


1-dodecanol
0.5
0.9
1.6
2.8
4.2


1-nonanal
0.5
0.7
0.7
0.8
1.8


1-decanol
0.5
0.5
0.7
0.6
0.8
















TABLE 4







Glucose Results













Glucose
Glucose
Glucose
Glucose
Glucose



(g/L)
(g/L)
(g/L)
(g/L)
(g/L)


Solvent
t = 0
t = 2 h
t = 4 h
t = 6 h
t = 24 h















none (control)
17.5
14.9
9.3
2.1
0.0


oleic acid
17.5
19.0
10.2
2.2
0.0


oleyl alcohol
17.5
15.3
10.4
3.4
0.0


octanoic acid
17.5
17.1
17.6
19.2
18.5


nonanol
17.5
17.0
16.9
18.1
17.8


1-dodecanol
17.5
15.4
11.6
3.9
0.0


1-nonanal
17.5
16.6
17.0
17.8
17.6


1-decanol
17.5
16.6
17.0
17.8
17.6









As can been from the results in Tables 3 and 4, the S. cerevisiae grown in the presence of oleic acid, oleyl alcohol, and 1-dodecanol exhibited about the same level of glucose utilization and growth as the control without solvent, indicating that these solvents are biocompatible with the S. cerevisiae strain tested. The solvents octanoic acid, 1-nonanal, 1-decanol, and nonanol all inhibited growth and glucose utilization of the S. cerevisiae strain.
Example 2 And Example 3
Comparative
Growth of Saccharomyces cerevisiae in the Presence of Isobutanol and Oleyl Alcohol
The purpose of these Examples was to demonstrate that oleyl alcohol mitigates the toxicity of isobutanol to Saccharomyces cerevisiae. The glucose consumption rate and the growth rate of wild-type Saccharomyces cerevisiae BY4741 strain were measured in shake flask cultures containing a high concentration of isobutanol in the presence of oleyl alcohol (Example 2) and the absence of oleyl alcohol (Example 3, Comparative).
Three seed shake flasks containing 600 mL of YPD medium were inoculated with 100, 300, and 1000 μL of Saccharomyces cerevisiae BY4741 inoculum, respectively. The flasks were incubated overnight at 30° C. with shaking at 250 rpm until the OD600 reached about 0.1. Samples were withdrawn from each culture and the OD600 and glucose concentration were measured as described above.
To a 125 mL flask was added 100 mL of the culture which was derived from the 300 μL inoculum (Example 3, Comparative). To another 125 mL flask was added 75 mL of the culture and 25 mL of oleyl alcohol (i.e., 25 vol %) (Example 2). The flasks were incubated at 30° C. with shaking at 100 rpm. Samples were withdrawn for OD600 and glucose measurement. When the OD600 reached about 0.4, isobutanol was added to both flasks to a final concentration of 30 g/L of the aqueous phase. A third flask containing 75 mL of the culture and 25 vol % of oleyl alcohol, but no added isobutanol, served as a positive control. Samples were withdrawn from all flasks at various times for the determination of OD600 and glucose concentration. The optical density results and the glucose results are given in Tables 5 and 6, respectively.







TABLE 5







Optical Density Results in the Presence and Absence of Oleyl Alcohol














Example 3




Time
Example 2
(Comparative)
Control



(h)
OD600
OD600
OD600







0 (isobutanol
0.4
0.4
0.4



addition)



3
0.5
0.6
0.6



4
1.3
0.7
1.7



5
1.7
0.6
2.5



14 
3.1
0.6
3.3

















TABLE 6







Glucose Concentration in the Presence and Absence of Oleyl Alcohol












Example 3



Time
Example 2
(Comparative)
Control


(h)
Glucose (g/L)
Glucose (g/L)
Glucose (g/L)













0 (isobutanol
18.9
18.9
19.7


addition)


3
16.6
17.5
16.0


4
14.4
17.7
12.9


5
12.0
17.4
9.2


14 
2.7
17.4
0.3









As can be seen from the data in Tables 5 and 6, isobutanol at a concentration of 30 g/L in the absence of oleyl alcohol (Example 3, Comparative) almost completely inhibited glucose utilization and biomass growth. However, the culture was able to grow and utilize glucose In the presence of isobutanol at a concentration of 30 g/L when oleyl alcohol was added to the culture. The growth and glucose utilization rate of the culture containing isobutanol and oleyl alcohol were comparable to those of the control containing only oleyl alcohol. These results demonstrate that oleyl alcohol mitigates the toxicity of isobutanol to the strain of Saccharomyces cerevisiae studied. It would be reasonable to expect that oleyl alcohol could be used to mitigate the toxicity of isobutanol, as well as other butanols, to other strains of Saccharomyces cerevisiae, including recombinant strains.
Example 4 And Example 5
Comparative
Growth of Lactobacillus plantarum in the Presence of Isobutanol and Oleyl Alcohol
The purpose of these Examples was to demonstrate that oleyl alcohol mitigates the toxicity of isobutanol to Lactobacillus plantarum. The glucose consumption rate and the growth rate of Lactobacillus plantarum strain PN0512 were measured in shake flask cultures containing a high concentration of isobutanol in the presence of oleyl alcohol (Example 4) and the absence of oleyl alcohol (Example 5, Comparative).
Three seed shake flasks containing 50 mL of de Man-Rogosa-Sharpe (MRS) medium were inoculated with 200, 500, and 1000 μL, respectively, of Lactobacillus plantarum strain PN0512 inoculum (ATCC: PTA-7727, biological deposit made Jul. 12, 2006 for U.S. patent application Ser. No. 11/761,497). The flasks were incubated overnight at 30° C. with shaking at 250 rpm until the OD600 was between 2 and 5. A 1-L flask containing 600 mL of MRS medium was inoculated from one of the above seed flasks having an OD600=3 to an initial OD600=0.1 and cultivated at 30° C. with shaking at 180 rpm. After 4 to 5 hours of cultivation, the OD600 was between 0.5 and 1.0. Samples were withdrawn from each culture and the OD600 and glucose concentration were measured as described above.
To a 125 mL flask was added 100 mL of the culture from the above mentioned 1-L flask (Example 3, Comparative). To another 125 mL flask was added 75 mL of the culture and 25 mL of oleyl alcohol (i.e., 25 vol %) (Example 2). The flasks were incubated at 30° C. with shaking at 100 rpm. Samples were withdrawn for OD600 and glucose measurement. When the OD600 reached about 1.5, isobutanol was added to both flasks to a final concentration of 30 g/L of the aqueous phase. A third flask containing 75 mL of the culture and 25 vol % of oleyl alcohol, but no added isobutanol, served as a positive control. Samples were withdrawn from all flasks at various times for the determination of OD600 and glucose concentration. The optical density results and the glucose results are given in Tables 7 and 8, respectively.







TABLE 7







Optical Density Results in the Presence and Absence of Oleyl Alcohol














Example 5




Time
Example 4
(Comparative)
Control



(h)
OD600
OD600
OD600
















0 (isobutanol
6.2
5.7
5.6



addition)



3
8.8
6.8
8.9



4
10.4
6.9
10.4



5
12.6
7.2
14.1



14 
7.4
5.3
8.8

















TABLE 8







Glucose Concentration in the Presence and Absence of Oleyl Alcohol












Example 5



Time
Example 4
(Comparative)
Control


(h)
Glucose (g/L)
Glucose (g/L)
Glucose (g/L)













0 (isobutanol
12.9
12.7
12.9


addition)


3
7.8
11.7
7.4


4
5.2
11.7
4.5


5
2.8
11.7
1.9


14 
0
11.6
0









As can be seen from the data in Tables 7 and 8, isobutanol at a concentration of 30 g/L in the absence of oleyl alcohol (Example 3, Comparative) almost completely inhibited glucose utilization and biomass growth of the Lactobacillus strain. However, the culture was able to grow and utilize glucose in the presence of isobutanol at a concentration of 30 g/L when oleyl alcohol was added to the culture. The growth and glucose utilization rate of the culture containing isobutanol and oleyl alcohol were comparable to those of the control containing only oleyl alcohol. These results demonstrate that oleyl alcohol mitigates the toxicity of isobutanol to the strain of Lactobacillus plantarum studied. It would be reasonable to expect that oleyl alcohol could be used to mitigate the toxicity of isobutanol, as well as other butanols, to other strains of Lactobacillus plantarum, including recombinant strains.
Example 6
Production of Isobutanol By Recombinant Escherichia coli Using Extractive Fermentation
The purpose of this Example was to demonstrate the production of isobutanol by a recombinant strain of Escherichia coli that contains an isobutanol biosynthetic pathway using extractive fermentation with oleyl alcohol as the water immiscible, organic extractant.
The strain used was Escherichia coli Strain NGCI-031, constructed as described in the General Methods Section herein above. All seed cultures for inoculum preparation were grown in Luria-Bertani (LB) medium with ampicillin (100 mg/L) as the selection antibiotic. The fermentation medium was a semi-synthetic medium, the composition of which is given in Table 9.







TABLE 9







Fermentation Medium Composition










Ingredient
Amount/L







Phosphoric Acid 85%
0.75 mL



Sulfuric Acid (18 M)
0.30 mL



Balch's w/ Cobalt - 1000X (composition given in
1.00 mL



Table 10)



Potassium Phosphate Monobasic
1.40 g



Citric Acid Monohydrate
 200 g



Magnesium Sulfate, heptahydrate
 200 g



Ferric Ammonium Citrate
0.33 g



Calcium chloride, dihydrate
0.20 g



Yeast Extracta
5.00 g



Antifoam 204b
0.20 mL



Thiamince•HCl, 5 g/L stock
1.00 mL



Ampicillin, 25 mg/mL stock
4.00 mL



Glucose 50 wt % stock
33.3 mL







aObtained from BD Diagnostic Systems, Sparks, MD



bObtained from Sigma-Aldrich













TABLE 10







Balch's Modified Trace Metals - 1000X










Ingredient
Concentration (g/L)














Citric Acid Monohydrate
40.0



MnSO4•H2O
30.0



NaCl
10.0



FeSO4•7H2O
1.0



COCl2•6H2O
1.0



ZnSO4•7H2O
1.5



CuSO4•5H2O
0.1



Boric Acid (H3BO3)
0.1



Sodium Molybnate (NaMoO4•2H2O)
0.1










Ingredients 1-10 from Table 9 were added to water at the prescribed concentration to make a final volume of 1.5 L in the fermentor. The contents of the fermentor were sterilized by autoclaving. Components 11-13 were mixed, filter sterilized and added to the fermentor after the autoclaved medium had cooled. The total final volume of the fermentation medium (the aqueous phase) was about 1.6 L.
The fermentation was done using a Biostat-B DCU-3 fermentor (Braun Biotech International, Melesungen, Germany) with a working volume of 2.0 L. The temperature was maintained at 30° C. during the entire fermentation and the pH was maintained at 6.8 using ammonium hydroxide. Following inoculation of the sterile fermentation medium with seed culture (2-10 vol %), the fermentor was operated aerobically at a 30% dissolved oxygen (DO) set point with 0.5 vvm of air flow by automatic control of the agitation rate (rpm). Once the desired optical density (OD600) was reached (i.e., OD600=10), the culture was induced with the addition of 0.4-0.5 mM IPTG to overexpress the isobutanol biosynthetic pathway. Four hours post induction, fermentation conditions were switched to microaerobic conditions by decreasing the stirrer speed to 200 rpm. The shift to microaerobic conditions initiated isobutanol production while minimizing the amount of carbon going to biomass production, thereby uncoupling biomass formation from isobutanol production. Oleyl alcohol (about 780 mL) was added during the isobutanol production phase to alleviate the problem of inhibition due to build up of isobutanol in the aqueous phase. Glucose was added as a bolus (50 wt % stock solution) to the fermentor to keep levels of glucose between 30 g/L and 2 g/L.
Because efficient production of isobutanol requires microaerobic conditions to enable redox balance in the biosynthetic pathway, air was continuously supplied to the fermentor at 0.5 vvm. Continuous aeration led to significant stripping of isobutanol from the aqueous phase of the fermentor. To quantify the loss of isobutanol due to stripping, the off-gas from the fermentor was sparged through a chilled (6.5° C.) water trap to condense the isobutanol, which was then quantified using the GC Method described in the General Methods Section herein above. Alternatively, the air stream exiting the fermentor was directly sent to a mass spectrometer (Prima dB mass spectrometer, Thermo Electron Corp., Madison, Wis.) to quantify the amount of isobutanol in the gas stream. The isobutanol peaks at mass to charge ratios of 74 or 42 were monitored continuously to quantify the amount of isobutanol in the gas stream.
For isobutanol production, the effective titer, the effective rate, and the effective yield, all corrected for the isobutanol lost due to stripping, were 37 g/L, 0.40 g/L/h, and 0.33 g/g, respectively. As can be seen by comparing these results to those obtained without oleyl alcohol as extractant (as shown in Example 7, Comparative below), the use of oleyl alcohol in an extractive fermentation for isobutanol production results in significantly higher effective titer, effective rate, and effective yield. The isobutanol product, which is toxic to the bacterial host, is continuously extracted into the oleyl alcohol phase, decreasing its concentration in the aqueous phase, thereby reducing the toxicity to the microorganism. Additionally, oleyl alcohol appears to have another, unexpected beneficial effect on isobutanol production. This can also be seen by comparison with Example 7 (Comparative). In that Example, gas stripping alone continuously removed isobutanol from the fermentation medium and limited the isobutanol concentration in the fermentation medium to a maximum of about 8-10 g/L, a level that is comparable to the level observed using a combination of extraction with oleyl alcohol and gas stripping (see FIG. 1). In both Examples, the isobutanol concentration was below inhibitory levels for most of the fermentation. However, significantly higher isobutanol effective titer, effective rate, and effective yield were obtained with a combination of oleyl alcohol extractive fermentation and gas stripping (Example 6) than with gas stripping alone (Example 7), suggesting the oleyl alcohol has another, unexpected beneficial effect on isobutanol production.
Example 7
Comparative
Production of Isobutanol By Recombinant Escherichia coli Using Fermentation Without Addition of an Extractant
The purpose of this Comparative Example was to demonstrate that without the addition of oleyl alcohol the production of isobutanol is inhibited due to the toxicity of the product.
The fermentation was done as described in Example 6, except that oleyl alcohol was not added to the fermentation medium during the isobutanol production phase. The following changes were also made. The culture was induced by addition of IPTG when the OD600 reached 6, and the switch to microaerobic conditions was made 3 hours post induction. To quantify the amount of isobutanol lost due to stripping, the off-gas from the fermentor was sent directly to a mass spectrometer (Prima dB, Thermo Electron Corp., Madison, Wis.). The isobutanol peaks at mass to charge ratios of 74 or 42 were continuously monitored to quantify isobutanol loss due to stripping.
For isobutanol production, the effective titer, the effective rate, and the effective yield, all corrected for the isobutanol lost due to stripping, were 13 g/L, 0.21 g/L/h, and 0.22 g/g, respectively. Because isobutanol was continuously removed from the fermentation medium by gas stripping, the isobutanol concentration in the aqueous phase during fermentation was limited to a maximum of 8-10 g/L, and was actually below inhibitory levels for most of the fermentation (see FIG. 1).
Example 8
Production of Isobutanol By Recombinant Saccharomyces cerevisiae Using Extractive Fermentation
The purpose of this Example was to demonstrate the production of isobutanol by a recombinant strain of Saccharomyces cerevisiae that contains an isobutanol biosynthetic pathway using extractive fermentation with oleyl alcohol as the water immiscible, organic extractant.
The strain used was Saccharomyces cerevisiae Strain NGCI-049, constructed as described in the General Methods Section herein above. All seed cultures for inoculum preparation were grown in Yeast Nitrogen Base (YNB) without amino acids medium (6.7 g/L), supplemented with amino acid dropout mix (1.4 g/L), leucine (100 mg/L) and tryptophan (20 mg/L). Ethanol at 1% (v/v) was used as the sole carbon source for all seed cultures. The fermentation medium was a semi-synthetic medium, the composition of which is given in Table 11.







TABLE 11







Fermentation Medium Composition










Ingredient
Amount/L















1. YNB w/o amino acidsa
6.7
g



2. Sigma Dropout Mix (Y2001)b
2.8
g



3. Leucine (10 g/L)
20
mL



4. Tryptophan (10 g/L)
4
mL



5. Ethanol
10
mL



6. Glucose 50 wt % stock
4
g







aObtained from BD Diagnostic Systems, Sparks, MD



bObtained from Sigma-Aldrich, St. Louis, MO






Ingredients 1-4 from Table 11 were added to water at the prescribed concentration to make a final volume of 0.54 L in the fermentor. The contents of the fermentor were sterilized by autoclaving. Components 5 and 6 were mixed, filter sterilized and added to the fermentor after the autoclaved medium had cooled. The total final volume of the fermentation medium (the aqueous phase) was about 0.54 L.
The fermentation was done using a 1 L autoclavable bioreactor, Bio Console ADI 1025 (Applikon, Inc, Holland) with a working volume of 900 mL. The temperature was maintained at 30° C. during the entire fermentation and the pH was maintained at 5.5 using sodium hydroxide. Following inoculation of the sterile fermentation medium with seed culture (10 vol %), the fermentor was operated aerobically at a 30% dissolved oxygen (DO) set point with 0.3 vvm of air flow by automatic control of the agitation rate (rpm). Once the initial batched glucose of 2 g/L was consumed, glucose was fed using a pump at an exponential rate such that glucose never accumulated above 0.2 g/L in the fermentor. Once the desired optical density (OD600) was reached (i.e., OD600=6), the culture was induced to isobutanol production phase by feeding glucose such that excess glucose (>2 g/L) was maintained at all times during fermentation. Two hours post glucose excess, 60 mL of filter sterilized 10× Yeast Extract Peptone stock solution (10×YEP=100 g/L of yeast extract and 200 g/L of peptone) was added. Two hours post addition of YEP, Oleyl alcohol (about 300 mL) was added during the isobutanol production phase to alleviate the problem of inhibition due to build up of isobutanol and other byproducts in the aqueous phase. Glucose was fed (50 wt % stock solution) to the fermentor to keep levels of glucose greater than 2 g/L.
Because efficient production of isobutanol requires microaerobic conditions to enable redox balance in the biosynthetic pathway, air was continuously supplied to the fermentor at 0.3 vvm. Continuous aeration led to significant stripping of isobutanol from the aqueous phase of the fermentor. To quantify the loss of isobutanol due to stripping, the off-gas from the fermentor was directly sent to a mass spectrometer (Prima dB mass spectrometer, Thermo Electron Corp., Madison, Wis.) to quantify the amount of isobutanol in the gas stream. The isobutanol peaks at mass to charge ratios of 74 or 42 were monitored continuously to quantify the amount of isobutanol in the gas stream.
Glucose and organic acids in the aqueous phase were monitored during the fermentation using HPLC. Glucose was also monitored quickly using a glucose analyzer (YSI, Inc., Yellow Springs, Ohio). Isobutanol in the aqueous phase was quantified by HPLC and isobutanol in the oleyl alcohol phase were monitored using the GC method described in the General Method Section herein above after the two phases were removed periodically from the fermentor and separated by centrifugation. The concentration of isobutanol in the aqueous phase during the fermentation is shown in FIG. 2, where the closed squares (▪) refer to the concentrations of Example 8, fermentation using oleyl alcohol as the organic extractant with gas stripping, and the closed circles (●) refer to the concentrations of Example 9 (Comparative), fermentation with gas stripping alone.
For isobutanol production, the effective titer, the effective rate, and the effective yield, all corrected for the isobutanol lost due to stripping, were 5 g/L, 0.06 g/L/h, and 0.16 g/g, respectively. As can be seen by comparing these results to those obtained without oleyl alcohol as extractant (as shown in Example 9, Comparative below), the use of oleyl alcohol in an extractive fermentation for isobutanol production results in significantly higher effective titer, effective rate, and effective yield. The isobutanol product, which is toxic to the host, is continuously extracted into the oleyl alcohol phase, decreasing its concentration in the aqueous phase, thereby reducing the toxicity to the microorganism. Additionally, oleyl alcohol appears to have another, unexpected beneficial effect on isobutanol production. This can also be seen by comparison with Example 9 (Comparative). In that Example, gas stripping alone continuously removed isobutanol from the fermentation medium and limited the isobutanol concentration in the fermentation medium to a maximum of about 4 g/L, a level that is comparable to the level observed using a combination of extraction with oleyl alcohol and gas stripping (see FIG. 2). In both Examples, the isobutanol concentration was below inhibitory levels for most of the fermentation. However, significantly higher isobutanol effective titer, effective rate, and effective yield were obtained with a combination of oleyl alcohol extractive fermentation and gas stripping (Example 8) than with gas stripping alone (Example 9), suggesting the oleyl alcohol has another, unexpected beneficial effect on isobutanol production.
Example 9
Comparative
Production of Isobutanol By Recombinant Saccharomyces cerevisiae Using Fermentation Without Addition of an Extractant
The purpose of this Comparative Example was to demonstrate that without the addition of oleyl alcohol the production of isobutanol is inhibited due to the toxicity of the product.
The fermentation was done as described in Example 8, except that oleyl alcohol was not added to the fermentation medium during the isobutanol production phase. To quantify the amount of isobutanol lost due to stripping, the off-gas from the fermentor was sent directly to a mass spectrometer (Prima dB, Thermo Electron Corp., Madison, Wis.). The isobutanol peaks at mass to charge ratios of 74 or 42 were continuously monitored to quantify isobutanol loss due to stripping.
For isobutanol production, the effective titer, the effective rate, and the effective yield, all corrected for the isobutanol lost due to stripping, were 3 g/L, 0.04 g/L/h, and 0.16 g/g, respectively. Because isobutanol was continuously removed from the fermentation medium by gas stripping, the isobutanol concentration in the aqueous phase during fermentation was limited to a maximum of 2-3 g/L, and was actually below inhibitory levels for most of the fermentation (see FIG. 2).
Example 10
A 2 L fermentor can be used to run a fermentation with recombinant yeast producing isobutanol as in Example 8 except that a portion of the initial batched oleyl alcohol is removed and replaced with fresh oleyl alcohol. This process can be run in a continuous mode where fresh oleyl alcohol is slowly trickled in and spent oleyl alcohol is pumped out such that the amount of oleyl alcohol in the fermentor remains constant during the entire fermentation process. Such continuous extraction of products and byproducts from the fermentation can increase effective rate, titer and yield.

## Claims

1. A method for recovering butanol from a fermentation medium, the method comprising:
a) providing a fermentation medium comprising butanol, water, and a genetically modified yeast that produces butanol from a fermentation medium comprising at least one fermentable carbon source;
b) providing oleic acid and adding steric acid to form an extractant;
c) contacting the fermentation medium with the extractant comprising oleic acid and stearic acid to form a two-phase mixture comprising an aqueous phase and a butanol-containing organic phase;
d) separating the butanol-containing organic phase from the aqueous phase; and
e) recovering the butanol from the butanol-containing organic phase to produce recovered butanol;
wherein the extractant is recycled.

2. The method according to claim 1, wherein the butanol is isobutanol.

3. The method of claim 1, wherein the fermentation medium further comprises ethanol, and the butanol-containing organic phase contains ethanol.

4. The method of claim 1, wherein the yeast is Saccharomyces cerevisiae.

5. A method for the production butanol, comprising:
a) providing a genetically modified yeast that produces butanol from a fermentation medium comprising at least one fermentable carbon source;
b) providing oleic acid and adding steric acid to form an extractant;
c) growing the yeast in a biphasic fermentation medium comprising an aqueous phase and the extractant comprising oleic acid and stearic acid, wherein said biphasic fermentation medium comprises from about 3% to about 60% by volume of the extractant, for a time sufficient to allow extraction of the butanol into the extractant to form a butanol-containing organic phase;
d) separating the butanol-containing organic phase from the aqueous phase; and
e) recovering the butanol from the butanol-containing organic phase to produce recovered butanol;
wherein the extractant is recycled.

6. The method according to claim 5, wherein the butanol is isobutanol.

7. The method of claim 5, wherein the fermentation medium further comprises ethanol, and the butanol-containing organic phase comprises ethanol.

8. The method of claim 5, wherein the yeast is Saccharomyces cerevisiae.

9. A method for the production of butanol comprising the steps of:
a) providing a genetically modified yeast that produces butanol from a fermentation medium comprising at least one fermentable carbon source;
b) growing the microorganism in a fermentation medium wherein the yeast produces said butanol into the fermentation medium to produce a butanol-containing fermentation medium;
c) providing oleic acid and adding steric acid to form an extractant;
d) contacting the butanol-containing fermentation medium with the extractant comprising oleic acid and stearic to form a two-phase mixture comprising an aqueous phase and a butanol-containing organic phase;
e) separating the butanol-containing organic phase from the aqueous phase; and
f) recovering the butanol from the butanol-containing organic phase;
wherein the extractant is recycled.

10. The method according to claim 9, wherein the butanol is isobutanol.

11. The method of claim 9, wherein the yeast is Saccharomyces cerevisiae.

12. A method for the production of butanol comprising the steps of:
a) providing a genetically modified yeast that produces butanol from a fermentation medium comprising at least one fermentable carbon source;
b) growing the yeast in a fermentation medium under aerobic conditions for a time sufficient to reach a preselected growth level;
c) switching to microaerobic or anaerobic conditions to stimulate butanol production into the fermentation medium to form a butanol-containing fermentation medium;
d) providing oleic acid and adding steric acid to form an extractant;
e) contacting the butanol-containing fermentation medium with the extractant comprising oleic acid and stearic acid to form a two-phase mixture comprising an aqueous phase and a butanol-containing organic phase;
f) separating the butanol-containing organic phase from the aqueous phase; and
g) recovering the butanol from the butanol-containing organic phase;
wherein the extractant is recycled.

13. The method according to claim 12 wherein the butanol is isobutanol.

14. The method of claim 12, wherein the yeast is Saccharomyces cerevisiae.

15. A method for the production of butanol comprising the steps of:
a) providing a fermentation medium comprising butanol, water, and a genetically modified yeast that produces butanol from a fermentation medium comprising at least one fermentable carbon source;
b) providing oleic acid and adding steric acid to form an extractant;
c) contacting the fermentation medium via a co-current or counter-current extractant stream with the extractant comprising oleic acid and stearic acid to form a two-phase mixture comprising an aqueous phase and a butanol-containing organic phase;
d) separating the butanol-containing organic phase from the aqueous phase; and
e) recovering the butanol from the butanol-containing organic phase to produce recovered butanol;
wherein the extractant is recycled.

16. The method according to claim 15, wherein the butanol is isobutanol.

17. The method of claim 15, wherein the yeast is Saccharomyces cerevisiae.

18. The method according to any of claims 1, 5, 9, 12, and 15, wherein a portion of the butanol is concurrently removed from the fermentation medium by a process comprising the steps of:
a) stripping butanol from the fermentation medium with a gas, forming a butanol-containing gas phase; and
b) recovering butanol from the butanol-containing gas phase.