U.S. patent application number 15/348160 was filed with the patent office on 2017-03-02 for method for producing butanol using extractive fermentation.
The applicant listed for this patent is Butamax Advanced Biofuels LLC. Invention is credited to Thomas Basham, Leslie William Bolton, Ian David Dobson, Sarah Richardson Hanson, Aidan Hurley, Karen Kustedjo, Andrew Richard Lucy, Liang Song.
Application Number | 20170057894 15/348160 |
Document ID | / |
Family ID | 54929765 |
Filed Date | 2017-03-02 |
United States Patent
Application |
20170057894 |
Kind Code |
A1 |
Basham; Thomas ; et
al. |
March 2, 2017 |
Method for Producing Butanol Using Extractive Fermentation
Abstract
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. The invention also relates to a method for producing
butanol through microbial fermentation, in which the butanol
product is removed during the fermentation by extraction into a
water-immiscible extractant composition. The invention further
relates to compositions comprising a solution of butanol in a water
immiscible organic extractant composition.
Inventors: |
Basham; Thomas; (San Diego,
CA) ; Bolton; Leslie William; (Hampshire, GB)
; Dobson; Ian David; (London, GB) ; Hanson; Sarah
Richardson; (San Marcus, CA) ; Hurley; Aidan;
(East Riding of Yorkshire, GB) ; Kustedjo; Karen;
(La Jolla, CA) ; Lucy; Andrew Richard; (Brough
East Yorkshire, GB) ; Song; Liang; (San Diego,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Butamax Advanced Biofuels LLC |
Wilmington |
DE |
US |
|
|
Family ID: |
54929765 |
Appl. No.: |
15/348160 |
Filed: |
November 10, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14772705 |
Sep 3, 2015 |
9517985 |
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PCT/US14/29142 |
Mar 14, 2014 |
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15348160 |
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61788213 |
Mar 15, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07C 31/12 20130101;
C07C 29/86 20130101; Y02E 50/10 20130101; C12P 7/16 20130101; C07C
29/86 20130101; C07C 29/76 20130101; C07C 31/12 20130101 |
International
Class: |
C07C 29/86 20060101
C07C029/86; C07C 31/12 20060101 C07C031/12 |
Claims
1. A method for recovering butanol from a fermentation medium, the
method comprising: (a) providing a fermentation medium comprising
butanol, water, and a recombinant microorganism comprising a
butanol biosynthetic pathway, wherein the recombinant microorganism
produces butanol; (b) contacting the fermentation medium with a
water immiscible organic extractant composition comprising a
solvent selected from the group consisting of C.sub.12 to C.sub.22
fatty alcohols, C.sub.12 to C.sub.22 ethers, esters of C.sub.12 to
C.sub.22 fatty acids, C.sub.10 to C.sub.22 alkanes, and mixtures
thereof, to form a butanol-containing organic phase and an aqueous
phase, wherein the solvent is biocompatible with the microorganism
such that at least about 90% of the microorganisms are viable after
exposure to the organic extractant composition, and wherein the
solvent has a boiling point less than about 300.degree. C., with
the proviso that the organic extractant is not oleyl alcohol,
1-dodecanol, behenyl alcohol, cetyl alcohol, myristyl alcohol, or
stearyl alcohol; and (c) recovering the butanol from the
butanol-containing organic phase.
2. The method of claim 1, wherein the solvent is trimethylnonanol,
methyl laurate, di-n-octyl ether, dodecane, n-undecane, ethyl
decanoate, ethyl laurate, or mixtures thereof.
3. A method for recovering butanol from a fermentation medium, the
method comprising: a) providing a fermentation medium comprising
butanol, water, and a recombinant microorganism comprising a
butanol biosynthetic pathway, wherein the recombinant microorganism
produces butanol; b) contacting the fermentation medium with a
water immiscible organic extractant composition comprising a
solvent is trimethylnonanol, methyl laurate, di-n-octyl ether,
dodecane, n-undecane, ethyl decanoate, ethyl laurate, or mixtures
thereof, to form a butanol-containing organic phase and an aqueous
phase; and c) recovering the butanol from the butanol-containing
organic phase.
4. The method of claim 1, wherein the contacting of the organic
extractant composition with the fermentation medium occurs in a
fermentor.
5. The method of claim 1, further comprising: transferring a
portion of the fermentation medium from the fermentor to a vessel,
wherein the contacting of the organic extractant composition with
the fermentation medium occurs in the vessel.
6. The method of claim 1, wherein the butanol is isobutanol.
7. The method of claim 1, wherein the organic extractant
composition further comprises an additional solvent, wherein the
additional solvent is n-hexanol, methyl isobutyl carbinol,
2-ethyl-1-hexanol, 2,6-dimethylheptan-4-ol, or mixtures
thereof.
8. The method of claim 7, wherein the additional solvent is
2,6-dimethylheptan-4-ol.
9. The method of claim 1, wherein the organic extractant
composition further comprises an additional solvent with a butanol
partition coefficient greater than about 4.
10. The method of claim 1, wherein the organic extractant
composition further comprises an additional solvent with a butanol
partition coefficient greater than about 5.
11. The method of claim 1, the organic extractant composition
further comprises an additional solvent with a butanol partition
coefficient greater than about 6.
12. The method of claim 1, wherein the organic extractant
composition further comprises an additional solvent with a butanol
partition coefficient greater than about 7.
13. The method according to claim 9, wherein the organic extractant
composition further comprises an additional solvent, wherein the
additional solvent is n-hexanol, methyl isobutyl carbinol,
2-ethyl-1-hexanol, 2,6-dimethylheptan-4-ol, or mixtures
thereof.
14. The method of claim 13, wherein the additional solvent is
2,6-dimethylheptan-4-ol.
15. The method of claim 1, wherein the contacting comprises
contacting the fermentation medium via a co-current or
counter-current stream of the organic extractant composition.
16. The method of claim 1, wherein the recovered butanol has an
effective titer from about 22 g per liter to about 40 g per liter
of the fermentation medium.
17. The method of claim 1, wherein the recovered butanol has an
effective titer of at least about 37 g per liter of the
fermentation medium.
18. A composition comprising a solution of butanol in a water
immiscible organic extractant composition, wherein the organic
extractant composition comprises a solvent selected from the group
consisting of C.sub.12 to C.sub.22 fatty alcohols, C.sub.12 to
C.sub.22 ethers, esters of C.sub.12 to C.sub.22 fatty acids,
C.sub.10 to C.sub.22 alkanes, and mixtures thereof, wherein the
solvent is biocompatible with a recombinant microorganism
comprising a butanol biosynthetic pathway, wherein at least 90% of
the recombinant microorganisms are viable after exposure to the
organic extractant composition, and wherein the solvent has a
boiling point less than about 300.degree. C., with the proviso that
the solvent is not oleyl alcohol, 1-dodecanol, behenyl alcohol,
cetyl alcohol, myristyl alcohol, or stearyl alcohol.
19. The composition of claim 18, wherein the solvent is
trimethylnonanol, methyl laurate, di-n-octyl ether, dodecane,
n-undecane, ethyl decanoate, ethyl laurate, or mixtures
thereof.
20. A composition comprising a solution of butanol in a water
immiscible organic extractant composition comprising a solvent,
wherein the solvent is trimethylnonanol, methyl laurate, di-n-octyl
ether, dodecane, n-undecane, ethyl decanoate, ethyl laurate, or
mixtures thereof.
21. The composition of claim 18, wherein the organic extractant
composition further comprises an additional solvent, wherein the
additional solvent is n-hexanol, methyl isobutyl carbinol,
2-ethyl-1-hexanol, 2,6-dimethylheptan-4-ol, or mixtures
thereof.
22. The composition of claim 21, wherein the additional solvent is
2,6-dimethylheptan-4-ol.
23. The composition of claim 18, wherein the butanol is isobutanol.
Description
CROSS-REFERENCE
[0001] This application claims priority to U.S. Provisional Patent
Application No. 61/788,213, filed on 15 Mar. 2013, entitled Method
for Production of Butanol Using Extractive Fermentation, which is
hereby incorporated by reference in its entirety. Additionally,
this application incorporates by reference in their entireties U.S.
Provisional Patent Application No. 61/790,828, filed on 15 Mar.
2013, entitled Method for Production of Butanol Using Extractive
Fermentation and U.S. Provisional Patent Application No.
61/790,401, filed on 15 Mar. 2013, entitled Method for Production
of Butanol Using Extractive Fermentation.
FIELD OF THE INVENTION
[0002] 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
[0003] 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 food grade 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.
[0004] 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).
[0005] 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.
With these recombinant hosts, the biological production of butanol
appears to be limited by the butanol toxicity thresholds of the
host microorganism used in the fermentation. U.S. Patent
Application Publication Nos. 2009/0305370 and 2011/0097773, each of
which is incorporated by reference herein in its entirety, disclose
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.
[0006] Improved methods for producing and recovering butanol from a
fermentation medium are continually sought. Lower cost processes
and improvements to process operability are also desired.
Identification of improved extractants for use with fermentation
media, such as extractants exhibiting higher partition
coefficients, lower viscosity, lower density, commercially useful
boiling points, and sufficient microbial biocompatibility, is a
continual need.
BRIEF SUMMARY OF THE INVENTION
[0007] Provided herein are methods for recovering butanol from a
fermentation medium. The methods comprise (a) providing a
fermentation medium comprising butanol, water, and a recombinant
microorganism comprising a butanol biosynthetic pathway, wherein
the recombinant microorganism produces butanol; (b) contacting the
fermentation medium with a water immiscible organic extractant
composition comprising a solvent selected from the group consisting
of C.sub.12 to C.sub.22 fatty alcohols, C.sub.12 to C.sub.22
ethers, esters of C.sub.12 to C.sub.22 fatty acids, C.sub.10 to
C.sub.22 alkanes, and mixtures thereof, to form a
butanol-containing organic phase and an aqueous phase, wherein the
solvent is biocompatible with the microorganism such that at least
about 75% of the microorganisms are viable after exposure to the
organic extractant composition, and wherein the solvent has a
boiling point less than about 300.degree. C., with the proviso that
the organic extractant is not oleyl alcohol, 1-dodecanol, behenyl
alcohol, cetyl alcohol, myristyl alcohol, or stearyl alcohol; and
(c) recovering the butanol from the butanol-containing organic
phase.
[0008] In some embodiments, the solvent is trimethylnonanol, methyl
laurate, di-n-octyl ether, dodecane, n-undecane, ethyl decanoate,
ethyl laurate, or mixtures thereof.
[0009] In certain embodiments, methods for recovering butanol from
a fermentation medium comprise (a) providing a fermentation medium
comprising butanol, water, and a recombinant microorganism
comprising a butanol biosynthetic pathway, wherein the recombinant
microorganism produces butanol; (b) contacting the fermentation
medium with a water immiscible organic extractant composition
comprising a solvent, wherein the solvent is trimethylnonanol,
methyl laurate, di-n-octyl ether, dodecane, n-undecane, ethyl
decanoate, ethyl laurate, or mixtures thereof, to form a
butanol-containing organic phase and an aqueous phase; and (c)
recovering the butanol from the butanol-containing organic
phase.
[0010] In some embodiments, the contacting of the organic
extractant composition with the fermentation medium occurs in the
fermentor. In other embodiments, the contacting of the organic
extractant composition with the fermentation medium occurs outside
the fermentor. In some embodiments, the butanol is recovered after
transferring a portion of the fermentation medium from the
fermentor to a vessel, wherein the contacting of the organic
extractant composition with the fermentation medium occurs in the
vessel. In some embodiments, the butanol is isobutanol.
[0011] In certain embodiments, the organic extractant composition
further comprises an additional solvent, wherein the second solvent
is n-hexanol, methyl isobutyl carbinol, 2-ethyl-1-hexanol,
2,6-dimethylheptan-4-ol, or mixtures thereof. In other embodiments,
the organic extractant composition further comprises
2,6-dimethylheptan-4-ol. In some embodiments, the additional
solvent has a butanol partition coefficient greater than about 4,
4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8.
[0012] In some embodiments, the contacting of the organic
extractant composition with the fermentation medium comprises
contacting the fermentation medium via a co-current or
counter-current stream of the organic extractant composition.
[0013] In some embodiments, the recovered butanol has an effective
titer from about 20 g per liter to about 50 g per liter of the
fermentation medium. In some embodiments, the recovered butanol has
an effective titer from about 22 g per liter to about 50 g per
liter. In some embodiments, the recovered butanol has an effective
titer from about 25 g per liter to about 50 g per liter. In
embodiments, the recovered butanol has an effective titer of at
least 25 g, at least 30 g, at least 35 g, at least 37 g, at least
40 g, or at least 45 g per liter of the fermentation medium.
[0014] Also provided herein is a composition comprising butanol in
a water immiscible organic extractant composition, wherein the
organic extractant composition comprises a solvent selected from
the group consisting of C.sub.12 to C.sub.22 fatty alcohols,
C.sub.12 to C.sub.22 ethers, esters of C.sub.12 to C.sub.22 fatty
acids, C.sub.10 to C.sub.22 alkanes, and mixtures thereof, wherein
the solvent is biocompatible with a recombinant microorganism
comprising a butanol biosynthetic pathway, wherein at least 75% of
the recombinant microorganism is viable after exposure to the
organic extractant composition, and wherein the solvent has a
boiling point less than about 300.degree. C., with the proviso that
the solvent is not oleyl alcohol, 1-dodecanol, behenyl alcohol,
cetyl alcohol, myristyl alcohol, or stearyl alcohol.
[0015] In certain embodiments, the boiling point of the solvent is
less than about 275.degree. C., less than about 250.degree. C.,
less than about 225.degree. C., or less than about 200.degree.
C.
[0016] In certain embodiments, provided herein is a composition,
comprising a solution of butanol in a water immiscible organic
extractant composition, wherein the organic extractant composition
comprises a solvent, wherein the solvent is trimethylnonanol,
methyl laurate, di-n-octyl ether, dodecane, n-undecane, ethyl
decanoate, ethyl laurate, or mixtures thereof.
[0017] In other embodiments, the composition further comprises an
additional solvent, wherein the second solvent is n-hexanol, methyl
isobutyl carbinol, 2-ethyl-1-hexanol, 2,6-dimethylheptan-4-ol, or
mixtures thereof. In other embodiments, the organic extractant
composition further comprises 2,6-dimethylheptan-4-ol.
[0018] In some embodiments, the butanol in the composition is
isobutanol.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0019] FIG. 1 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.
[0020] FIG. 2 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.
[0021] 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
added separately to different fermentation vessels for contacting
of the fermentation medium with the extractant.
[0022] FIG. 4 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.
[0023] FIG. 5 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.
[0024] 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
added separately to different vessels for contacting of the
fermentation medium with the extractant.
[0025] FIG. 7 schematically illustrates one embodiment of the
methods of the invention, in which extraction of the product occurs
in at least on batch fermentor via co-current flow of a
water-immiscible extractant comprising a first solvent and an
optional second solvent 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.
[0026] FIG. 8 is a schematic work flow diagram for an automated
primary assay for solvent biocompatibility.
[0027] FIG. 9 is a schematic flow diagram for a secondary assay for
solvent biocompatibility.
[0028] FIG. 10 is a schematic flow diagram for a tertiary assay for
solvent biocompatibility.
[0029] FIG. 11 illustrates the production of isobutanol from an
isobutanologen in a tertiary screen for five biocompatible
solvents, methyl laurate, trimethylnonanol, di-n-octyl ether,
dodecane, and n-undecane using oleyl alcohol as a control (n=3).
Error bars denote 95% confidence intervals.
[0030] FIG. 12 illustrates the utilization of glucose by an
isobutanologen in a tertiary screen for five biocompatible
solvents, methyl laurate, trimethylnonanol, di-n-octyl ether,
dodecane, and n-undecane using oleyl alcohol as a control (n=3).
Error bars denote 95% confidence intervals.
[0031] FIG. 13 illustrates the utilization and consumption of
ethanol by an isobutanologen in a tertiary screen for five
biocompatible solvents, methyl laurate, trimethylnonanol,
di-n-octyl ether, dodecane, and n-undecane using oleyl alcohol as a
control (n=3). Error bars denote 95% confidence intervals.
[0032] FIG. 14 illustrates the effect of different aqueous media on
the solvent panel K.sub.d value (n=1 for minimal and rich media;
n=3 for water). A solvent panel was used to extract 3% isobutanol
prepared in water (left bar), minimal media (middle bar), and rich
media (right bar). Media properties, like ionic strength, enhances
organic phase extraction.
[0033] FIG. 15 illustrates the primary viability data for an
isobutanologen exposed to mixtures using three biocompatible
solvents, oleyl alcohol, methyl laurate, and trimethylnonanol, and
four high K.sub.d solvents: n-hexanol, methyl isobutyl carbinol,
2-ethyl-1-hexanol, and 2,6-dimethylhepta-4-ol. The ordinate axis
describes the level of mixture where 0% is defined as pure
biocompatible solvent and 100% is defined as pure high K.sub.d
solvent.
[0034] FIG. 16 is a scatter plot in which all primary viability
data were plotted against interpolated K.sub.d of all mixtures
studied with no regard to the chemical identity of the
solvents.
[0035] FIG. 17 illustrates the isobutanol production from an
isobutanologen in a tertiary screen of 75% oleyl alcohol and 25%
2,6-dimethylheptan-4-ol.
[0036] FIG. 18 illustrates the isobutanol production from an
isobutanologen of biocompatible methyl laurate chemical structure
analogs. Error bars denote 95% confidence intervals.
DETAILED DESCRIPTION OF THE INVENTION
[0037] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. In case
of conflict, the present application including the definitions will
control. Also, unless otherwise required by context, singular terms
shall include pluralities and plural terms shall include the
singular. All publications, patents and other references mentioned
herein are incorporated by reference in their entireties for all
purposes as if each individual publication or patent application
were specifically and individually indicated to be incorporated by
reference, unless only specific sections of patents or patent
publications are indicated to be incorporated by reference.
[0038] In order to further define this invention, the following
terms, abbreviations and definitions are provided.
[0039] As used herein, the terms "comprises," "comprising,"
"includes," "including," "has," "having," "contains" or
"containing," or any other variation thereof, will be understood to
imply the inclusion of a stated integer or group of integers but
not the exclusion of any other integer or group of integers and are
intended to be non-exclusive or open-ended. For example, a
composition, a mixture, a process, a method, an article, or an
apparatus that comprises a list of elements is not necessarily
limited to only those elements but can include other elements not
expressly listed or inherent to such composition, mixture, process,
method, article, or apparatus. Further, unless expressly stated to
the contrary, "or" refers to an inclusive or and not to an
exclusive or. For example, a condition A or B is satisfied by any
one of the following: A is true (or present) and B is false (or not
present), A is false (or not present) and B is true (or present),
and both A and B are true (or present).
[0040] As used herein, the term "consists of," or variations such
as "consist of" or "consisting of," as used throughout the
specification and claims, indicate the inclusion of any recited
integer or group of integers, but that no additional integer or
group of integers can be added to the specified method, structure,
or composition.
[0041] As used herein, the term "consists essentially of," or
variations such as "consist essentially of" or "consisting
essentially of," as used throughout the specification and claims,
indicate the inclusion of any recited integer or group of integers,
and the optional inclusion of any recited integer or group of
integers that do not materially change the basic or novel
properties of the specified method, structure or composition. See
M.P.E.P. .sctn.2111.03.
[0042] Also, the indefinite articles "a" and "an" preceding an
element or component of the invention are intended to be
nonrestrictive regarding the number of instances, i.e., occurrences
of the element or component. Therefore "a" or "an" should be read
to include one or at least one, and the singular word form of the
element or component also includes the plural unless the number is
obviously meant to be singular.
[0043] The term "invention" or "present invention" as used herein
is a non-limiting term and is not intended to refer to any single
embodiment of the particular invention but encompasses all possible
embodiments as described in the claims as presented or as later
amended and supplemented, or in the specification.
[0044] As used herein, the term "about" modifying the quantity of
an ingredient or reactant of the invention employed refers to
variation in the numerical quantity that can occur, for example,
through typical measuring and liquid handling procedures used for
making concentrates or solutions in the real world; through
inadvertent error in these procedures; through differences in the
manufacture, source, or purity of the ingredients employed to make
the compositions or to carry out the methods; and the like. The
term "about" also encompasses amounts that differ due to different
equilibrium conditions for a composition resulting from a
particular initial mixture. Whether or not modified by the term
"about", the claims include equivalents to the quantities. In one
embodiment, the term "about" means within 10% of the reported
numerical value, or within 5% of the reported numerical value.
[0045] The term "butanol biosynthetic pathway" as used herein
refers to the enzymatic pathway to produce 1-butanol, 2-butanol, or
isobutanol.
[0046] The term "l-butanol biosynthetic pathway" refers to an
enzymatic pathway to produce 1-butanol. A "1-butanol biosynthetic
pathway" can refer to an enzyme pathway to produce 1-butanol from
acetyl-coenzyme A (acetyl-CoA). For example, 1-butanol biosynthetic
pathways are disclosed in U.S. Patent Application Publication No.
2008/0182308 and International Publication No. WO 2007/041269,
which are herein incorporated by reference in their entireties.
[0047] The term "2-butanol biosynthetic pathway" refers to an
enzymatic pathway to produce 2-butanol. A "2-butnaol biosynthetic
pathway" can refer to an enzyme pathway to produce 2-butanol from
pyruvate. For example, 2-butanol biosynthetic pathways are
disclosed in U.S. Pat. No. 8,206,970, U.S. Patent Application
Publication No. 2007/0292927, International Publication Nos. WO
2007/130518 and WO 2007/130521, which are herein incorporated by
reference in their entireties.
[0048] The term "isobutanol biosynthetic pathway" refers to an
enzymatic pathway to produce isobutanol. An "isobutanol
biosynthetic pathway" can refer to an enzyme pathway to produce
isobutanol from pyruvate. For example, isobutanol biosynthetic
pathways are disclosed in U.S. Pat. No. 7,851,188, U.S. Application
Publication No. 2007/0092957, and International Publication No. WO
2007/050671, which are herein incorporated by reference in their
entireties. From time to time "isobutanol biosynthetic pathway" is
used synonymously with "isobutanol production pathway."
[0049] The term "butanol" as used herein refers to the butanol
isomers 1-butanol (1-BuOH), 2-butanol (2-BuOH), tert-butanol
(t-BuOH), and/or isobutanol (iBuOH or i-BuOH, also known as
2-methyl-1-propanol), either individually or as mixtures thereof.
From time to time, as used herein the terms "biobutanol" and
"bio-produced butanol" may be used synonymously with "butanol."
[0050] Uses for butanol can include, but are not limited to, fuels
(e.g., biofuels), a fuel additive, an alcohol used for the
production of esters that can be used as diesel or biodiesel fuel,
as a chemical in the plastics industry, an ingredient in formulated
products such as cosmetics, and a chemical intermediate. Butanol
may also be used as a solvent for paints, coatings, varnishes,
resins, gums, dyes, fats, waxes, resins, shellac, rubbers, and
alkaloids.
[0051] As used herein, the term "bio-produced" means that the
molecule (e.g., butanol) is produced from a renewable source (e.g.,
the molecule can be produced during a fermentation process from a
renewable feedstock). Thus, for example, bio-produced isobutanol
can be isobutanol produced by a fermentation process from a
renewable feedstock. Molecules produced from a renewable source can
further be defined by the .sup.14C/.sup.12C isotope ratio. A
.sup.14C/.sup.12C isotope ratio in range of from 1:0 to greater
than 0:1 indicates a bio-produced molecule, whereas a ratio of 0:1
indicates that the molecule is fossil derived.
[0052] "Product alcohol" as used herein, refers to any alcohol that
can be produced by a microorganism in a fermentation process that
utilizes biomass as a source of fermentable carbon substrate.
Product alcohols include, but are not limited to, C.sub.1 to
C.sub.8 alkyl alcohols, and mixtures thereof. In some embodiments,
the product alcohols are C.sub.2 to C.sub.8 alkyl alcohols. In
other embodiments, the product alcohols are C.sub.2 to C.sub.5
alkyl alcohols. It will be appreciated that C.sub.1 to C.sub.8
alkyl alcohols include, but are not limited to, methanol, ethanol,
propanol, butanol, pentanol, and mixtures thereof. Likewise C.sub.2
to C.sub.8 alkyl alcohols include, but are not limited to, ethanol,
propanol, butanol, and pentanol. "Alcohol" is also used herein with
reference to a product alcohol.
[0053] A recombinant host cell comprising an "engineered alcohol
production pathway" (such as an engineered butanol or isobutanol
production pathway) refers to a host cell containing a modified
pathway that produces alcohol in a manner different than that
normally present in the host cell. Such differences include
production of an alcohol not typically produced by the host cell,
or increased or more efficient production.
[0054] The term "heterologous biosynthetic pathway" as used herein
refers to an enzyme pathway to produce a product in which at least
one of the enzymes is not endogenous to the host cell containing
the biosynthetic pathway.
[0055] The term "butanologen" as used herein refers to a
microorganism capable of producing butanol. The term
"isobutanologen" as used herein refers to a microorganism capable
of producing isobutanol.
[0056] The term "ethanologen" as used herein refers to a
microorganism capable of producing ethanol.
[0057] The term "extractant" as used herein refers to one or more
organic solvents which can be used to extract a product alcohol.
From time to time as used herein, the term "extractant" may be used
synonymously with "solvent."
[0058] The term "effective isobutanol productivity" as used herein
refers to the total amount in grams of isobutanol produced per gram
of cells.
[0059] The term "effective titer" as used herein, refers to the
total amount of a particular alcohol (e.g., butanol) produced by
fermentation per liter of fermentation medium. The total amount of
butanol includes: (i) the amount of butanol in the fermentation
medium; (ii) the amount of butanol recovered from the organic
extractant; and (iii) the amount of butanol recovered from the gas
phase, if gas stripping is used.
[0060] The term "effective rate" as used herein, refers to the
total amount of butanol produced by fermentation per liter of
fermentation medium per hour of fermentation.
[0061] The term "effective yield" as used herein, refers to the
amount of butanol produced per unit of fermentable carbon substrate
consumed by the biocatalyst.
[0062] The term "separation" as used herein is synonymous with
"recovery" and refers to removing a chemical compound from an
initial mixture to obtain the compound in greater purity or at a
higher concentration than the purity or concentration of the
compound in the initial mixture.
[0063] The term "In Situ Product Removal" (ISPR) as used herein
refers to the selective removal of a fermentation product from a
biological process such as fermentation to control the product
concentration as the product is produced.
[0064] The term "aqueous phase," as used herein, refers to the
aqueous phase of a biphasic mixture obtained by contacting a
fermentation broth with a water-immiscible organic extractant. In
an embodiment of a process described herein that includes
fermentative extraction, the term "fermentation broth" then
specifically refers to the aqueous phase in biphasic fermentative
extraction, and the terms "solvent-poor phase" may be used
synonymously with "aqueous phase" and "fermentation broth."
[0065] The term "organic phase," as used herein, refers to the
non-aqueous phase of a biphasic mixture obtained by contacting a
fermentation broth with a water-immiscible organic extractant. From
time to time, as used herein the terms "solvent-rich phase" may be
used synonymously with "organic phase."
[0066] The term "aqueous phase titer" as used herein, refers to the
concentration of product alcohol (e.g., butanol) in the
fermentation broth.
[0067] The term "water-immiscible" as used herein refers to a
chemical component such as an extractant or a solvent, which is
incapable of mixing with an aqueous solution such as a fermentation
broth, in such a manner as to form one liquid phase.
[0068] The term "biphasic fermentation medium" as used herein
refers to a two-phase growth medium comprising a fermentation
medium (i.e., an aqueous phase) and a suitable amount of a
water-immiscible organic extractant.
[0069] The term "carbon substrate" or "fermentable carbon
substrate" refers to a carbon source capable of being metabolized
by host organisms of the present invention and particularly carbon
sources selected from the group consisting of monosaccharides,
oligosaccharides, polysaccharides, and one-carbon substrates or
mixtures thereof. Non-limiting examples of carbon substrates are
provided herein and include, but are not limited to,
monosaccharides, disaccharides, oligosaccharides, polysaccharides,
ethanol, lactate, succinate, glycerol, carbon dioxide, methanol,
glucose, fructose, lactose, sucrose, xylose, arabinose, dextrose,
cellulose, methane, amino acids, or mixtures thereof.
[0070] "Fermentation broth" as used herein means the mixture of
water, sugars (fermentable carbon sources), dissolved solids (if
present), microorganisms producing alcohol, product alcohol and all
other constituents of the material in which product alcohol is
being made by the reaction of sugars to alcohol, water and carbon
dioxide (CO.sub.2) by the microorganisms present. From time to
time, as used herein the term "fermentation medium" and "fermented
mixture" can be used synonymously with "fermentation broth."
[0071] As used herein a "fermentor" refers to any container,
containers, or apparatus that are used to ferment a substrate. A
fermentor can contain a fermentation medium and microorganism
capable of fermentation. The term "fermentation vessel" refers to
the vessel in which the fermentation reaction is carried out
whereby alcohol such as butanol is made from sugars. "Fermentor"
can be used herein interchangeable with "fermentation vessel."
[0072] The term "fermentation product" includes any desired product
of interest, including, but not limited to 1-butanol, 2-butanol,
isobutanol, etc.
[0073] The term "sugar" as used herein, refers to oligosaccharides,
disaccharides, monosaccharides, and/or mixtures thereof. The term
"saccharide" also includes carbohydrates including starches,
dextrans, glycogens, cellulose, pentosans, as well as sugars.
[0074] The term "fermentable sugar" as used herein, refers to one
or more sugars capable of being metabolized by the microorganisms
disclosed herein for the production of fermentative alcohol.
[0075] The term "undissolved solids" as used herein, means
non-fermentable portions of feedstock, for example, germ, fiber,
and gluten. For example, the non-fermentable portions of feedstock
include the portion of feedstock that remains as solids and can
absorb liquid from the fermentation broth.
[0076] "Biomass" as used herein refers to a natural product
containing a hydrolysable starch that provides a fermentable sugar,
including any cellulosic or lignocellulosic material and materials
comprising cellulose, and optionally further comprising
hemicellulose, lignin, starch, oligosaccharides, disaccharides,
and/or monosaccharides. Biomass can also comprise additional
components, such as protein and/or lipids. Biomass can be derived
from a single source, or biomass can comprise a mixture derived
from more than one source. For example, biomass can comprise a
mixture of corn cobs and corn stover, or a mixture of grass and
leaves. Biomass includes, but is not limited to, bioenergy crops,
agricultural residues, municipal solid waste, industrial solid
waste, sludge from paper manufacture, yard waste, wood, and
forestry waste. Examples of biomass include, but are not limited
to, corn grain, corn cobs, crop residues such as corn husks, corn
stover, grasses, wheat, rye, wheat straw, barley, barley straw,
hay, rice straw, switchgrass, waste paper, sugar cane bagasse,
sorghum, soy, components obtained from milling of grains, trees,
branches, roots, leaves, wood chips, sawdust, shrubs and bushes,
vegetables, fruits, flowers, animal manure, and mixtures
thereof.
[0077] "Feedstock" as used herein means a product containing a
fermentable carbon source. Suitable feedstock include, but are not
limited to, rye, wheat, corn, corn mash, cane, cane mash, sugar
cane, barley, cellulosic material, lignocellulosic material, and
mixtures thereof.
[0078] The term "aerobic conditions" as used herein means growth
conditions in the presence of oxygen.
[0079] The term "microaerobic conditions" as used herein means
growth conditions with low levels of oxygen (i.e., below normal
atmospheric oxygen levels).
[0080] The term "anaerobic conditions" as used herein means growth
conditions in the absence of oxygen.
[0081] 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, phosphorous, and sulfur to allow the microorganism to
synthesize proteins and nucleic acids.
[0082] 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.
[0083] The term "biocompatibility" as used herein refers to the
measure of the ability of a microorganism to utilize glucose in the
presence of an extractant. A biocompatible extractant permits the
microorganism to utilize glucose. A non-biocompatible (i.e., a
biotoxic) extractant does not permit the microorganism to utilize
glucose, for example, at a rate greater than about 25% of the rate
when the extractant is not present.
[0084] The term "toxicity" of solvent as used herein refers to the
percentage of butanol-producing microorganisms killed after
exposure to the solvent for a prolonged time, for example 24
hours.
[0085] The term "fatty acid" as used herein, refers to a carboxylic
acid (e.g., aliphatic monocarboxylic acid) having C.sub.4 to
C.sub.28 carbon atoms (most commonly C.sub.12 to C.sub.24 carbon
atoms), which is either saturated or unsaturated. Fatty acids may
also be branched or unbranched. Fatty acids may be derived from, or
contained in esterified form, in an animal or vegetable fat, oil,
or wax. Fatty acids may occur naturally in the form of glycerides
in fats and fatty oils or may be obtained by hydrolysis of fats or
by synthesis. The term fatty acid may describe a single chemical
species or a mixture of fatty acids. In addition, the term fatty
acid also encompasses free fatty acids.
[0086] The term "fatty alcohol" as used herein, refers to an
alcohol having an aliphatic chain of C.sub.4 to C.sub.22 carbon
atoms, which is either saturated or unsaturated.
[0087] The term "fatty aldehyde" as used herein, refers to an
aldehyde having an aliphatic chain of C.sub.4 to C.sub.22 carbon
atoms, which is either saturated or unsaturated.
[0088] The term "fatty amide" as used herein, refers to an amide
having a long, aliphatic chain of C.sub.4 to C.sub.22 carbon atoms,
which is either saturated or unsaturated.
[0089] The term "fatty ester" as used herein, refers to an ester
having a long aliphatic chain of C.sub.4 to C.sub.22 carbon atoms,
which is either saturated or unsaturated.
[0090] The term "carboxylic acid" as used herein, refers to any
organic compound with the general chemical formula --COOH in which
a carbon atom is bonded to an oxygen atom by a double bond to make
a carbonyl group (--C.dbd.O) and to a hydroxyl group (--OH) by a
single bond. A carboxylic acid may be in the form of the protonated
carboxylic acid, in the form of a salt of a carboxylic acid (e.g.,
an ammonium, sodium, or potassium salt), or as a mixture of
protonated carboxylic acid and salt of a carboxylic acid. The term
carboxylic acid may describe a single chemical species (e.g., oleic
acid) or a mixture of carboxylic acids as can be produced, for
example, by the hydrolysis of biomass-derived fatty acid esters or
triglycerides, diglycerides, monoglycerides, and phospholipids.
[0091] The term "alkane" as used herein refers to a saturated
hydrocarbon.
[0092] "Portion" as used herein, includes a part of a whole or the
whole. For example, a portion of fermentation broth includes a part
of the fermentation broth as well as the whole (or all) the
fermentation broth.
[0093] "Partition coefficient" or "K.sub.d" refers to the ratio of
the concentration of a compound in the two phases of a mixture of
two immiscible solvents at equilibrium. A partition coefficient is
a measure of the differential solubility of a compound between two
immiscible solvents. Partition coefficient, as used herein, is
synonymous with the term distribution coefficient.
[0094] The term "gene" refers to a nucleic acid fragment that is
capable of being expressed as a specific protein, optionally
including regulatory sequences preceding (5' non-coding sequences)
and following (3' non-coding sequences) the coding sequence.
"Native gene" refers to a gene as found in nature with its own
regulatory sequences. "Chimeric gene" refers to any gene that is
not a native gene, comprising regulatory and coding sequences that
are not found together in nature. Accordingly, a chimeric gene can
comprise regulatory sequences and coding sequences that are derived
from different sources, or regulatory sequences and coding
sequences derived from the same source, but arranged in a manner
different than that found in nature. "Endogenous gene" refers to a
native gene in its natural location in the genome of a
microorganism. A "foreign" gene refers to a gene not normally found
in the host microorganism, but that is introduced into the host
microorganism by gene transfer. Foreign genes can comprise native
genes inserted into a non-native microorganism, or chimeric genes.
A "transgene" is a gene that has been introduced into the genome by
a transformation procedure.
[0095] As used herein, "native" refers to the form of a
polynucleotide, gene, or polypeptide as found in nature with its
own regulatory sequences, if present.
[0096] As used herein the term "coding sequence" or "coding region"
refers to a DNA sequence that encodes for a specific amino acid
sequence.
[0097] As used herein, "endogenous" refers to the native form of a
polynucleotide, gene or polypeptide in its natural location in the
organism or in the genome of an organism. "Endogenous
polynucleotide" includes a native polynucleotide in its natural
location in the genome of an organism. "Endogenous gene" includes a
native gene in its natural location in the genome of an organism.
"Endogenous polypeptide" includes a native polypeptide in its
natural location in the organism transcribed and translated from a
native polynucleotide or gene in its natural location in the genome
of an organism.
[0098] The term "heterologous" when used in reference to a
polynucleotide, a gene, or a polypeptide refers to a
polynucleotide, gene, or polypeptide not normally found in the host
organism. "Heterologous" also includes a native coding region, or
portion thereof, that is reintroduced into the source organism in a
form that is different from the corresponding native gene, e.g.,
not in its natural location in the organism's genome. The
heterologous polynucleotide or gene can be introduced into the host
organism by, e.g., gene transfer. A heterologous gene can include a
native coding region with non-native regulatory regions that is
reintroduced into the native host. For example, a heterologous gene
can include a native coding region that is a portion of a chimeric
gene including non-native regulatory regions that is reintroduced
into the native host. "Heterologous polypeptide" includes a native
polypeptide that is reintroduced into the source organism in a form
that is different from the corresponding native polypeptide. A
"heterologous" polypeptide or polynucleotide can also include an
engineered polypeptide or polynucleotide that comprises a
difference from the "native" polypeptide or polynucleotide, e.g., a
point mutation within the endogenous polynucleotide can result in
the production of a "heterologous" polypeptide. As used herein a
"chimeric gene," a "foreign gene," and a "transgene," can all be
examples of "heterologous" genes.
[0099] A "transgene" is a gene that has been introduced into the
genome by a transformation procedure.
Microorganisms
[0100] Microbial hosts for butanol production can 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.
[0101] 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 can 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)).
[0102] 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.
[0103] Suitable host strains with a tolerance for butanol can be
identified by screening based on the intrinsic tolerance of the
strain. The intrinsic tolerance of microbes to butanol can 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 can be determined using
methods known in the art. For example, the microbes of interest can
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 can 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 can be determined from a graph of the percent
inhibition of growth versus the butanol concentration. In one
embodiment, the host strain has an IC50 for butanol of greater than
about 0.5%. In another embodiment, the host strain has an IC50 for
butanol that is greater than about 1.5%. In yet another embodiment,
the host strain has an IC50 for butanol that is greater than about
2.5%.
[0104] 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.
[0105] The ability to genetically modify the host is essential for
the production of any recombinant microorganism. Modes of gene
transfer technology that can be used include, for example,
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.
[0106] The microbial host also can 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 can undergo improvements in intrinsic butanol tolerance
through chemical mutagenesis and mutant screening.
[0107] 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. In some embodiments, the host can be: 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 or Saccharomyces
cerevisiae.
Recombinant Microorganisms
[0108] While not wishing to be bound by theory, it is believed that
the processes described herein are useful in conjunction with any
alcohol producing microorganism, particularly recombinant
microorganisms which produce alcohol.
[0109] Recombinant microorganisms which produce alcohol are also
known in the art (e.g., Ohta et al., Appl. Environ. Microbiol.
57:893-900 (1991); Underwood et al., Appl. Envrion. Microbiol.
68:1071-81 (2002); Shen and Liao, Metab. Eng. 10:312-20 (2008);
Hahnai et al., Appl. Environ. 73:7814-8 (2007); U.S. Pat. No.
5,514,583; U.S. Pat. No. 5,712,133; International Publication No.
WO 1995/028476; Feldmann et al., Appl. Microbiol. Biotechnol.
38:354-61 (1992); Zhang et al., Science 267:240-3 (1995); U.S.
Patent Publication No. 2007/0031918A1; U.S. Pat. No. 7,223,575;
U.S. Pat. No. 7,741,119; U.S. Patent Publication No.
2009/0203099A1; U.S. Patent Publication No. 2009/0246846A1; and
International Publication No. WO 2010/075241, which are herein
incorporated by reference).
[0110] For example, the metabolic pathways of microorganisms may be
genetically modified to produce butanol. These pathways may also be
modified to reduce or eliminate undesired metabolites, and thereby
improve yield of the product alcohol. The production of butanol by
a microorganism is disclosed, for example, in U.S. Pat. Nos.
7,851,188; 7,993,889; 8,178,328, 8,206,970; U.S. Patent Application
Publication Nos. 2007/0292927; 2008/0182308; 2008/0274525;
2009/0305363; 2009/0305370; 2011/0250610; 2011/0313206;
2011/0111472; 2012/0258873; and U.S. patent application Ser. No.
13/428,585, the entire contents of each are herein incorporated by
reference. In some embodiments, microorganisms comprise a butanol
biosynthetic pathway or a biosynthetic pathway for a butanol isomer
such as 1-butanol, 2-butanol, or isobutanol. In some embodiments,
the biosynthetic pathway converts pyruvate to a fermentative
product. In some embodiments, the biosynthetic pathway converts
pyruvate as well as amino acids to a fermentative product. In some
embodiments, at least one, at least two, at least three, or at
least four polypeptides catalyzing substrate to product conversions
of a pathway are encoded by heterologous polynucleotides in the
microorganism. In some embodiments, all polypeptides catalyzing
substrate to product conversions of a pathway are encoded by
heterologous polynucleotides in the microorganism.
[0111] In some embodiments, the microorganism may be bacteria,
cyanobacteria, filamentous fungi, or yeasts. Suitable
microorganisms capable of producing product alcohol (e.g., butanol)
via a biosynthetic pathway include a member of the genera
Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia,
Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus,
Lactobacillus, Enterococcus, Alcaligenes, Paenibacillus,
Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces,
Kluveromyces, Yarrowia, Pichia, Zygosaccharomyces, Debaryomyces,
Candida, Brettanomyces, Pachysolen, Hansenula, Issatchenkia,
Trichosporon, Yamadazyma, or Saccharomyces. In one embodiment,
recombinant microorganisms may be selected from the group
consisting of Escherichia coli, Alcaligenes eutrophus, Bacillus
lichenifonnis, Paenibacillus macerans, Rhodocuccus erythropolis,
Pseudomonas putida, Lactobacillus plantarum, Enterococcus faecium,
Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis,
Candida sonorensis, Candida methanosorbosa, Kluyveromyces lactis,
Kluyveromyces marxianus, Kluveromyces thermotolerans, Issatchenkia
orientalis, Debaryomyces hansenii, and Saccharomyces cerevisiae. In
one embodiment, the genetically modified microorganism is yeast. In
one embodiment, the genetically modified microorganism is a
crabtree-positive yeast selected from Saccharomyces,
Zygosaccharomyces, Schizosaccharomyces, Dekkera, Torulopsis,
Brettanomyces, and some species of Candida. Species of
crabtree-positive yeast include, but are not limited to,
Saccharomyces cerevisiae, Saccharomyces kluyveri,
Schizosaccharomyces pombe, Saccharomyces bayanus, Saccharomyces
mikitae, Saccharomyces paradoxus, Saccharomyces uvarum,
Saccharomyces castelli, Zygosaccharomyces rouxii, Zygosaccharomyces
bailli, and Candida glabrata.
[0112] In some embodiments, the host cell is Saccharomyces
cerevisiae. Saccharomyces cerevisiae are known in the art and are
available from a variety of sources including, but not limited to,
American Type Culture Collection (Rockville, Md.), Centraalbureau
voor Schimmelcultures (CBS) Fungal Biodiversity Centre, LeSaffre,
Gert Strand AB, Ferm Solutions, North American Bioproducts,
Martrex, and Lallemand. S. cerevisiae include, but are not limited
to, BY4741, CEN.PK 113-7D, Ethanol Red.RTM. yeast, Ferm Pro.TM.
yeast, Bio-Ferm.RTM. XR yeast, Gert Strand Prestige Batch Turbo
alcohol yeast, Gert Strand Pot Distillers yeast, Gert Strand
Distillers Turbo yeast, FerMax.TM. Green yeast, FerMax.TM. Gold
yeast, Thermosacc.RTM. yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960,
and CBS7961.
[0113] In some embodiments, the microorganism may be immobilized or
encapsulated. For example, the microorganism may be immobilized or
encapsulated using alginate, calcium alginate, or polyacrylamide
gels, or through the induction of biofilm formation onto a variety
of high surface area support matrices such as diatomite, celite,
diatomaceous earth, silica gels, plastics, or resins. In some
embodiments, ISPR may be used in combination with immobilized or
encapsulated microorganisms. This combination may improve
productivity such as specific volumetric productivity, metabolic
rate, product alcohol yields, tolerance to product alcohol. In
addition, immobilization and encapsulation may minimize the effects
of the process conditions such as shearing on the
microorganisms.
[0114] Biosynthetic pathways for the production of isobutanol that
may be used include those as described by Donaldson et al. in U.S.
Pat. No. 7,851,188; U.S. Pat. No. 7,993,388; and International
Publication No. WO 2007/050671, which are incorporated herein by
reference. In one embodiment, the isobutanol biosynthetic pathway
comprises the following substrate to product conversions:
[0115] a) pyruvate to acetolactate, which may be catalyzed, for
example, by acetolactate synthase;
[0116] b) the acetolactate from step a) to
2,3-dihydroxyisovalerate, which may be catalyzed, for example, by
acetohydroxy acid reductoisomerase;
[0117] c) the 2,3-dihydroxyisovalerate from step b) to
.alpha.-ketoisovalerate, which may be catalyzed, for example, by
acetohydroxy acid dehydratase;
[0118] d) the .alpha.-ketoisovalerate from step c) to
isobutyraldehyde, which may be catalyzed, for example, by a
branched-chain .alpha.-keto acid decarboxylase; and,
[0119] e) the isobutyraldehyde from step d) to isobutanol, which
may be catalyzed, for example, by a branched-chain alcohol
dehydrogenase.
[0120] In another embodiment, the isobutanol biosynthetic pathway
comprises the following substrate to product conversions:
[0121] a) pyruvate to acetolactate, which may be catalyzed, for
example, by acetolactate synthase;
[0122] b) the acetolactate from step a) to
2,3-dihydroxyisovalerate, which may be catalyzed, for example, by
ketol-acid reductoisomerase;
[0123] c) the 2,3-dihydroxyisovalerate from step b) to
.alpha.-ketoisovalerate, which may be catalyzed, for example, by
dihydroxyacid dehydratase;
[0124] d) the .alpha.-ketoisovalerate from step c) to valine, which
may be catalyzed, for example, by transaminase or valine
dehydrogenase;
[0125] e) the valine from step d) to isobutylamine, which may be
catalyzed, for example, by valine decarboxylase;
[0126] f) the isobutylamine from step e) to isobutyraldehyde, which
may be catalyzed by, for example, omega transaminase; and,
[0127] g) the isobutyraldehyde from step f) to isobutanol, which
may be catalyzed, for example, by a branched-chain alcohol
dehydrogenase.
[0128] In another embodiment, the isobutanol biosynthetic pathway
comprises the following substrate to product conversions:
[0129] a) pyruvate to acetolactate, which may be catalyzed, for
example, by acetolactate synthase;
[0130] b) the acetolactate from step a) to
2,3-dihydroxyisovalerate, which may be catalyzed, for example, by
acetohydroxy acid reductoisomerase;
[0131] c) the 2,3-dihydroxyisovalerate from step b) to
.alpha.-ketoisovalerate, which may be catalyzed, for example, by
acetohydroxy acid dehydratase;
[0132] d) the .alpha.-ketoisovalerate from step c) to
isobutyryl-CoA, which may be catalyzed, for example, by
branched-chain keto acid dehydrogenase;
[0133] e) the isobutyryl-CoA from step d) to isobutyraldehyde,
which may be catalyzed, for example, by acylating aldehyde
dehydrogenase; and,
[0134] f) the isobutyraldehyde from step e) to isobutanol, which
may be catalyzed, for example, by a branched-chain alcohol
dehydrogenase.
[0135] Biosynthetic pathways for the production of 1-butanol that
may be used include those described in U.S. Patent Application
Publication No. 2008/0182308 and WO2007/041269, which are
incorporated herein by reference. In one embodiment, the 1-butanol
biosynthetic pathway comprises the following substrate to product
conversions:
[0136] a) acetyl-CoA to acetoacetyl-CoA, which may be catalyzed,
for example, by acetyl-CoA acetyltransferase;
[0137] b) the acetoacetyl-CoA from step a) to 3-hydroxybutyryl-CoA,
which may be catalyzed, for example, by 3-hydroxybutyryl-CoA
dehydrogenase;
[0138] c) the 3-hydroxybutyryl-CoA from step b) to crotonyl-CoA,
which may be catalyzed, for example, by crotonase;
[0139] d) the crotonyl-CoA from step c) to butyryl-CoA, which may
be catalyzed, for example, by butyryl-CoA dehydrogenase;
[0140] e) the butyryl-CoA from step d) to butyraldehyde, which may
be catalyzed, for example, by butyraldehyde dehydrogenase; and,
[0141] f) the butyraldehyde from step e) to 1-butanol, which may be
catalyzed, for example, by butanol dehydrogenase.
[0142] Biosynthetic pathways for the production of 2-butanol that
may be used include those described by Donaldson et al. in U.S.
Pat. No. 8,206,970; U.S. Patent Application Publication Nos.
2007/0292927 and 2009/0155870; International Publication Nos. WO
2007/130518 and WO 2007/130521, all of which are incorporated
herein by reference. In one embodiment, the 2-butanol biosynthetic
pathway comprises the following substrate to product
conversions:
[0143] a) pyruvate to alpha-acetolactate, which may be catalyzed,
for example, by acetolactate synthase;
[0144] b) the alpha-acetolactate from step a) to acetoin, which may
be catalyzed, for example, by acetolactate decarboxylase;
[0145] c) the acetoin from step b) to 3-amino-2-butanol, which may
be catalyzed, for example, acetonin aminase;
[0146] d) the 3-amino-2-butanol from step c) to 3-amino-2-butanol
phosphate, which may be catalyzed, for example, by aminobutanol
kinase;
[0147] e) the 3-amino-2-butanol phosphate from step d) to
2-butanone, which may be catalyzed, for example, by aminobutanol
phosphate phosphorylase; and,
[0148] f) the 2-butanone from step e) to 2-butanol, which may be
catalyzed, for example, by butanol dehydrogenase.
[0149] In another embodiment, the 2-butanol biosynthetic pathway
comprises the following substrate to product conversions:
[0150] a) pyruvate to alpha-acetolactate, which may be catalyzed,
for example, by acetolactate synthase;
[0151] b) the alpha-acetolactate from step a) to acetoin, which may
be catalyzed, for example, by acetolactate decarboxylase;
[0152] c) the acetoin to 2,3-butanediol from step b), which may be
catalyzed, for example, by butanediol dehydrogenase;
[0153] d) the 2,3-butanediol from step c) to 2-butanone, which may
be catalyzed, for example, by dial dehydratase; and,
[0154] e) the 2-butanone from step d) to 2-butanol, which may be
catalyzed, for example, by butanol dehydrogenase.
[0155] Biosynthetic pathways for the production of 2-butanone that
may be used include those described in U.S. Pat. No. 8,206,970 and
U.S. Patent Application Publication Nos. 2007/0292927 and
2009/0155870, which are incorporated herein by reference. In one
embodiment, the 2-butanone biosynthetic pathway comprises the
following substrate to product conversions:
[0156] a) pyruvate to alpha-acetolactate, which may be catalyzed,
for example, by acetolactate synthase;
[0157] b) the alpha-acetolactate from step a) to acetoin, which may
be catalyzed, for example, by acetolactate decarboxylase;
[0158] c) the acetoin from step b) to 3-amino-2-butanol, which may
be catalyzed, for example, acetonin aminase;
[0159] d) the 3-amino-2-butanol from step c) to 3-amino-2-butanol
phosphate, which may be catalyzed, for example, by aminobutanol
kinase; and,
[0160] e) the 3-amino-2-butanol phosphate from step d) to
2-butanone, which may be catalyzed, for example, by aminobutanol
phosphate phosphorylase.
[0161] In another embodiment, the 2-butanone biosynthetic pathway
comprises the following substrate to product conversions:
[0162] a) pyruvate to alpha-acetolactate, which may be catalyzed,
for example, by acetolactate synthase;
[0163] b) the alpha-acetolactate from step a) to acetoin which may
be catalyzed, for example, by acetolactate decarboxylase;
[0164] c) the acetoin from step b) to 2,3-butanediol, which may be
catalyzed, for example, by butanediol dehydrogenase;
[0165] d) the 2,3-butanediol from step c) to 2-butanone, which may
be catalyzed, for example, by diol dehydratase.
[0166] The terms "acetohydroxyacid synthase," "acetolactate
synthase," and "acetolactate synthetase" (abbreviated "ALS") are
used interchangeably herein to refer to an enzyme that catalyzes
the conversion of pyruvate to acetolactate and CO.sub.2. Example
acetolactate synthases are known by the EC number 2.2.1.6 (Enzyme
Nomenclature 1992, Academic Press, San Diego). These enzymes are
available from a number of sources, including, but not limited to,
Bacillus subtilis (GenBank Nos: CAB07802.1, Z99122, NCBI (National
Center for Biotechnology Information) amino acid sequence, NCBI
nucleotide sequence, respectively), CAB 15618, Klebsiella
pneumoniae (GenBank Nos: AAA25079, M73842), and Lactococcus lactis
(GenBank Nos: AAA25161, L16975)
[0167] The term "ketol-acid reductoisomerase" ("KARI"),
"acetohydroxy acid isomeroreductase," and "acetohydroxy acid
reductoisomerase" will be used interchangeably and refer to enzymes
capable of catalyzing the reaction of (S)-acetolactate to
2,3-dihydroxyisovalerate. Example KARI enzymes may be classified as
EC number EC 1.1.1.86 (Enzyme Nomenclature 1992, Academic Press,
San Diego), and are available from a vast array of microorganisms,
including, but not limited to, Escherichia coli (GenBank Nos:
NP_418222, NC_000913), Saccharomyces cerevisiae (GenBank Nos:
NP_013459, NC_001144), Methanococcus maripaludis (GenBank Nos:
CAF30210, BX957220), Bacillus subtilis (GenBank Nos: CAB14789,
Z99118), and Anaerostipes caccae. Ketol-acid reductoisomerase
(KARI) enzymes are described in U.S. Pat. Nos. 7,910,342 and
8,129,162; U.S. Patent Application Publication Nos. 2008/0261230,
2009/0163376, 2010/0197519, PCT Application Publication No.
WO/2011/041415, PCT Application Publication No. WO2012/129555; and
U.S. Provisional Application No. 61/705,977, filed on Sep. 26,
2012, all of which are incorporated herein by reference. Examples
of KARIs disclosed therein are those from Lactococcus lactis,
Vibrio cholera, Pseudomonas aeruginosa PAO1, and Pseudomonas
fluorescens PF5 mutants. In some embodiments, the KARI utilizes
NADH. In some embodiments, the KARI utilizes NADPH. In some
embodiments, the KARI utilizes NADH or NADPH.
[0168] The term "acetohydroxy acid dehydratase" and "dihydroxyacid
dehydratase" ("DHAD") refers to an enzyme that catalyzes the
conversion of 2,3-dihydroxyisovalerate to .alpha.-ketoisovalerate.
Example acetohydroxy acid dehydratases are known by the EC number
4.2.1.9. Such enzymes are available from a vast array of
microorganisms, including, but not limited to, E. coli (GenBank
Nos: YP_026248, NC000913), Saccharomyces cerevisiae (GenBank Nos:
NP_012550, NC 001142), M maripaludis (GenBank Nos: CAF29874,
BX957219), B. subtilis (GenBank Nos: CAB14105, Z99115), L. lactis,
and N. crassa. U.S. Patent Application Publication No.
2010/0081154, U.S. Pat. No. 7,851,188, and U.S. Pat. No. 8,241,878,
which are incorporated herein by reference in their entireties,
describe dihydroxyacid dehydratases (DHADs), including a DHAD from
Streptococcus mutans and variants thereof.
[0169] The term "branched-chain .alpha.-keto acid decarboxylase,"
".alpha.-ketoacid decarboxylase," ".alpha.-ketoisovalerate
decarboxylase," or "2-ketoisovalerate decarboxylase" ("KIVD")
refers to an enzyme that catalyzes the conversion of
.alpha.-ketoisovalerate to isobutyraldehyde and CO.sub.2. Example
branched-chain .alpha.-keto acid decarboxylases are known by the EC
number 4.1.1.72 and are available from a number of sources,
including, but not limited to, Lactococcus lactis (GenBank Nos:
AAS49166, AY548760; CAG34226, AJ746364), Salmonella typhimurium
(GenBank Nos: NP_461346, NC_003197), Clostridium acetobutylicum
(GenBank Nos: NP_149189, NC_001988), M caseolyticus, and L.
grayi.
[0170] The term "branched-chain alcohol dehydrogenase" ("ADH")
refers to an enzyme that catalyzes the conversion of
isobutyraldehyde to isobutanol. Example branched-chain alcohol
dehydrogenases are known by the EC number 1.1.1.265, but may also
be classified under other alcohol dehydrogenases (specifically, EC
1.1.1.1 or 1.1.1.2). Alcohol dehydrogenases may be NADPH dependent
or NADH dependent. Such enzymes are available from a number of
sources, including, but not limited to, S. cerevisiae (GenBank Nos:
NP_010656, NC_001136, NP_014051, NC_001145), E. coli (GenBank Nos:
NP_417484, NC_000913), C. acetobutylicum (GenBank Nos: NP_349892,
NC_003030; NP_349891, NC_003030). U.S. Patent Application
Publication No. 2009/0269823 describes SadB, an alcohol
dehydrogenase (ADH) from Achromobacter xylosoxidans. Alcohol
dehydrogenases can also include horse liver ADH and Beijerinkia
indica ADH, as described by U.S. Patent Application Publication No.
2011/0269199, which is incorporated herein by reference in its
entirety.
[0171] The term "butanol dehydrogenase" refers to a polypeptide (or
polypeptides) having an enzyme activity that catalyzes the
conversion of isobutyraldehyde to isobutanol or the conversion of
2-butanone and 2-butanol. Butanol dehydrogenases are a subset of a
broad family of alcohol dehydrogenases. Butanol dehydrogenase may
be NAD- or NADP-dependent. The NAD-dependent enzymes are known as
EC 1.1.1.1 and are available, for example, from Rhodococcus ruber
(GenBank Nos: CAD36475, AJ491307). The NADP dependent enzymes are
known as EC 1.1.1.2 and are available, for example, from Pyrococcus
furiosus (GenBank Nos: AAC25556, AF013169). Additionally, a butanol
dehydrogenase is available from Escherichia coli (GenBank Nos: NP
417484, NC_000913) and a cyclohexanol dehydrogenase is available
from Acinetobacter sp. (GenBank Nos: AAG10026, AF282240). The term
"butanol dehydrogenase" also refers to an enzyme that catalyzes the
conversion of butyraldehyde to 1-butanol, using either NADH or
NADPH as cofactor. Butanol dehydrogenases are available from, for
example, C. acetobutylicum (GenBank NOs: NP_149325, NC_001988;
note: this enzyme possesses both aldehyde and alcohol dehydrogenase
activity); NP_349891, NC_003030; and NP_349892, NC_003030) and E.
coli (GenBank NOs: NP_417-484, NC_000913).
[0172] The term "branched-chain keto acid dehydrogenase" refers to
an enzyme that catalyzes the conversion of .alpha.-ketoisovalerate
to isobutyryl-CoA (isobutyryl-coenzyme A), typically using
NAD.sup.+ (nicotinamide adenine dinucleotide) as an electron
acceptor. Example branched-chain keto acid dehydrogenases are known
by the EC number 1.2.4.4. Such branched-chain keto acid
dehydrogenases are comprised of four subunits and sequences from
all subunits are available from a vast array of microorganisms,
including, but not limited to, B. subtilis (GenBank Nos: CAB14336,
Z99116; CAB14335, Z99116; CAB14334, Z99116; and CAB14337, Z99116)
and Pseudomonas putida (GenBank Nos: AAA65614, M57613; AAA65615,
M57613; AAA65617, M57613; and AAA65618, M57613).
[0173] The term "acylating aldehyde dehydrogenase" refers to an
enzyme that catalyzes the conversion of isobutyryl-CoA to
isobutyraldehyde, typically using either NADH or NADPH as an
electron donor. Example acylating aldehyde dehydrogenases are known
by the EC numbers 1.2.1.10 and 1.2.1.57. Such enzymes are available
from multiple sources, including, but not limited to, Clostridium
beijerinckii (GenBank Nos: AAD31841, AF157306), C. acetobutylicum
(GenBank Nos: NP_149325, NC_001988; NP_149199, NC_001988), P.
putida (GenBank Nos: AAA89106, U13232), and Thermus thermophilus
(GenBank Nos: YP_145486, NC_006461).
[0174] The term "transaminase" refers to an enzyme that catalyzes
the conversion of .alpha.-ketoisovalerate to L-valine, using either
alanine or glutamate as an amine donor. Example transaminases are
known by the EC numbers 2.6.1.42 and 2.6.1.66. Such enzymes are
available from a number of sources. Examples of sources for
alanine-dependent enzymes include, but are not limited to, E. coli
(GenBank Nos: YP_026231, NC_000913) and Bacillus licheniformis
(GenBank Nos: YP_093743, NC_006322). Examples of sources for
glutamate-dependent enzymes include, but are not limited to, E.
coli (GenBank Nos: YP_026247, NC_000913), Saccharomyces cerevisiae
(GenBank Nos: NP_012682, NC_001142) and Methanobacterium
thermoautotrophicum (GenBank Nos: NP_276546, NC_000916).
[0175] The term "valine dehydrogenase" refers to an enzyme that
catalyzes the conversion of .alpha.-ketoisovalerate to L-valine,
typically using NAD(P)H as an electron donor and ammonia as an
amine donor. Example valine dehydrogenases are known by the EC
numbers 1.4.1.8 and 1.4.1.9 and such enzymes are available from a
number of sources, including, but not limited to, Streptomyces
coelicolor (GenBank Nos: NP_628270, NC_003888) and B. subtilis
(GenBank Nos: CAB14339, Z99116).
[0176] The term "valine decarboxylase" refers to an enzyme that
catalyzes the conversion of L-valine to isobutylamine and CO.sub.2.
Example valine decarboxylases are known by the EC number 4.1.1.14.
Such enzymes are found in Streptomyces, such as for example,
Streptomyces viridifaciens (GenBank Nos: AAN10242, AY116644).
[0177] The term "omega transaminase" refers to an enzyme that
catalyzes the conversion of isobutylamine to isobutyraldehyde using
a suitable amino acid as an amine donor. Example omega
transaminases are known by the EC number 2.6.1.18 and are available
from a number of sources, including, but not limited to,
Alcaligenes denitrificans (AAP92672, AY330220), Ralstonia eutropha
(GenBank Nos: YP_294474, NC_007347), Shewanella oneidensis (GenBank
Nos: NP_719046, NC_004347), and P. putida (GenBank Nos: AAN66223,
AE016776).
[0178] The term "acetyl-CoA acetyltransferase" refers to an enzyme
that catalyzes the conversion of two molecules of acetyl-CoA to
acetoacetyl-CoA and coenzyme A (CoA). Example acetyl-CoA
acetyltransferases are acetyl-CoA acetyltransferases with substrate
preferences (reaction in the forward direction) for a short chain
acyl-CoA and acetyl-CoA and are classified as E.C. 2.3.1.9 [Enzyme
Nomenclature 1992, Academic Press, San Diego]; although, enzymes
with a broader substrate range (E.C. 2.3.1.16) will be functional
as well. Acetyl-CoA acetyltransferases are available from a number
of sources, for example, Escherichia coli (GenBank Nos: NP_416728,
NC_000913; NCBI (National Center for Biotechnology Information)
amino acid sequence, NCBI nucleotide sequence), Clostridium
acetobutylicum (GenBank Nos: NP_349476.1, NC_003030; NP_149242,
NC_001988, Bacillus subtilis (GenBank Nos: NP_390297, NC_000964),
and Saccharomyces cerevisiae (GenBank Nos: NP_015297,
NC_001148).
[0179] The term "3-hydroxybutyryl-CoA dehydrogenase" refers to an
enzyme that catalyzes the conversion of acetoacetyl-CoA to
3-hydroxybutyryl-CoA. 3-Example hydroxybutyryl-CoA dehydrogenases
may be reduced nicotinamide adenine dinucleotide (NADH)-dependent,
with a substrate preference for (S)-3-hydroxybutyryl-CoA or
(R)-3-hydroxybutyryl-CoA. Examples may be classified as E.C.
1.1.1.35 and E.C. 1.1.1.30, respectively. Additionally,
3-hydroxybutyryl-CoA dehydrogenases may be reduced nicotinamide
adenine dinucleotide phosphate (NADPH)-dependent, with a substrate
preference for (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA
and are classified as E.C. 1.1.1.157 and E.C. 1.1.1.36,
respectively. 3-Hydroxybutyryl-CoA dehydrogenases are available
from a number of sources, for example, C. acetobutylicum (GenBank
NOs: NP_349314, NC_003030), B. subtilis (GenBank NOs: AAB09614,
U29084), Ralstonia eutropha (GenBank NOs: YP_294481, NC_007347),
and Alcaligenes eutrophus (GenBank NOs: AAA21973, J04987).
[0180] The term "crotonase" refers to an enzyme that catalyzes the
conversion of 3-hydroxybutyryl-CoA to crotonyl-CoA and H.sub.2O.
Example crotonases may have a substrate preference for
(S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and may be
classified as E.C. 4.2.1.17 and E.C. 4.2.1.55, respectively.
Crotonases are available from a number of sources, for example, E.
coli (GenBank NOs: NP_415911, NC_000913), C. acetobutylicum
(GenBank NOs: NP_349318, NC_003030), B. subtilis (GenBank NOs:
CAB13705, Z99113), and Aeromonas caviae (GenBank NOs: BAA21816,
D88825).
[0181] The term "butyryl-CoA dehydrogenase" refers to an enzyme
that catalyzes the conversion of crotonyl-CoA to butyryl-CoA.
Example butyryl-CoA dehydrogenases may be NADH-dependent,
NADPH-dependent, or flavin-dependent and may be classified as E.C.
1.3.1.44, E.C. 1.3.1.38, and E.C. 1.3.99.2, respectively.
Butyryl-CoA dehydrogenases are available from a number of sources,
for example, C. acetobutylicum (GenBank NOs: NP_347102, NC_003030),
Euglena gracilis (GenBank NOs: Q5EU90, AY741582), Streptomyces
collinus (GenBank NOs: AAA92890, U37135), and Streptomyces
coelicolor (GenBank NOs: CAA22721, AL939127).
[0182] The term "butyraldehyde dehydrogenase" refers to an enzyme
that catalyzes the conversion of butyryl-CoA to butyraldehyde,
using NADH or NADPH as cofactor. Butyraldehyde dehydrogenases with
a preference for NADH are known as E.C. 1.2.1.57 and are available
from, for example, Clostridium beijerinckii (GenBank NOs: AAD31841,
AF157306) and C. acetobutylicum (GenBank NOs: NP_149325,
NC_001988).
[0183] The term "isobutyryl-CoA mutase" refers to an enzyme that
catalyzes the conversion of butyryl-CoA to isobutyryl-CoA. This
enzyme uses coenzyme B.sub.12 as cofactor. Example isobutyryl-CoA
mutases are known by the EC number 5.4.99.13. These enzymes are
found in a number of Streptomyces, including, but not limited to,
Streptomyces cinnamonensis (GenBank Nos: AAC08713, U67612;
CAB59633, AJ246005), S. coelicolor (GenBank Nos: CAB70645,
AL939123; CAB92663, AL939121), and Streptomyces avermitilis
(GenBank Nos: NP_824008, NC_003155; NP_824637, NC_003155).
[0184] The term "acetolactate decarboxylase" refers to a
polypeptide (or polypeptides) having an enzyme activity that
catalyzes the conversion of alpha-acetolactate to acetoin. Example
acetolactate decarboxylases are known as EC 4.1.1.5 and are
available, for example, from Bacillus subtilis (GenBank Nos:
AAA22223, L04470), Klebsiella terrigena (GenBank Nos: AAA25054,
L04507) and Klebsiella pneumoniae (GenBank Nos: AAU43774,
AY722056).
[0185] The term "acetoin aminase" or "acetoin transaminase" refers
to a polypeptide (or polypeptides) having an enzyme activity that
catalyzes the conversion of acetoin to 3-amino-2-butanol. Acetoin
aminase may utilize the cofactor pyridoxal 5'-phosphate or NADH
(reduced nicotinamide adenine dinucleotide) or NADPH (reduced
nicotinamide adenine dinucleotide phosphate). The resulting product
may have (R) or (S) stereochemistry at the 3-position. The
pyridoxal phosphate-dependent enzyme may use an amino acid such as
alanine or glutamate as the amino donor. The NADH- and
NADPH-dependent enzymes may use ammonia as a second substrate. A
suitable example of an NADH dependent acetoin aminase, also known
as amino alcohol dehydrogenase, is described by Ito, et al. (U.S.
Pat. No. 6,432,688). An example of a pyridoxal-dependent acetoin
aminase is the amine:pyruvate aminotransferase (also called
amine:pyruvate transaminase) described by Shin and Kim (J. Org.
Chem. 67:2848-2853, 2002).
[0186] The term "acetoin kinase" refers to a polypeptide (or
polypeptides) having an enzyme activity that catalyzes the
conversion of acetoin to phosphoacetoin. Acetoin kinase may utilize
ATP (adenosine triphosphate) or phosphoenolpyruvate as the
phosphate donor in the reaction. Enzymes that catalyze the
analogous reaction on the similar substrate dihydroxyacetone, for
example, include enzymes known as EC 2.7.1.29 (Garcia-Alles, et
al., Biochemistry 43:13037-13046, 2004).
[0187] The term "acetoin phosphate aminase" refers to a polypeptide
(or polypeptides) having an enzyme activity that catalyzes the
conversion of phosphoacetoin to 3-amino-2-butanol O-phosphate.
Acetoin phosphate aminase may use the cofactor pyridoxal
5'-phosphate, NADH or NADPH. The resulting product may have (R) or
(S) stereochemistry at the 3-position. The pyridoxal
phosphate-dependent enzyme may use an amino acid such as alanine or
glutamate. The NADH and NADPH-dependent enzymes may use ammonia as
a second substrate. Although there are no reports of enzymes
catalyzing this reaction on phosphoacetoin, there is a pyridoxal
phosphate-dependent enzyme that is proposed to carry out the
analogous reaction on the similar substrate serinol phosphate
(Yasuta, et al., Appl. Environ. Microbial. 67:4999-5009, 2001).
[0188] The term "aminobutanol phosphate phospholyase," also called
"amino alcohol O-phosphate lyase," refers to a polypeptide (or
polypeptides) having an enzyme activity that catalyzes the
conversion of 3-amino-2-butanol O-phosphate to 2-butanone. Amino
butanol phosphate phospho-lyase may utilize the cofactor pyridoxal
5'-phosphate. There are reports of enzymes that catalyze the
analogous reaction on the similar substrate 1-amino-2-propanol
phosphate (Jones, et al., Biochem J. 134:167-182, 1973). U.S.
Patent Application Publication No. 2007/0259410 describes an
aminobutanol phosphate phospho-lyase from the organism Erwinia
carotovora.
[0189] The term "aminobutanol kinase" refers to a polypeptide (or
polypeptides) having an enzyme activity that catalyzes the
conversion of 3-amino-2-butanol to 3-amino-2-butanol O-phosphate.
Amino butanol kinase may utilize ATP as the phosphate donor.
Although there are no reports of enzymes catalyzing this reaction
on 3-amino-2-butanol, there are reports of enzymes that catalyze
the analogous reaction on the similar substrates ethanolamine and
1-amino-2-propanol (Jones, et al., supra). U.S. Patent Application
Publication No. 2009/0155870 describes, in Example 14, an amino
alcohol kinase of Erwinia carotovora subsp. Atroseptica.
[0190] The term "butanediol dehydrogenase" also known as "acetoin
reductase" refers to a polypeptide (or polypeptides) having an
enzyme activity that catalyzes the conversion of acetoin to
2,3-butanediol. Butanedial dehydrogenases are a subset of the broad
family of alcohol dehydrogenases. Butanediol dehydrogenase enzymes
may have specificity for production of (R)- or (S)-stereochemistry
in the alcohol product. (S)-specific butanediol dehydrogenases are
known as EC 1.1.1.76 and are available, for example, from
Klebsiella pneumoniae (GenBank Nos: BBA13085, D86412). (R)-specific
butanediol dehydrogenases are known as EC 1.1.1.4 and are
available, for example, from Bacillus cereus (GenBank Nos. NP
830481, NC_004722; AAP07682, AE017000), and Lactococcus lactis
(GenBank Nos. AAK04995, AE006323).
[0191] The term "butanediol dehydratase," also known as "dial
dehydratase" or "propanediol dehydratase" refers to a polypeptide
(or polypeptides) having an enzyme activity that catalyzes the
conversion of 2,3-butanediol to 2-butanone. Butanediol dehydratase
may utilize the cofactor adenosyl cobalamin (also known as coenzyme
Bw or vitamin B.sub.12; although vitamin B12 may refer also to
other forms of cobalamin that are not coenzyme B12). Adenosyl
cobalamin-dependent enzymes are known as EC 4.2.1.28 and are
available, for example, from Klebsiella oxytoca (GenBank Nos:
AA08099 (alpha subunit), D45071; BAA08100 (beta subunit), D45071;
and BBA08101 (gamma subunit), D45071 (Note all three subunits are
required for activity), and Klebsiella pneumonia (GenBank Nos:
AAC98384 (alpha subunit), AF102064; GenBank Nos: AAC98385 (beta
subunit), AF102064, GenBank Nos: AAC98386 (gamma subunit),
AF102064). Other suitable dial dehydratases include, but are not
limited to, B12-dependent dial dehydratases available from
Salmonella typhimurium (GenBank Nos: AAB84102 (large subunit),
AF026270; GenBank Nos: AAB84103 (medium subunit), AF026270; GenBank
Nos: AAB84104 (small subunit), AF026270); and Lactobacillus
collinoides (GenBank Nos: CAC82541 (large subunit), AJ297723;
GenBank Nos: CAC82542 (medium subunit); AJ297723; GenBank Nos:
CAD01091 (small subunit), AJ297723); and enzymes from Lactobacillus
brevis (particularly strains CNRZ 734 and CNRZ 735, Speranza, et
al., J. Agric. Food Chem. 45:3476-3480, 1997), and nucleotide
sequences that encode the corresponding enzymes. Methods of diol
dehydratase gene isolation are well known in the art (e.g., U.S.
Pat. No. 5,686,276).
[0192] The term "pyruvate decarboxylase" refers to an enzyme that
catalyzes the decarboxylation of pyruvic acid to acetaldehyde and
carbon dioxide. Pyruvate dehydrogenases are known by the EC number
4.1.1.1. These enzymes are found in a number of yeast, including
Saccharomyces cerevisiae (GenBank Nos: CAA97575, CAA97705,
CAA97091).
[0193] It will be appreciated that host cells comprising an
isobutanol biosynthetic pathway as provided herein may further
comprise one or more additional modifications. U.S. Patent
Application Publication No. 2009/0305363 (incorporated by
reference) discloses increased conversion of pyruvate to
acetolactate by engineering yeast for expression of a
cytosol-localized acetolactate synthase and substantial elimination
of pyruvate decarboxylase activity. In some embodiments, the host
cells comprise modifications to reduce glycerol-3-phosphate
dehydrogenase activity and/or disruption in at least one gene
encoding a polypeptide having pyruvate decarboxylase activity or a
disruption in at least one gene encoding a regulatory element
controlling pyruvate decarboxylase gene expression as described in
U.S. Patent Application Publication No. 2009/0305363 (incorporated
herein by reference), modifications to a host cell that provide for
increased carbon flux through an Entner-Doudoroff Pathway or
reducing equivalents balance as described in U.S. Patent
Application Publication No. 2010/0120105 (incorporated herein by
reference). Other modifications include integration of at least one
polynucleotide encoding a polypeptide that catalyzes a step in a
pyruvate-utilizing biosynthetic pathway.
[0194] Other modifications include at least one deletion, mutation,
and/or substitution in an endogenous polynucleotide encoding a
polypeptide having acetolactate reductase activity. As used herein,
"acetolactate reductase activity" refers to the activity of any
polypeptide having the ability to catalyze the conversion of
acetolactate to DHMB. Such polypeptides can be determined by
methods well known in the art and disclosed herein. As used herein,
"DHMB" refers to 2,3-dihydroxy-2-methyl butyrate. DHMB includes
"fast DHMB," which has the 2S, 3S configuration, and "slow DHMB,"
which has the 2S, 3R configurate. See Kaneko et al., Phytochemistry
39: 115-120 (1995), which is herein incorporated by reference in
its entirety and refers to fast DHMB as anglyceric acid and slow
DHMB as tiglyceric acid. In embodiments, the polypeptide having
acetolactate reductase activity is YMR226C of Saccharomyces
cerevisiae or a homolog thereof.
[0195] Additional modifications include a deletion, mutation,
and/or substitution in an endogenous polynucleotide encoding a
polypeptide having aldehyde dehydrogenase and/or aldehyde oxidase
activity. As used herein, "aldehyde dehydrogenase activity" refers
to any polypeptide having a biological function of an aldehyde
dehydrogenase. Such polypeptides include a polypeptide that
catalyzes the oxidation (dehydrogenation) of aldehydes. Such
polypeptides include a polypeptide that catalyzes the conversion of
isobutyraldehyde to isobutyric acid. Such polypeptides also include
a polypeptide that corresponds to Enzyme Commission Numbers EC
1.2.1.3, EC 1.2.1.4 or EC 1.2.1.5. Such polypeptides can be
determined by methods well known in the art and disclosed herein.
As used herein, "aldehyde oxidase activity" refers to any
polypeptide having a biological function of an aldehyde oxidase.
Such polypeptides include a polypeptide that catalyzes production
of carboxylic acids from aldehydes. Such polypeptides include a
polypeptide that catalyzes the conversion of isobutyraldehyde to
isobutyric acid. Such polypeptides also include a polypeptide that
corresponds to Enzyme Commission Number EC 1.2.3.1. Such
polypeptides can be determined by methods well known in the art and
disclosed herein. In some embodiments, the polypeptide having
aldehyde dehydrogenase activity is ALD6 from Saccharomyces
cerevisiae or a homolog thereof.
[0196] A genetic modification which has the effect of reducing
glucose repression wherein the yeast production host cell is pdc-
is described in U.S. Patent Application Publication No.
2011/0124060, incorporated herein by reference. In some
embodiments, the pyruvate decarboxylase that is deleted or
down-regulated is selected from the group consisting of: PDC1,
PDC5, PDC6, and combinations thereof. In some embodiments, the
pyruvate decarboxylase is selected from PDC1 pyruvate decarboxylase
from Saccharomyces cerevisiae, PDC5 pyruvate decarboxylase from
Saccharomyces cerevisiae, PDC6 pyruvate decarboxylase from
Saccharomyces cerevisiae, pyruvate decarboxylase from Candida
glabrata, PDC1 pyruvate decarboxylase from Pichia stipites, PDC2
pyruvate decarboxylase from Pichia stipites, pyruvate decarboxylase
from Kluveromyces lactis, pyruvate decarboxylase from Yarrowia
lipolytica, pyruvate decarboxylase from Schizosaccharomyces pombe,
and pyruvate decarboxylase from Zygosaccharomyces rouxii. In some
embodiments, host cells contain a deletion or down-regulation of a
polynucleotide encoding a polypeptide that catalyzes the conversion
of glyceraldehyde-3-phosphate to glycerate 1,3, bisphosphate. In
some embodiments, the enzyme that catalyzes this reaction is
glyceraldehyde-3-phosphate dehydrogenase.
[0197] WIPO publication number WO 2001/103300 discloses recombinant
host cells comprising (a) at least one heterologous polynucleotide
encoding a polypeptide having dihydroxy-acid dehydratase activity;
and (b)(i) at least one deletion, mutation, and/or substitution in
an endogenous gene encoding a polypeptide affecting Fe--S cluster
biosynthesis; and/or (ii) at least one heterologous polynucleotide
encoding a polypeptide affecting Fe--S cluster biosynthesis. In
embodiments, the polypeptide affecting Fe--S cluster biosynthesis
is encoded by AFT1, AFT2, FRA2, GRX3, or CCC1. In embodiments, the
polypeptide affecting Fe--S cluster biosynthesis is constitutive
mutant AFT1 L99A, AFT1 L102A, AFT1 C291F, or AFT1 C293F.
[0198] Additionally, host cells may comprise heterologous
polynucleotides encoding a polypeptide with phosphoketolase
activity and/or a heterologous polynucleotide encoding a
polypeptide with phosphotransacetylase activity.
[0199] In some embodiments, any particular nucleic acid molecule or
polypeptide may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% identical to a nucleotide sequence or polypeptide sequence
described herein. The term "percent identity" as known in the art,
is a relationship between two or more polypeptide sequences or two
or more polynucleotide sequences, as determined by comparing the
sequences. In the art, "identity" also means the degree of sequence
relatedness between polypeptide or polynucleotide sequences, as the
case may be, as determined by the match between strings of such
sequences. "Identity" and "similarity" can be readily calculated by
known methods, including but not limited to those disclosed in: 1.)
Computational Molecular Biology (Lesk, A. M., Ed.) Oxford
University: NY (1988); 2.) Biocomputing: Informatics and Genome
Projects (Smith, D. W., Ed.) Academic: NY (1993); 3.) Computer
Analysis of Sequence Data, Part I (Griffin, A. M., and Griffin, H.
G., Eds.) Humania: NJ (1994); 4.) Sequence Analysis in Molecular
Biology (von Heinje, G., Ed.) Academic (1987); and 5.) Sequence
Analysis Primer (Gribskov, M. and Devereux, J., Eds.) Stockton: NY
(1991).
[0200] Standard recombinant DNA and molecular cloning techniques
are well known in the art and are described by Sambrook, et al.
(Sambrook, J., Fritsch, E. F. and Maniatis, T. (Molecular Cloning:
A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, 1989, here in referred to as Maniatis) and by
Ausubel, et al. (Ausubel, et al., Current Protocols in Molecular
Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience,
1987). Examples of methods to construct microorganisms that
comprise a butanol biosynthetic pathway are disclosed, for example,
in U.S. Pat. No. 7,851,188, and U.S. Patent Application Publication
Nos. 2007/0092957; 2007/0259410; 2007/0292927; 2008/0182308;
2008/0274525; 2009/0155870; 2009/0305363; and 2009/0305370, the
entire contents of each are herein incorporated by reference.
Organic Extractants
[0201] A product alcohol may be recovered from fermentation broth
using a number of methods including liquid-liquid extraction. In
some embodiments of the processes and systems described herein, an
extractant may be used to recover product alcohol from fermentation
broth. Extractants used herein may be have, for example, one or
more of the following properties and/or characteristics: (i)
biocompatible with the microorganisms, (ii) immiscible with the
fermentation medium, (iii) a high partition coefficient (K.sub.d)
for the extraction of product alcohol, (iv) a low partition
coefficient for the extraction of nutrients and other side
products, (v) a low spreading coefficient, (vi) a high interfacial
tension with water, (vii) low viscosity (i), (viii) high
selectivity for product alcohol as compared to, for example, water,
(ix) low density (p) relative to the fermentation medium, (x)
boiling point suitable for downstream processing of the extractant
and product alcohol, (xi) melting point lower than ambient
temperature, (xii) minimal solubility in solids, (xiii) a low
tendency to form emulsions with the fermentation medium, (xiv)
stability over the fermentation process, (xv) low cost, (xvi)
commercial availability, and (xvii) nonhazardous.
[0202] In some embodiments, the extractant may be selected based
upon certain properties and/or characteristics as described above.
For example, viscosity of the extractant can influence the mass
transfer properties of the system, for example, the efficiency with
which the product alcohol may be extracted from the aqueous phase
to the extractant phase (i.e., organic phase). The density of the
extractant can affect phase separation. In some embodiments, the
extractant may be liquid at the temperatures of the fermentation
process. In some embodiments, selectivity refers to the relative
amounts of product alcohol to water taken up by the extractant. The
boiling point can affect the cost and method of product alcohol
recovery. For example, in the case where butanol is recovered from
the extractant phase by distillation, the boiling point of the
extractant should be sufficiently low as to enable separation of
butanol while minimizing any thermal degradation or side reactions
of the extractant, or the need for vacuum in the distillation
process.
[0203] The extractant can be biocompatible with the microorganism,
that is, nontoxic to the microorganism or toxic only to such an
extent that the microorganism is impaired to an acceptable level.
In some embodiments, biocompatible refers to the measure of the
ability of a microorganism to utilize fermentable carbon sources in
the presence of an extractant. The extent of biocompatibility of an
extractant may be determined, for example, by the glucose
utilization rate of the microorganism in the presence of the
extractant and product alcohol. In some embodiments, a
non-biocompatible extractant refers to an extractant that
interferes with the ability of a microorganism to utilize
fermentable carbon sources. For example, a non-biocompatible
extractant does not permit the microorganism to utilize glucose at
a rate greater than about 25%, greater than about 30%, greater than
about 35%, greater than about 40%, greater than about 45%, or
greater than about 50% of the rate when the extractant is not
present.
[0204] One skilled in the art may select an extractant to maximize
the desired properties and/or characteristics as described above
and to optimize recovery of a product alcohol. One of skill in the
art can also appreciate that it may be advantageous to use a
mixture of extractants. For example, extractant mixtures may be
used to increase the partition coefficient for the product alcohol.
Additionally, extractant mixtures may be used to adjust and
optimize physical characteristics of the extractant, such as the
density, boiling point, and viscosity. For example, the appropriate
combination may provide an extractant which has a sufficient
partition coefficient for the product alcohol, sufficient
biocompatibility to enable its economical use for removing product
alcohol from a fermentative broth, and sufficient selectivity to
enable the selective removal of the product alcohol over, for
example, water.
[0205] Suitable organic extractants for use in the methods
disclosed herein are selected from the group consisting of C.sub.12
to C.sub.22 fatty alcohols, C.sub.12 to C.sub.22 ethers, C.sub.12
to C.sub.22 fatty acids, esters of C.sub.12 to C.sub.22 fatty
acids, C.sub.12 to C.sub.22 fatty aldehydes, C.sub.12 to C.sub.22
fatty amides, C.sub.10 to C.sub.22 alkanes, and mixtures thereof.
In some embodiments, the solvent is trimethylnonanol, methyl
laurate, di-n-octyl ether, dodecane, n-undecane, ethyl decanoate,
ethyl laurate, or mixtures thereof. In some embodiments, the
solvent is not oleyl alcohol, 1-dodecanol, behenyl alcohol, cetyl
alcohol, myristyl alcohol, or stearyl alcohol. As used herein, the
term "mixtures thereof" encompasses both mixtures within and
mixtures between these group members, including structural
homologs, for example mixtures within C.sub.12 to C.sub.22 fatty
alcohols, C.sub.12 to C.sub.22 ethers, C.sub.12 to C.sub.22 fatty
acids, esters of C.sub.12 to C.sub.22 fatty acids, C.sub.12 to
C.sub.22 fatty aldehydes, C.sub.12 to C.sub.22 fatty amides, and
C.sub.10 to C.sub.22 alkanes.
[0206] In some embodiments, the solvent has a boiling point less
than about 320.degree. C., less than about 310.degree. C., less
than about 300.degree. C., less than about 290.degree. C., less
than about 280.degree. C., less than about 270.degree. C., less
than about 260.degree. C., less than about 250.degree. C., less
than about 240.degree. C., less than about 230.degree. C., less
than about 220.degree. C., less than about 210.degree. C., or less
than about 200.degree. C.
[0207] In some embodiments, the solvent is biocompatible with the
microorganism such that at least about 70%, at least about 75%, at
least about 80%, at least about 85%, at least about 90%, or at
least about 95% of the microorganism is viable after exposure to
the organic extractant composition. In some embodiments, at least
90%, at least 80%, at least 70%, or at least 60% of the
butanol-producing microorganisms are viable after exposure of the
fermentation medium to the organic extractant for 25 hours. In
other embodiments, at least 90%, at least 80%, at least 70%, or at
least 60% of the butanol-producing microorganisms are viable after
exposure of the fermentation medium to the organic extractant for
30 hours.
[0208] Suitable organic extractant compositions can also include a
mixture of a first solvent and a second solvent. A suitable first
solvent can include a solvent having one or more of the
characteristics described in the preceding paragraph. For example,
the first solvent can be trimethylnonanol, methyl laurate,
di-n-octyl ether, dodecane, n-undecane, ethyl decanoate, ethyl
laurate. In certain embodiments, suitable second solvents include
solvents having a higher butanol partition coefficient than the
first solvent. Optionally, the second solvent may have a higher
toxicity to a recombinant microorganism comprising a butanol
biosynthetic pathway than the first solvent. In some embodiments,
the second solvent has a butanol partition coefficient greater than
about 4, greater than about 4.5, greater than about 5, greater than
about 5.5, greater than about 6, greater than about 6.5, greater
than about 7, greater than about 7.5, or greater than about 8.
Examples of a suitable second solvent in the organic extractant
include n-hexanol, methyl isobutyl carbinol, 2-ethyl-1-hexanol,
2,6-dimethylheptan-4-ol, and mixtures thereof. Additional examples
of a suitable second solvent can include, but is not limited to, an
organic solvent such as oleic acid, lauric acid, myristic acid,
stearic acid, octanoic acid, decanoic acid, undecanoic acid, methyl
myristate, methyl oleate, 1-nonanol, 1-decanol, 2-undecanol,
1-nonanal, 1-undecanol, undecanal, lauric aldehyde,
2-methylundecanal, oleamide, linoleamide, palmitamide,
stearylamide, 2-hexyl-1-decanol, 2-octyl-1-dodecanol, and mixtures
thereof.
[0209] In some embodiments, the extractant may be a mixture of
biocompatible and non-biocompatible extractants. Examples of
mixtures of biocompatible and non-biocompatible extractants
include, but are not limited to, trimethylnonanol and n-hexanol,
trimethylnonanol and methyl isobutyl carbinol, trimethylnonanol and
2-ethyl-1-hexanol, trimethylnonanol and 2,6-dimethylheptan-4-ol,
methyl laurate and n-hexanol, methyl laurate and methyl isobutyl
carbinol, methyl laurate and 2-ethyl-1-hexanol, methyl laurate and
2,6-dimethylheptan-4-ol, di-n-octyl ether and n-hexanol, di-n-octyl
ether and methyl isobutyl carbinol, di-n-octyl ether and
2-ethyl-1-hexanol, di-n-octyl ether and 2,6-dimethylheptan-4-ol,
dodecane and n-hexanol, dodecane and methyl isobutyl carbinol,
dodecane and 2-ethyl-1-hexanol, dodecane 1 and
2,6-dimethylheptan-4-ol, n-undecane and n-hexanol, n-undecane and
methyl isobutyl carbinol, n-undecane and 2-ethyl-1-hexanol,
n-undecane and 2,6-dimethylheptan-4-ol, ethyl decanoate and
n-hexanol, ethyl decanoate and methyl isobutyl carbinol, ethyl
decanoate and 2-ethyl-1-hexanol, ethyl decanoate and
2,6-dimethylheptan-4-ol, ethyl laurate and n-hexanol, ethyl laurate
and methyl isobutyl carbinol, ethyl laurate and 2-ethyl-1-hexanol,
and ethyl laurate and 2,6-dimethylheptan-4-ol. Additional examples
of biocompatible and non-biocompatible extractants are described in
U.S. Patent Application Publication No. 2009/0305370 and U.S.
Patent Application Publication No. 2011/0097773; the entire
contents of each herein incorporated by reference. In some
embodiments, biocompatible extractants may have high atmospheric
boiling points. For example, biocompatible extractants may have
atmospheric boiling points greater than the atmospheric boiling
point of water.
[0210] The relative amounts of the first and second solvents which
form the extractant can vary within a suitable range. In some
embodiments, the extractant composition can contain about 30
percent to about 90 percent of the first solvent, based on the
total volume of the first and second solvents. In some embodiments,
the extractant can contain about 40 percent to about 80 percent
first solvent. In some embodiments, the extractant can contain
about 45 percent to about 75 percent first solvent. In another
embodiment, the extractant can contain about 50 percent to about 70
percent first solvent. The optimal range reflects maximization of
the extractant characteristics, for example balancing a relatively
high partition coefficient for butanol with an acceptable level of
biocompatibility. For a two-phase extractive fermentation for the
production or recovery of butanol, the temperature, contacting
time, butanol concentration in the fermentation medium, relative
amounts of extractant and fermentation medium, specific solvent(s)
used, relative amounts of the first and second solvents (when more
than one solvent is used), presence of other organic solutes, and
the amount and type of microorganism are related; thus these
variables can be adjusted as necessary within appropriate limits to
optimize the extraction process as described herein.
[0211] These organic extractants are available commercially from
various sources, such as Sigma-Aldrich (St. Louis, Mo.), in various
grades, many of which can 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.
Growth for Production
[0212] Recombinant host cells disclosed herein are contacted with
suitable carbon substrates, typically in fermentation media.
Additional carbon substrates may include, but are not limited to,
monosaccharides such as fructose, oligosaccharides such as lactose,
maltose, galactose, or sucrose, polysaccharides such as starch or
cellulose or mixtures thereof and unpurified mixtures from
renewable feedstocks such as cheese whey permeate, cornsteep
liquor, sugar beet molasses, and barley malt. Other carbon
substrates can include ethanol, lactate, succinate, or
glycerol.
[0213] Additionally the carbon substrate may also be one-carbon
substrates such as carbon dioxide, or methanol for which metabolic
conversion into key biochemical intermediates has been
demonstrated. In addition to one and two carbon substrates,
methylotrophic organisms are also known to utilize a number of
other carbon containing compounds such as methylamine, glucosamine
and a variety of amino acids for metabolic activity. For example,
methylotrophic yeasts are known to utilize the carbon from
methylamine to form trehalose or glycerol (Bellion et al., Microb.
Growth C1 Compd., [Int. Symp.], 7.sup.th (1993), 415-32, Editors:
Murrell, J. Collin, Kelly, Don P.; Publisher: Intercept, Andover,
UK). Similarly, various species of Candida will metabolize alanine
or oleic acid (Sulter et al., Arch. Microbiol. 153:485-489 (1990)).
Hence it is contemplated that the source of carbon utilized in the
present invention may encompass a wide variety of carbon containing
substrates and will only be limited by the choice of organism.
[0214] Although it is contemplated that all of the above mentioned
carbon substrates and mixtures thereof are suitable in the present
invention, in some embodiments, the carbon substrates are glucose,
fructose, and sucrose, or mixtures of these with C5 sugars such as
xylose and/or arabinose for yeasts cells modified to use C5 sugars.
Sucrose may be derived from renewable sugar sources such as sugar
cane, sugar beets, cassava, sweet sorghum, and mixtures thereof.
Glucose and dextrose can be derived from renewable grain sources
through saccharification of starch based feedstocks including
grains such as corn, wheat, rye, barley, oats, and mixtures
thereof. In addition, fermentable sugars can be derived from
renewable cellulosic or lignocellulosic biomass through processes
of pretreatment and saccharification, as described, for example, in
U.S. Patent Application Publication No. 2007/0031918 A1, which is
herein incorporated by reference. Biomass, when used in reference
to carbon substrate, refers to any cellulosic or lignocellulosic
material and includes materials comprising cellulose, and
optionally further comprising hemicellulose, lignin, starch,
oligosaccharides and/or monosaccharides. Biomass can also comprise
additional components, such as protein and/or lipid. Biomass can be
derived from a single source, or biomass can comprise a mixture
derived from more than one source; for example, biomass may
comprise a mixture of corn cobs and corn stover, or a mixture of
grass and leaves. Biomass includes, but is not limited to,
bioenergy crops, agricultural residues, municipal solid waste,
industrial solid waste, sludge from paper manufacture, yard waste,
wood and forestry waste. Examples of biomass include, but are not
limited to, corn grain, corn cobs, crop residues such as corn
husks, corn stover grasses, wheat, wheat straw, barley, barley
straw, hay, rice straw, switchgrass, waste paper, sugar cane
bagasse, sorghum, soy, components obtained from milling of grains,
trees, branches, roots, leaves, wood chips, sawdust, shrubs and
bushes, vegetables, fruits, flowers, animal manure, and mixtures
thereof.
[0215] In addition to an appropriate carbon source, fermentation
media must contain suitable minerals, salts, cofactors, buffers and
other components, known to those skilled in the art, suitable for
the growth of the cultures and promotion of an enzymatic pathway
described herein.
Culture Conditions
[0216] Typically cells are grown at a temperature in the range of
about 20.degree. C. to about 40.degree. C. in an appropriate
medium. Suitable growth media in the present invention are common
commercially prepared media such as Luria Bertani (LB) broth,
Sabouraud Dextrose (SD) broth or Yeast Medium (YM) broth or broth
that includes yeast nitrogen base, ammonium sulfate, and dextrose
(as the carbon/energy source) or YPD Medium, a blend of peptone,
yeast extract, and dextrose in optimal proportions for growing most
Saccharomyces cerevisiae strains. Other defined or synthetic growth
media can also be used, and the appropriate medium for growth of
the particular microorganism will be known by one skilled in the
art of microbiology or fermentation science. The use of agents
known to modulate catabolite repression directly or indirectly,
e.g., cyclic adenosine 2',3'-monophosphate (cAMP), can also be
incorporated into the fermentation medium.
[0217] Suitable pH ranges for the fermentation are between pH 5.0
to pH 9.0, where pH 6.0 to pH 8.0 is preferred for the initial
condition. Suitable pH ranges for the fermentation of yeast are
typically between about pH 3.0 to about pH 9.0. In one embodiment,
about pH 5.0 to about pH 8.0 is used for the initial condition.
Suitable pH ranges for the fermentation of other microorganisms are
between about pH 3.0 to about pH 7.5. In one embodiment, about pH
4.5 to about pH 6.5 is used for the initial condition.
[0218] Fermentations can be performed under aerobic or anaerobic
conditions. In one embodiment, anaerobic or microaerobic conditions
are used for fermentation.
Industrial Batch and Continuous Fermentations
[0219] Butanol, or other products, can be produced using a batch
method of fermentation. A classical batch fermentation is a closed
system where the composition of the medium is set at the beginning
of the fermentation and not subject to artificial alterations
during the fermentation. A variation on the standard batch system
is the fed-batch system. Fed-batch fermentation processes are also
suitable in the present invention and comprise a typical batch
system with the exception that the substrate is added in increments
at the fermentation progresses. Fed-batch systems are useful when
catabolite repression is apt to inhibit the metabolism of the cells
and where it is desirable to have limited amounts of substrate in
the media. Batch and fed-batch fermentations are common and well
known in the art and examples can be found in Thomas D. Brock in
Biotechnology: A Textbook of Industrial Microbiology, Second
Edition (1989) Sinauer Associates, Inc., Sunderland, Mass., or
Deshpande, Mukund V., Appl. Biochem. Biotechnol., 36:227, (1992),
herein incorporated by reference.
[0220] Butanol, or other products, may also be produced using
continuous fermentation methods. Continuous fermentation is an open
system where a defined fermentation medium is added continuously to
a bioreactor and an equal amount of conditioned media is removed
simultaneously for processing. Continuous fermentation generally
maintains the cultures at a constant high density where cells are
primarily in log phase growth. Continuous fermentation allows for
the modulation of one factor or any number of factors that affect
cell growth or end product concentration. Methods of modulating
nutrients and growth factors for continuous fermentation processes
as well as techniques for maximizing the rate of product formation
are well known in the art of industrial microbiology and a variety
of methods are detailed by Brock, supra.
[0221] It is contemplated that the production of butanol, or other
products, can be practiced using batch, fed-batch or continuous
processes and that any known mode of fermentation would be
suitable. Additionally, it is contemplated that cells can be
immobilized on a substrate as whole cell catalysts and subjected to
fermentation conditions for butanol production.
Methods for Recovering Butanol Using Two-Phase Extractive
Fermentation
[0222] Bioproduced 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. The first
step in the process is contacting the fermentation medium with a
water immiscible organic extractant composition comprising a
solvent, as 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 composition or its solvent component(s) 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.
[0223] In certain embodiments where more than one solvent is used
for the extraction, the contacting may be performed with the
solvents of the extractant composition having been previously
combined. For example, the first and second solvents may be
combined in a vessel such as a mixing tank to form the extractant,
which is then added to a vessel containing the fermentation medium.
Alternatively, the contacting may be performed with the first and
second solvents becoming combined during the contacting. For
example, the first and second solvents may be added separately to a
vessel which contains the fermentation medium. In one embodiment,
contacting the fermentation medium with the organic extractant
composition further comprises contacting the fermentation medium
with the first solvent prior to contacting the fermentation medium
and the first solvent with the second solvent. In one embodiment,
the contacting with the second solvent occurs in the same vessel as
the contacting with the first solvent. In one embodiment, the
contacting with the second solvent occurs in a different vessel
from the contacting with the first solvent. For example, the first
solvent may be contacted with the fermentation medium in one
vessel, and the contents transferred to another vessel in which
contacting with the second solvent occurs.
[0224] The organic extractant composition can contact the
fermentation medium at the start of the fermentation forming a
biphasic fermentation medium. Alternatively, the organic extractant
composition can 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.
[0225] Further, the organic extractant composition can 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 can be monitored during the fermentation using
methods known in the art, such as gas chromatography or high
performance liquid chromatography.
[0226] Fermentation can 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
can 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, for
example, in detail in Example 6 of US Patent Application
Publication No. 2009/0305370. The extractant is added after the
switch to microaerobic or anaerobic conditions.
[0227] Through contacting the fermentation medium with the organic
extractant composition, 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 ratio of the
extractant to the fermentation medium is from about 1:20 to about
20:1 on a volume:volume basis, for example from about 1:15 to about
15:1, or from about 1:12 to about 12:1, or from about 1:10 to about
10:1, or from about 1:9 to about 9:1, or from about 1:8 to about
8:1.
[0228] 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, and membrane-assisted
phase splitting. 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, and pervaporation.
Specifically, distillation can be used to recover the butanol from
the butanol-containing organic phase.
[0229] Gas stripping can be used concurrently with the solvents of
the organic extractant composition 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.
[0230] 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, including, but not limited
to distillation, azeotropic distillation, liquid-liquid extraction,
adsorption, gas stripping, membrane evaporation, pervaporation, and
the like.
[0231] 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 composition 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
composition 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 composition 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 carry out 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.
[0232] 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 composition is added to the fermentor and the extractant
is not removed during the process. The organic extractant
composition may be formed in the fermentor by separate addition of
the first and the second solvents, or the solvents may be combined
to form the extractant composition prior to the addition of the
extractant composition to the fermentor. Although this mode is
simpler than the continuous or the entirely continuous modes
described above, it requires a larger volume of organic extractant
composition 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 extractant composition 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.
[0233] 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 extractant
composition used and its methods of addition in this mode is the
same as that used in the batchwise mode, described above.
[0234] 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
composition 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
composition 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, the first solvent, and/or the second solvent may be
recycled for use in the extraction process. Alternatively, fresh
extractant may be used. In this mode the extractant is not present
in the fermentor, so the toxicity of the extractant is much less of
a problem. If the cells are separated from the fermentation medium
before contacting with the extractant, the problem of extractant
toxicity is further reduced. Furthermore, using this external mode
there is less chance of forming an emulsion and evaporation of the
extractant is minimized, alleviating environmental concerns.
[0235] An improved 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 composition, as described
above, wherein the biphasic fermentation medium comprises from
about 3% to about 60% by volume of the organic extractant. The
microorganism can be grown in the biphasic fermentation medium for
a time sufficient to extract butanol into the extractant
composition to form a butanol-containing organic phase. In the case
where the fermentation medium further comprises ethanol, the
butanol-containing organic phase can 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.
[0236] Also provided is an improved method for the production of
butanol wherein a microorganism that has been genetically modified
to produce butanol via a biosynthetic pathway from at least one
carbon source, is grown in a fermentation medium wherein the
microorganism produces the butanol into the fermentation medium to
produce a butanol-containing fermentation medium. At least a
portion of the butanol-containing fermentation medium is contacted
with a water immiscible organic extractant composition, as defined
herein, to form a two-phase mixture comprising an aqueous phase and
a butanol-containing organic phase. In some embodiments, the
fermentation medium further comprises ethanol, and the
butanol-containing organic phase can 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. At
least a portion of the aqueous phase is returned to the
fermentation medium.
[0237] Isobutanol can be produced by extractive fermentation with
the use of a modified Escherichia coli strain in combination with
an oleyl alcohol as the organic extractant, as disclosed, for
example, in US Patent Application Publication No. 2009/0305370. The
method yields a higher effective titer for isobutanol (i.e., 37
g/L) compared to using conventional fermentation techniques (see
Example 6 of US Patent Application Publication No. 2009/0305370).
For example, 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. The higher butanol titer obtained
with the extractive fermentation method disclosed in US Patent
Application Publication No. 2009/0305370 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. It is reasonable to assume that the present
extractive fermentation method employing a water-immiscible organic
extractant composition, as defined herein, would be used in a
similar way and provide similar results.
[0238] Butanol produced by the method disclosed herein can have an
effective titer of greater than about 20 g per liter of the
fermentation medium, greater than about 22 g per liter of the
fermentation medium, greater than about 25 g per liter of the
fermentation medium, greater than about 30 g per liter of the
fermentation medium, greater than about 35 g per liter of the
fermentation medium, greater than about 37 g per liter of the
fermentation medium, greater than about 40 g per liter of the
fermentation medium, greater than about 45 g per liter of the
fermentation medium, greater than about 50 g per liter of the
fermentation medium. In some embodiments, the recovered butanol has
an effective titer from about 22 g per liter to about 50 g per
liter, about 22 g per liter to 40 g per liter, about 22 g per liter
to about 30 g per liter, about 25 g per liter to about 50 g per
liter, about 25 g per liter to 40 g per liter, about 25 g per liter
to about 30 g per liter, about 30 g per liter to about 50 g per
liter, about 40 g per liter to about 50 g per liter, about 22 g per
liter to about 60 g per liter, about 30 g per liter to about 60 g
per liter, about 40 g per liter to about 60 g per liter, about 22 g
per liter to about 80 g per liter, about 40 g per liter to about 80
g per liter, about 50 g per liter to about 80 g per liter, about 65
g per liter to about 80 g per liter.
[0239] The present methods are generally described below with
reference to a FIG. 1 through FIG. 7.
[0240] Referring now to FIG. 1, 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
recombinant microorganism (not shown) capable of converting the at
least one fermentable carbon source into butanol. A stream of a
first solvent 12 and a stream of an optional second solvent 14 are
introduced to a vessel 16, in which the solvents are combined to
form the extractant 18. A stream of the extractant 18 is introduced
into the fermentor 20, in which contacting of the fermentation
medium with 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.
[0241] Referring now to FIG. 2, 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
recombinant microorganism (not shown) capable of converting the at
least one fermentable carbon source into butanol. A stream of the
first solvent 12 and a stream of the optional second solvent 14 of
which the extractant is comprised are introduced separately to the
fermentor 20, in which contacting of the fermentation medium with
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.
[0242] 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 first fermentor 20, which contains at least one
recombinant microorganism (not shown) capable of converting the at
least one fermentable carbon source into butanol. A stream of the
first solvent 12 of which the extractant is comprised 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 solvent 14 of
which the extractant is comprised is introduced into the second
fermentor 24, in which contacting of the fermentation medium with
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.
[0243] Referring now to FIG. 4, 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 recombinant
microorganism (not shown) capable of converting the at least one
fermentable carbon source into butanol. A stream of the first
solvent 112 and a stream of the optional second solvent 114 are
introduced to a vessel 116, in which the solvents 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, in which contacting of the fermentation medium with 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.
[0244] Referring now to FIG. 5, 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 recombinant
microorganism (not shown) capable of converting the at least one
fermentable carbon source into butanol. A stream of the first
solvent 112 and a stream of the optional second solvent 114 of
which the extractant is comprised are introduced separately to a
vessel 124, in which the solvents are combined to form the
extractant. At least a portion, shown as stream 122, of the
fermentation medium in fermentor 120 is also introduced into vessel
124, in which contacting of the fermentation medium with 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.
[0245] 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 recombinant
microorganism (not shown) capable of converting the at least one
fermentable carbon source into butanol. A stream of the first
solvent 112 of which the extractant is comprised 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 solvent and the
contents of fermentor 120 is introduced into a second vessel 132. A
stream of the optional second solvent 114 of which the extractant
is comprised is introduced into the second vessel 132, in which
contacting of the fermentation medium with 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.
[0246] 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.
[0247] 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
recombinant microorganism in a continuous fashion one after another
for as long as the plant is operating. Referring to FIG. 7, 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.
[0248] 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. 7,
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 be 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.
[0249] 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.
[0250] 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.
ADVANTAGES OF THE PRESENT METHODS
[0251] The present extractive fermentation methods provide butanol
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 CO.sub.2 and little or no
SO.sub.x or NO.sub.x when burned in the standard internal
combustion engine. Additionally, butanol is less corrosive than
ethanol, the most preferred fuel additive to date.
[0252] In addition to its utility as a biofuel or fuel additive,
the butanol produced according to 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. Furthermore, the present methods
produce butanol from plant derived carbon sources, avoiding the
negative environmental impact associated with standard
petrochemical processes for butanol production.
[0253] One of the advantages of the present methods is the higher
butanol partition coefficient compared to solvents used in the art.
Combinations of solvents obtained by the appropriate combination of
a first and a second solvent as described herein, can provide
extractants having a higher partition coefficient. Extractants
having higher partition coefficients can provide more effective
extraction of butanol from the fermentation medium. Another
advantage of the present method is the ability to use a solvent
which has a desirably higher partition coefficient but undesirably
lower biocompatibility, and to mitigate the lower biocompatibility
by the combination with a solvent having higher biocompatibility.
As a result, a more effective extractant is obtained, an extractant
which can be used in the presence of the microorganism with
continued viability of the microorganism.
[0254] Further advantages of the present methods include the
improved process operability characteristics of the extractant
relative to those characteristics of oleyl alcohol. The extractant
of the present methods has lower viscosity, lower density, and
lower boiling point than oleyl alcohol, which provides improvements
to the extraction process using such an extractant. Improved
viscosity and density of the extractant can lead to improved
efficiency of extraction and ease of phase separation. A lower
boiling point can reduce the energy required for distillative
separations, reduce the energy for removing the extractant from
DDGS (dried distiller's grains with solubles), and can lower the
bottoms temperatures in a distillation column separating the
butanol from the extractant. Together these characteristics can
provide an economic advantage for extractive fermentation using an
extractant as disclosed herein.
Confirmation of Isobutanol Production
[0255] The presence and/or concentration of isobutanol in the
culture medium can be determined by a number of methods known in
the art (see, for example, U.S. Pat. No. 7,851,188, incorporated by
reference). For example, a specific high performance liquid
chromatography (HPLC) method utilizes a Shodex SH-1011 column with
a Shodex SHG guard column, both may be purchased from Waters
Corporation (Milford, Mass.), with refractive index (RI) detection.
Chromatographic separation is achieved using 0.01 M H.sub.2SO.sub.4
as the mobile phase with a flow rate of 0.5 mL/min and a column
temperature of 50.degree. C. Isobutanol has a retention time of
46.6 min under the conditions used.
[0256] Alternatively, gas chromatography (GC) methods are
available. For example, a specific GC method utilizes an HP-INNOWax
column (30 m.times.0.53 mm id, 1 .mu.m film thickness, Agilent
Technologies, Wilmington, Del.), with a flame ionization detector
(FID). The carrier gas is helium at a flow rate of 4.5 mL/min,
measured at 150.degree. C. with constant head pressure; injector
split is 1:25 at 200.degree. C.; oven temperature is 45.degree. C.
for 1 min, 45 to 220.degree. C. at 10.degree. C./min, and
220.degree. C. for 5 min; and FID detection is employed at
240.degree. C. with 26 mL/min helium makeup gas. The retention time
of isobutanol is 4.5 min.
[0257] While various embodiments of the present invention have been
described herein, it should be understood that they have been
presented by way of example only, and not limitation. It will be
apparent to persons skilled in the relevant art that various
changes in form and detail can be made therein without departing
from the spirit and scope of the invention. Thus, the breadth and
scope of the present invention should not be limited by any of the
above-described exemplary embodiments, but should be defined only
in accordance with the claims and their equivalents.
[0258] All publications, patents, and patent applications mentioned
in this specification are indicative of the level of those skilled
in the art to which this invention pertains, and are herein
incorporated by reference to the same extent as if each individual
publication, patent, or patent application was specifically and
individually indicated to be incorporated by reference.
EXAMPLES
[0259] The present invention is further defined in the following
Examples. It should be understood that these Examples, while
indicating 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.
Materials
[0260] The following materials were used in the examples. All
commercial reagents were used as received.
[0261] All solvents 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: 1-nonanol, 98%; 1-decanol,
98%; 1-undecanol, 98%; 2-undecanol, 98%; dodecanol, 98%; 1-nonanal,
98%. Isobutanol (purity 99.5%) was obtained from Sigma-Aldrich and
was used without further purification.
Growth Protocols
Primary and Secondary Screen Growth Protocol
[0262] Minimal Media:
[0263] 6.7 g Yeast nitrogen base without amino acids; 1.4 g Yeast
synthetic drop-out medium without histidine, leucine, tryptophan,
uracil; 2 g/L D-(+)-Glucose; 10 ml 1% Leucine stock; 2 mL 1%
Tryptophan stock; 2 mL Ethyl Alcohol; make up to 1 L with DI water
and filter sterilize.
[0264] Rich Media:
[0265] The following was added to 3.0 L warm deionized water: 6.7
g/L Yeast nitrogen base w/o amino acids; 30 g/L dextrose (glucose);
6.3 mL/L ethanol, anhydrous (200 proof); 4.0 g/L peptone, Bacto;
2.0 g/L yeast extract, Difco; 38.4 g/L MES Buffer (0.2 M); 3.7 g/L
ForMedium (2.times.); or 2.8 g/L yeast dropout mix (Sigma Y2001);
20 mL/L of 10 g/L leucine solution; 4.0 mL/L of 10 g/L tryptophan
solution. 2 L warm DI water gently heated; components mixed until
dissolved; cooled to room temperature; titrated to pH 5.8 using 2M
NaOH; brought up to 3.0 L; final pH measured; and solution filter
sterilized in sterile filter apparatus.
[0266] To grow the cells, one working stock of cells was thawed on
ice for 30 minutes. 50 mL of minimal media were warmed in a 250 mL
autoclaved flask at 30.degree. C. 400 uL of thawed working stock
was inoculated into 50 mL minimal media (at 30.degree. C.). The
resulting culture was incubated for 72 hr at 30.degree. C., 250
rpm. The extraction flask was prepared by adding 300 mL of rich
media and 100 mL of sterile filtered Oleyl alcohol to 2 L
polycarbonate flask and warming to 30.degree. C. 300 mL of rich
media were measured and autoclaved in a graduated cylinder. 100 mL
of sterile filtered oleyl alcohol was measured out with serological
pipette. 30 mL of the 24 hr minimal media culture was inoculated in
an extraction flask. The culture was incubated for 48 hrs at
30.degree. C., 200 rpm.
[0267] To prepare the cells for a pre-run, from 48 hr 2 L
extraction flask, 40 mL were aliquoted into 10.times.50 mL conical
tubes. The conical tubes were centrifuged for 10 minutes at
4.degree. C. at 19000 RPMs. The supernatant was decanted by pouring
and the pellet was resuspended in 10 mL of rich media. The
resuspension was distributed evenly into 4 conical tubes as
follows: 3 conicals @10 mL each into 1 conical (total volume 30
mL).times.2; 2 conicals @10 mL each into 1 conical (total volume 20
mL).times.2. The conical tubes were centrifuged for 10 min at
4.degree. C. at 19000 RPMs. The supernatant was decanted by pouring
and the pellet was resuspended in each of the 4 conicals to a final
volume of 25 mL using rich media. The conical tubes were
centrifuged, decanted, and resuspended again to a final volume of
25 mL using rich media. A sterilized 500 mL Erlenmeyer flask was
prepared by adding 250 mL of rich media. The resuspended cells from
the 4 conicals were added into the flask so that the final volume
of the cells in flask is 350 mL.
[0268] To run the time course, 25 mL from the 350 mL of
re-suspended cells was aliquoted into 12.times.125 mL Erlenmeyer
flasks. To each 125 mL erlenmeyer flask with 25 mL of cells, 25 mL
of solvent was added. The flasks were incubated at 30.degree. C.,
shaking at 250 RPMs for 24 hrs. Time points were taken at 4 hr and
24 hr.
[0269] For each time point, the cells were prepared as follows: the
flasks were allowed to sit at rest for 1 minute. From each flask 3
fractions of 1 mL samples were aliquoted into 1.5 mL microfuge
tubes, as follows: (a) 1 mL organic layer (analytical: iBuOH,
EtOH); (b) 1 mL aqueous layer (analytical: glucose, iBuOH, EtOH);
(c) 1 mL aqueous layer (Cellometer viability, OD.sub.600).
[0270] For fractions a) and b) analytical samples were prepared by
sterile filtering through a 0.22 um nylon filter Costar Spin-x
microfuge tube filter (Spin-x); the tubes were centrifuged for 5
min at 13,000 RPM, 4.degree. C. to separate aqueous and organic
layers; 500 uL of the organic layer was carefully aspirated from
fraction a) into a Spin-x; 500 uL of aqueous layer was carefully
aspirated from fraction b) into a Spin-x; the sample was spun at
13,000 RPM for 5 min, 4.degree. C.; and the resulting filtrate was
considered organism free and submitted for analysis for glucose,
iBuOH, EtOH.
[0271] For fraction c), the cells were washed in PBS pH 7.4 buffer
before assessing viability and OD600; the tubes were centrifuged
for 5 min at 13,000 RPM, 4.degree. C. to pellet cells' the
supernatant was decanted immediately and resuspend in 1 mL of PBS
by vortexing; the tubes were centrifuged again for 5 min at 13,000
RPM, 4.degree. C.; the supernatant was decanted immediately and
resuspend in 1 mL of PBS pH 7.4 buffer; the tubes were centrifuged
again for 5 min at 13,000 RPM, 4.degree. C.; the supernatant was
decanted immediately and resuspend in 1 mL of PBS pH 7.4 buffer.
The re-suspended samples were kept on ice, at 4.degree. C. until
ready to read on Cellometer. After Cellometer reads, dilutions were
made for OD.sub.600, blank with PBS.
[0272] To assess cell viability on Cellometer, each sample was
diluted Nexcelom yeast dilution buffer 5.times. (10:40 uL:sample:
buffer). 2.times. dilution to 20.times. dilution can be appropriate
depending on cell concentration. The resulting mixture was allowed
to incubate for 1 minute. The sample was diluted into AOPI
live-dead stain dye 4.times. (10:30 uL::sample: dye). The resulting
mixture was allowed to incubate at RT for 1 minute. 20 uL of the
resulting mixture was loaded into the disposable Nexcelom slide.
Using this assay, cells were counted and recorded (F1 (Fluorescein)
exposure: 1200 ms; F2 (Propidium Iodide) exposure: 4000 ms).
Tertiary Screen Growth Protocol
[0273] Minimal media: 6.7 g Yeast nitrogen base without amino
acids; 1.4 g yeast synthetic drop-out medium without histidine,
leucine, tryptophan, uracil; 2 g/L D-(+)-glucose; 10 ml 1% leucine
stock; 2 mL 1% tryptophan stock; 2 mL ethyl alcohol; made up to 1 L
with DI water and filter sterilized.
[0274] Rich media: The following was added to 3.0 L warm deionized
water: 6.7 g/L yeast nitrogen base w/o amino acids; 30 g/L dextrose
(glucose); 6.3 mL/L ethanol, anhydrous (200 proof); 4.0 g/L
peptone, Bacto; 2.0 g/L yeast extract, Difco; 38.4 g/L MES Buffer
(0.2 M); 3.7 g/L ForMedium (2.times.); or 2.8 g/L yeast dropout mix
(Sigma Y2001); 20 mL/L of 10 g/L leucine solution; 4.0 mL/L of 10
g/L tryptophan solution. 2 L warm DI water gently heated;
components mixed until dissolved; cooled to room temperature;
titrated to pH 5.8 using 2M NaOH; brought up to 3.0 L; final pH
measured; and solution filter sterilized in sterile filter
apparatus.
[0275] To grow the cells, 1 master stock vial from -80 C was thawed
on ice (or at 4.degree. C.) for 30 minutes. To 2.times.250 mL
sterile Erlenmeyer flasks, 50 mL minimal media was added. The
resulting media was warmed in flasks to 30.degree. C. 400 uL of
master stock was added into each 250 mL flask and incubated at
30.degree. C., 250 RPM, for 3-5 days.
[0276] To prepare the solvent and cells for pre-run, 12.5 mL of
each solvent (2 control+max 6 candidates) was measured into 100 mL
glass graduated cylinders in duplicate. 70 mL of minimal media
culture was inoculated into 700 mL of rich media at room
temperature. 37.5 mL of the inoculated rich media cell broth was
aliquoted into each 250 mL flask. 1 mL of the left over inoculated
rich media cell broth was aliquoted and stored at 4.degree. C.: the
O.D., cell concentration, and cell viability (Cellometer) were
recorded after starting the run. The rich media was not allowed to
sit at room temperature without solvent for longer than 30
minutes.
[0277] To run the time course, 12.5 mL of pre-measured solvent was
combined with 37.5 mL of inoculated rich media for each solvent in
duplicate. Oleyl Alcohol and 2-ethyl-1-hexanol were used as control
solvents. The solvent and inoculated rich media were incubated at
30.degree. C., 200 RPM for a 72 hr time course, taking time points
every 24 hr.
[0278] For each time point, the cells were prepared as follows: the
flasks were allowed to sit at rest for 1 minute. From each flask 3
fractions of 1 mL samples were aliquoted into 1.5 mL microfuge
tubes as follows: (a) 1 mL organic layer (analytical: iBuOH, EtOH);
(b) 1 mL aqueous layer (analytical: glucose, iBuOH, EtOH); and (c)
1 mL aqueous layer (Cellometer viability, OD.sub.600).
[0279] For fractions a) and b) analytical samples were prepared by
sterile filtering through a 0.22 um nylon filter Costar Spin-x
microfuge tube filter (Spin-x). The tubes were centrifuged 5 min at
13,000 RPM, 4.degree. C. to separate aqueous and organic layers.
500 uL of the organic layer was carefully aspirated from fraction
(a) into a Spin-x; 500 uL of the aqueous layer was carefully
aspirated from fraction (b) into a Spin-x; the samples were spun in
filled Spin-x tubes at 13,000 RPM for 5 min, 4.degree. C.; and the
resulting filtrate was considered "stopped" and organism free.
These samples were submitted for analysis of glucose, iBuOH, and
EtOH.
[0280] For fraction c), the cells were washed in PBS pH 7.4 buffer
before assessing viability and OD600. the tubes were centrifuged
for 5 min at 13,000 RPM, 4.degree. C. to pellet cells' the
supernatant was decanted immediately and resuspend in 1 mL of PBS
by vortexing; the tubes were centrifuged again for 5 min at 13,000
RPM, 4.degree. C.; the supernatant was decanted immediately and
resuspend in 1 mL of PBS pH 7.4 buffer; the tubes were centrifuged
again for 5 min at 13,000 RPM, 4.degree. C.; the supernatant was
decanted immediately and resuspend in 1 mL of PBS pH 7.4 buffer.
The re-suspended samples were kept on ice, at 4.degree. C. until
ready to read on Cellometer. After Cellometer reads, dilutions were
made for OD.sub.600, blank with PBS.
[0281] To assess cell viability on Cellometer, each sample was
diluted Nexcelom yeast dilution buffer 5.times. (10:40 uL::sample:
buffer). 2.times. dilution to 20.times. dilution can be appropriate
depending on cell concentration. The resulting mixture was allowed
to incubate for 1 minute. The sample was diluted into AOPI
live-dead stain dye 4.times. (10:30 uL::sample: dye). The resulting
mixture was allowed to incubate at RT for 1 minute. 20 uL of the
resulting mixture was loaded into the disposable Nexcelom slide.
Using this assay, cells were counted and recorded (F1 (Fluorescein)
exposure: 1200 ms; F2 (Propidium Iodide) exposure: 4000 ms).
Example 1
Identification of Biocompatible Solvents Capable of Isobutanol
Extraction Using a Three Stage Screening Process
[0282] Solvent extraction methods were investigated to support the
in situ product recovery (ISPR) process for isobutanol (iBuOH)
production and assist in reducing operating costs. Economical,
technically efficient, biocompatible solvents were sought. To
support this activity, a list of solvents of varied chemical
character was compiled to identify a biocompatible solvent that
could not be predicted a priori from published physical
characteristics. The solvents were screened for biocompatibility
and extractability. The following constraints were considered in
identifying the solvents to test experimentally: (a) the solvent is
minimally soluble/insoluble in water to improve recovery of
isobutanol; (b) the boiling point of the solvent is different from
isobutanol (74.degree. C.) for ease in distillation; (c) the
boiling point of the solvent is less than 250.degree. C. to
facilitate removal of the solvent from dried distillers grains
(DDGS); (d) the melting point of the solvent is less than
10.degree. C. to avoid handling problems; (e) the density of the
solvent is different from water to aid in phase separation; (f) the
Hildebrand solubility parameter is similar to isobutanol (22.7
MPa.sup.0.5); and (g) the solvent is commercially available. The
solvents that were tested are shown in Table 1.
TABLE-US-00001 TABLE 1 List of solvents experimentally screened.
Solvent name CAS no. Boiling point (.degree. C.) oleyl alcohol
143-28-2 333 n-decane 124-18-5 174 methyl laurate 111-82-0 260
di-n-hexyl ether 112-58-3 223 decalin 91-17-8 187 2-ethyl hexyl
acetate 103-09-3 199 2-ethylhexylglycol ether 1559-35-9 228
2-ethyl-1-hexanol 104-76-7 184 2,6-dimethylheptan-4-ol 108-82-7 178
ethyl n-butyrate 105-54-4 120 n-hexyl acetate 142-92-7 168
1,2-dibutoxyethane 112-48-1 203 n-butyl n-butyrate 109-21-7 166
2.6-dimethylheptan-4-one 108-83-8 169 ETBE 637-92-3 70 di-n-butyl
ether 142-96-1 142 ethylene glycol monohexyl ether 112-25-4 206
MTBE 1634-04-4 55 propylene glycol methyl ether acetate 108-65-6
146 isooctane 540-84-1 99 mesitylene 108-67-8 165 acetophenone
98-86-2 202 n-hexanol 111-27-3 157 phenyl acetate 122-79-2 196
1-octanol 111-87-5 194 8-methylquinoline 611-32-5 247
p-tolualdehyde 104-87-0 207 2-octanol 123-96-6 180 isophorone
78-59-1 215 methyl isobutyl carbinol 108-11-2 132 o-xylene 95-47-6
144 trimethylnonanol 123-17-1 225 n-butyl propionate 590-01-2 145
n-propyl propionate 106-36-5 122 n-butyl acetate 123-86-4 126
n-pentyl propionate 624-54-4 165 primary amyl acetate mixed isomers
628-63-7 149 isobutyl acetate 110-19-0 118 dodecanal 112-54-9 241
UCAR filmer IBT 25265-77-4 255 n-butyl valerate 591-68-4 187
di-n-octyl ether 629-82-3 287 isobutyl heptyl ketone 123-18-2 217
dodecane 112-40-3 218 n-undecane 1120-21-4 196 dodecanol 112-53-8
262 ethyl octanoate 106-32-1 208 ethyl decanoate 110-38-3 245 ethyl
laurate 106-33-2 269 methyl decanoate 110-42-9 224 methyl hexanoate
106-70-7 151 methyl heptanoate 106-73-0 172
Screening Strategy
[0283] Based on growth protocols described above, a strategy was
devised with a multi-stage screening process to optimize
throughput, resource usage, and information content. From the
protocols it was determined that a two-stage media process was
required for the isobutanologen. The first stage was a minimal
media stage deficient in uracil and leucine and low in glucose
followed by the second stage which was a rich media stage high in
glucose content that was conducive to isobutanol production in the
presence of a biocompatible solvent (e.g., oleyl alcohol).
[0284] For consistency, cells were grown in bulk by shake flask
using this two stage process (minimal media followed by rich media
and extractant (e.g., oleyl alcohol)) for use in the primary and
secondary screening stages. Cells were pelleted by centrifugation,
washed with phosphate buffered solution (PBS), and then dispensed
in the appropriate vessels (96 well plate for the primary screen,
50 ml total volume for the secondary screen) before exposure to
candidate solvents. This treatment was performed to ensure the
cells were in a representative metabolic stage before solvent
exposure. From the primary screen (discussed in more detail below),
viability data was collected by a fluorescence assay for all
solvents screened. FIG. 8 illustrates the work flow for the
automated primary screen/assay.
[0285] Solvents that passed the primary screen were analyzed by a
secondary screen (discussed in more detail below). A work flow
diagram illustrating the secondary screen is shown in FIG. 9. From
the secondary assay on viable hits identified from the primary
screen, two time points were taken from which viability, glucose,
ethanol, and isobutanol concentrations were analyzed.
[0286] For the tertiary screen (discussed in more detail below),
conditions were modeled that were more representative of a process
scenario; i.e., elimination of extractant treatment (e.g., oleyl
alcohol) and analysis of samples taken over a time course of at
least 48 hours. A work flow diagram illustrating the tertiary
screen is shown in FIG. 10. Cells were grown in bulk by shake flask
using only minimal media, and then transferred directly to rich
media pre-mixed with the reduced number of candidate solvents. The
candidate solvents were studied with greater replication (n=3, 50
ml total volume) by reducing the number of candidates that advanced
to the tertiary screening level. Key differences between the
primary, secondary, and tertiary screens are illustrated in Table
2.
TABLE-US-00002 TABLE 2 Summary of key differences at each screening
level of the multi-stage process. Total Exposure Volume Ratio time
Temp Repli- Data (mL) (aq:org) (hr) (.degree. C.) cates Collected
Primary 0.15 1:1 1 20 4 Viability Secondary 50 1:1 4 or 5; 30 1
Viability, 25 or 30 isobutanol, glucose, ethanol Tertiary 50 3:1
0-48.sup.+ 30 3 Viability, time isobutanol, course glucose,
ethanol
Primary Screen
[0287] An automated high throughput assay based on a 96 well plate
system was employed for the initial screen to identify solvents
that were biocompatible with the isobutanologen. Briefly, the
isobutanologen was pre-grown in shake flasks of minimal selection
media (low glucose) that were transferred to rich media (high
glucose) in the presence of oleyl alcohol. The cells were then
plated in a 96 well plate in both minimal selection media (low
glucose) and rich media (high glucose) in the presence of the
solvent. The cells were then washed to remove the media and solvent
and incubated for 1 hour at room temperature with shaking (1000
rpm). After three washes and a two-step dilution, these cells were
then treated with fluorescent dye (propidium iodide (PI)) to stain
for dead cells. The fluorescent signals were then compared to that
from cells treated with oleyl alcohol, which served as the live
cell control, and that from cells treated with at a 1:1 ratio of
cell broth to 100% ethanol, which served as a dead cell control, to
determine biocompatibility. FIG. 8 shows a schematic of the work
flow diagram of the automated primary screening assay. Twenty four
solvents were identified to be carried forward from primary
screening based on viability data obtained (Table 3).
TABLE-US-00003 TABLE 3 Solvents identified for secondary screening
based on high viability in 96 well plate primary screen. Proceed to
Solvent name Secondary Screen K.sub.d % Dead oleyl alcohol Live
Control 2.23 -2% n-decane YES 0.11 3% methyl laurate YES 1.12 -1%
di-n-hexyl ether YES 0.78 7% decalin YES 0.13 7% 2-ethyl hexyl
acetate YES 1.88 3% 2-ethylhexylglycol ether NO 4.95 79%
2-ethyl-1-hexanol Dead Control 5.51 73% 2,6-dimethylheptan-4-ol YES
4.25 11% ethyl n-butyrate NO 3.57 42% n-hexyl acetate YES 2.23 14%
1,2-dibutoxyethane YES 2.27 5% n-butyl n-butyrate YES 2.17 13%
2.6-dimethylheptan-4-one YES 2.12 5% ETBE YES 2.47 6% di-n-butyl
ether YES 1.26 7% ethylene glycol monohexyl ether NO 5.66 115% MTBE
YES 4.18 19% propylene glycol methyl ether acetate NO 3.15 79%
isooctane YES 0.18 8% mesitylene YES 0.47 11% acetophenone NO 2.95
73% n-hexanol NO 7.41 71% phenyl acetate NO 1.95 51% 1-octanol NO
5.19 90% 8-methylquinoline YES 5.03 2% p-tolualdehyde NO 2.41 41%
2-octanol NO 5.98 33% isophorone NO 5.68 37% methyl isobutyl
carbinol NO 7.78 71% o-xylene NO 0.85 55% trimethylnonanol YES 3.57
3% n-butyl propionate NO 3.02 45% n-propyl propionate NO 3.4 105%
n-butyl acetate YES 3.48 0% n-pentyl propionate NO 2.94 63% primary
amyl acetate mixed isomers NO 3.67 104% isobutyl acetate NO 3.33
71% dodecanal YES 5.38 2% UCAR filmer IBT YES 4.16 39% n-butyl
valerate YES 2.04 8% di-n-octyl ether YES 0.55 2% isobutyl heptyl
ketone YES 1.49 7% dodecane YES 0.18 2% n-undecane YES 0.20 2%
*UCAR filmer was carried directly to tertiary flasks for testing
after screening was completed because it shared structural
characteristics in common with confirmed solvents in which
isobutanol production was observed.
[0288] Favorable Kd values were greater than 1. Unfavorable Kd
values were less than 1. Kd values were defined as a concentration
of iBuOH in the organic phase/concentration of iBuOH in aqueous
phase. The % of dead cells was assessed by propidium iodide signal.
The solvent 2-ethyl-1-hexanol was identified as a kill control.
Secondary Screen
[0289] The secondary screen was designed to verify the solvents
identified in the primary screen under more process relevant
conditions. Cells were grown using seed flask protocols in rich
media and oleyl alcohol. The cells were then washed and split into
aliquots to be treated with the solvents identified in the primary
assay. The secondary screen was designed to ensure all the cells
began a standard treatment with solvent candidates in optimal
conditions. In a 125 mL flask, 25 mL of washed cell broth (oleyl
alcohol removed) was exposed to 25 mL of solvent at 30.degree. C.
for 4 h and 24 h at 20 rpm. The cell viability was checked by
fluorescent PI assay using a Cellometer (Nexcelom Vision, Lawrence,
Mass.) to count both live and dead cells. The primary screen had a
shorter exposure time (1 hour) at a lower temperature
(.about.22.degree. C., room temperature) as compared to the
secondary screen (4 and 24 hours at 30.degree. C.). The schematic
for the secondary screen is shown in FIG. 9.
[0290] From this screen, fifteen solvents demonstrated strong
viability at 4 or 5 hours/30.degree. C. solvent incubation (Table
4). Of the fifteen solvents demonstrating strong viability, only
five solvents demonstrated strong viability, isobutanol production
and complete glucose consumption at 25 or 30 hours/30.degree. C.
(Table 5).
TABLE-US-00004 TABLE 4 Secondary screen results for viability in
solvents identified in primary screen. 4 Hours 25 Hours % Via-
Glucose iBuOH % Via- Glucose iBuOH Solvent bility (g/L) (g/L)
bility (g/L) (g/L) oleyl alcohol 100% 5.7 1.8 100% 0.0 2.6 n-decane
107% 5.8 1.6 54% 0.0 2.3 methyl laurate 110% 3.9 2.1 94% 0.0 1.6
di-n-hexyl 106% 8.1 1.1 62% 0.9 1.9 ether decalin 76% 11.2 0.4 36%
10.3 0.5 2-ethyl hexyl 96% 10.5 0.6 42% 7.1 1.2 acetate 2-ethyl-1-
14% 13.1 0.0 6% 13.7 0.0 hexanol 2,6-dimeth- 42% 12.1 0.0 8% 11.8
0.0 ylheptan-4-ol n-hexyl 18% 12.8 0.0 3% 13.0 0.0 acetate 1,2- 63%
11.9 0.4 8% 10.7 0.4 dibutoxyethane n-butyl 48% 12.4 0.0 7% 11.7
0.0 n-butyrate 2.6-dimeth- 78% 10.9 0.6 26% 7.9 0.9 ylheptan-4-one
ETBE 16% 12.5 0.0 4% 12.8 0.0 di-n-butyl ether 90% 12.4 0.1 40%
11.3 0.5 MTBE 5% 12.7 0.0 1% 12.9 0.2 isooctane 90% 10.8 0.5 31%
8.5 0.8 mesitylene 64% 12.5 0.0 17% 12.1 0.1 % viability was
relative to oleyl alcohol and was based on fluorescence based cells
counts.
TABLE-US-00005 TABLE 5 Secondary screen results for viability for
additional solvents. 5 Hours 30 Hours % Via- Glucose iBuOH % Via-
Glucose iBuOH Solvent bility (g/L) (g/L) bility (g/L) (g/L) oleyl
alcohol 100% ND ND 100% 0.0 2.2 2-ethyl-1- 58% ND ND 47% 16.8 0.0
hexanol 8-meth- ND ND ND 47% 17.5 0.0 ylquinoline trimeth- 99% ND
ND 98% 0.0 2.1 ylnonanol n-butyl acetate 46% ND ND 35% 16.6 0.0
dodecanal 78% ND ND 62% 0.0 0.0 di-n-octyl ether 99% ND ND 99% 0.0
1.9 isobutyl heptyl 92% ND ND 56% 8.9 1.7 ketone dodecane 99% ND ND
99% 0.0 1.9 n-undecane 98% ND ND 100% 0.0 1.9 % viability was
relative to oleyl alcohol and was based on fluorescence based cell
counts.
Tertiary Screen
[0291] The tertiary screen was designed to examine the growth of
isobutanologen in the presence of the identified solvents from the
primary and secondary screens. The tertiary screen substitutes
solvent candidates for oleyl alcohol in a rich media seed protocol.
Briefly, a master stock was thawed and inoculated into minimal
selection media and grown for 3 days. From the master stock, 1.25
mL of 3-day-old minimal media cell broth was inoculated into a 250
mL flask with 37.5 mL rich media and 12.5 mL solvent candidate (1:3
solvent to broth). Cells were grown at 30.degree. C. and 200 RPM
over the course of 72 hours with time points every 24 hours.
Viability was checked by Cellometer, glucose consumption was
checked at each time point by high pressure liquid chromatography
(HPLC), and isobutanol (iBuOH) production was determined by gas
chromatography (GC). The schematic for the tertiary screen is shown
in FIG. 10.
[0292] All five solvents that demonstrated strong viability,
isobutanol production and complete glucose consumption at 25-30
hours in the secondary screen were confirmed in the tertiary screen
(Tables 3-5). Solvents that demonstrated weak glucose consumption
and isobutanol production at 25-30 hours in the secondary screen
were not confirmed in the tertiary screen. Methyl laurate,
trimethylnonanol, di-n-octyl ether, dodecane and n-undecane solvent
treated cells were observed to produce isobutanol (FIG. 11),
consume glucose (FIG. 12) and consume ethanol (FIG. 13) at levels
comparable to oleyl alcohol.
[0293] During the identification of solvents in the three step
screening process described above, it was determined that a
majority of the biocompatible solvents possessed more than eleven
carbons, boiling points between 196-300.degree. C., and log P
values >4. Biocompatible solvents were found amongst the esters,
alcohols, ethers and alkanes. Examination of the screened twelve
carbon (C12) compounds revealed that dodecanol, dodecanal, and UCAR
filmer IBT were toxic, while trimethylnonanol and dodecane were
biocompatible. None of the C10 or shorter compounds were
biocompatible.
Example 2
Identification of Biocompatible Methyl Laurate Analogs
[0294] Based on the solvent analyses by chemical and physical
characteristics, a pilot structure-viability analysis of esters
based on methyl laurate as a parent compound was performed. Two
more biocompatible hits identified by screening related structures
to methyl laurate. Ethyl decanoate and ethyl laurate were observed
to support similar levels of isobutanol production as methyl
laurate (FIG. 18).
TABLE-US-00006 TABLE 6 Chart of analogues of methyl laurate.
Solvent Structure Acid Alcohol methyl laurate ##STR00001## 12 1
ethyl laurate ##STR00002## 12 2 ethyl decanoate ##STR00003## 10 2
ethyl octanoate ##STR00004## 8 2 methyl decanoate ##STR00005## 10 1
methyl heptanoate ##STR00006## 7 1 methyl hexanoate ##STR00007## 6
1 "Acid" refers to the number of carbons that would be contributed
by a parent acid to the daughter ester compound; "Alcohol" likewise
to the parent alcohol.
Example 3
Determination of Solvent Partition Coefficients
[0295] Experiments were designed to obtain single point
partitioning values relevant to iBuOH toxicity and ISPR
biocompatibility. To approximate toxic iBuOH levels during
fermentation, aqueous standards were prepared at 3% iBuOH (30 g
iBuOH per aqueous L). In addition to pure water, we also examined
how composition of the aqueous phase might affect the extraction
capability of the solvents under process relevant conditions. To do
so, two additional aqueous standards were prepared: 3% iBuOH in
minimal growth media, and 3% iBuOH in rich growth media (growth
media prepared as per KRL protocol). In all cases, aqueous
solutions of 3% iBuOH were extracted at ambient temperature by the
solvent panel using an equal volume of aqueous to organic
components (1:1) to align with the extraction condition of the
primary automated biocompatibility screening. Extractions were
carried out in glass vials (containing 750 .mu.L aqueous and 750
.mu.L organic, 1.5 mL total volume) by vigorously mixing for 2
hours (vials were placed in a 96-well plate holder for mixing, 1000
rpm). The equilibration phase partition was verified by time-course
studies, wherein most solvents equilibrated in 15 minutes. Although
the relative extractability of the panel members at a given
condition remained similar, pilot studies indicated that variance
in absolute K.sub.d values can arise when keeping the 1:1 ratio
constant but changing extraction volumes (e.g., 5:5 mL vs. 750:750
.mu.L) and extraction containers (e.g., 50 mL centrifuge tubes vs.
5 mL vials). Notably, changing the extraction ratio (e.g., from 1:1
to 1:3) should change K.sub.d values more dramatically, as these
conditions represents a different point on the phase diagram. The
iBuOH content in each phase following extraction and phase
separation was determined using gas chromatography (GC method
available upon request). To prepare for GC analysis, a sample from
each phase was diluted 1:10 into methanol containing an internal
standard (0.2% n-propanol volume/volume). Concentrations of iBuOH
were determined using an external iBuOH standard curve correlated
to the internal n-propanol standard. Method development was used to
circumvent discrepancies with standards wherever possible; however,
co-elution with the internal standards persisted in the case of
ethyl n-butyrate, necessitating the use of only the external
standard curve.
[0296] Isobutanol concentrations from aqueous and organic phases
were used to calculate partition coefficients (K.sub.d) according
to the following Equation:
Kd = [ iBuOH ] ( g / L ) org [ iBuOH ] ( g / L ) aq
##EQU00001##
[0297] Table 7 lists the final K.sub.d values (all values generated
from triplicate extractions and GC measurements). Preliminary
studies indicated that variance in isobutanol measurements arise
when samples are directly injected onto the GC versus when they
were diluted 1/10 in methanol before GC analysis. All K.sub.d
values in Table 7 with an asterisk were determined using the
dilution method.
TABLE-US-00007 TABLE 7 Solvent panel K.sub.d values Solvent K.sub.d
Std. Error oleyl alcohol 2.23 0.15 n-decane 0.11 0.00 methyl
laurate 1.12 0.02 di-n-hexyl ether 0.78 0.02 decalin 0.13 0.00
2-ethyl hexyl acetate 1.88 0.06 2-ethylhexylglycol ether* 4.95 0.07
2-ethyl-1-hexanol 5.51 0.15 2,6-dimethylheptan-4-ol 4.25 0.13 ethyl
n-butyrate 3.57 0.44 n-hexyl acetate 2.23 0.04 1,2-dibutoxyethane
2.27 0.08 n-butyl n-butyrate 2.17 0.06 2.6-dimethylheptan-4-one
2.12 0.05 ETBE 2.47 0.05 di-n-butyl ether 1.26 0.03 ethylene glycol
monohexyl ether 5.66 0.14 MTBE 4.18 0.19 propylene glycol methyl
ether acetate 3.15 0.09 isooctane 0.18 0.02 mesitylene 0.47 0.02
acetophenone 2.95 0.07 n-hexanol 7.41 0.23 phenyl acetate* 1.95
0.05 1-octanol* 5.19 0.20 8-methylquinoline 5.03 0.20
p-tolualdehyde* 2.41 0.04 2-octanol 5.98 0.25 Isophorone 5.68 0.15
methyl isobutyl carbinol 7.78 0.15 o-xylene* 0.85 0.02
trimethylnonanol 3.57 0.08 n-butyl propionate* 3.02 0.13 n-propyl
propionate* 3.4 0.06 n-butyl acetate* 3.48 0.09 n-pentyl
propionate* 2.94 0.03 primary amyl acetate mixed isomers* 3.67 0.05
isobutyl acetate 3.33 0.12 dodecanal 5.38 0.35 UCAR filmer IBT 4.16
0.06 n-butyl valerate 2.04 0.01 di-n-octyl ether 0.55 0.01 isobutyl
heptyl ketone 1.49 0.01 dodecane 0.18 0.01 n-undecane 0.20 0.01
dodecanol 3.39 0.06 ethyl octanoate 1.81 0.02 ethyl decanoate 1.40
0.02 ethyl dodecanoate 1.26 0.01 methyl decanoate 1.42 0.02 methyl
hexanoate 2.47 0.04 methyl heptanoate 2.07 0.02 *indicates K.sub.d
values measured using the dilution technique. 3% iBuOH water,
extracted 1:1 with solvent (n = 3), a - Standard Error calculated
as (.sigma..sub.Kd/ n) where .sigma..sub.Kd = standard deviation of
K.sub.d and n = number of replicates. Formula for .sigma..sub.Kd =
[(.sigma..sub.Aq.sup.2/.mu..sub.Org.sup.2) +
(.mu..sub.Aq.sup.2/.mu..sub.Org.sup.4)] where .sigma. = standard
deviation, .mu. = average, Aq = value from the aqueous layer, and
Org = value from the organic layer.
Example 4
Enhancement of Extraction Efficiency of a Biocompatible Solvent by
Mixing with a High K.sub.d Solvent
[0298] In order to enhance the extraction efficiency of a
biocompatible solvent, it was determined that when mixed with a
high K.sub.d, low biocompatibility solvent, a high K.sub.d, high
biocompatibility extractant composition is obtained. A preliminary
test was performed with the automated primary screen protocol to
evaluate the feasibility of mixtures as a process strategy. A
factorial design was conducted using three biocompatible solvents
(oleyl alcohol, methyl laurate and trimethylnonanol) paired with
four high K.sub.d solvents (n-hexanol, methyl isobutyl carbinol,
2-ethyl-1-hexanol, and 2,6-dimethylheptan-4-ol) at five proportions
(0, 25, 50, 75 and 100% high K.sub.d solvent) in triplicate. Based
on the viability data, the most biocompatible combination was
carried forward to tertiary screening.
[0299] The primary screen indicated that mixtures of a
biocompatible solvent with 2,6-dimethylheptan-4-ol resulted in low
levels of cell death when used as the organic extractant (FIG. 15).
Among the solvent mixtures that were biocompatible, a combination
of oleyl alcohol and 2,6-dimethylheptan-4-ol in a 75%:25% ratio was
shown to support isobutanol production--1 g/L in 48 h (FIG. 17).
The solvent mixture has an interpolated K.sub.d value of 3.8, which
represents a 1.7 fold improvement when compared to oleyl alcohol
alone.
* * * * *