Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS FOR PRODUCING
CHEMICALS AND DERIVATIVES THEREOF
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority under U.S.C. 119(e) to continuation-
in-part of
U.S. Application Serial No. 14/222,453, filed March 21, 2014, continuation-in-
part of U.S.
Application Serial No. 14/033,300, filed September 20, 2013, which claims the
benefit of
priority under 35 U.S.C. 119(e) of U.S. provisional application serial number
61/704,408,
filed September 21, 2012, which are each hereby incorporated by reference in
their entireties,
including all tables, figures, and claims.
INCORPORATION OF SEQUENCE LISTING
[0002] The material in the accompanying Sequence Listing is hereby
incorporated by
reference into this application. The accompanying sequence listing text
file, name
SGI1660 3W0 Sequence Listing.txt, was created on March 20, 2015 and is 191 KB.
The file
can be assessed using Microsoft Word on a computer that uses Windows OS.
BACKGROUND OF THE INVENTION
[0003] In recent years, an increasing effort has been devoted to identify new
and
effective ways to use renewable feedstocks for the production of organic
chemicals. Among a
plethora of downstream chemical processing technologies, the conversion of
biomass-derived
sugars to value-added chemicals is considered very important. In particular,
six-carboned
carbohydrates, i.e. hexoses such as fructose and glucose, are widely
recognized the most
abundant monosaccharides existing in nature, therefore can be suitably and
economically used
as the chemical feedstocks.
[0004] The production of furans and furan derivatives from sugars has
attracted
increasing attention in chemistry and in catalysis studies, and is believed to
have the potential
to provide one of the major routes to achieving sustainable energy supply and
chemicals
production. Indeed, dehydration and/or oxidation of the sugars available
within biorefineries
with integrated biomass conversion processes can lead to a large family of
products including a
wide range of furans and furan derivatives.
[0005] Among the furans having the most commercial values, furan-2,5-
dicarboxylic
acid (also known as 2,5-furandicarboxylic acid, hereinafter abbreviated as
FDCA) is a valuable
intermediate with various uses in several industries including
pharmaceuticals, pesticides,
antibacterial agents, fragrances, agricultural chemicals, as well as in a wide
range of
manufacturing applications of polymer materials, e.g. bioplastic resins. As
such, FDCA is
considered a green alternative of terephthalic acid (TPA), a petroleum-based
monomer that is
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one of the largest-volume petrochemicals produced yearly worldwide. In fact,
the US
Department of Energy has identified FDCA as one of the top 12 priority
compounds made
from sugars into a value-added chemical for establishing the "green" chemistry
of the future,
and as such, it has been named one of the "sleeping giants" of the renewable
intermediate
chemicals (Werpy and Petersen, Top Value Added Chemicals from Biomass. US
Department of
Energy, Biomass, Vol 1, 2004).
[0006] Although various methods have been proposed for commercial scale
production
of FDCA (for review, see, e.g., Tong et al., Appl. Catalysis A: General, 385,
1-13, 2010), the
main industrial synthesis of FDCA currently relies on a chemical dehydration
of hexoses, such
as glucose or fructose, to the intermediate 5-hydroxymethylfurfural (5-HMF),
followed by a
chemical oxidation to FDCA. However, it has been reported that current FDCA
production
processes via dehydration are generally nonselective, unless immediately upon
their formation,
the unstable intermediate products can be transformed to more stable
materials. Thus, the
primary technical barrier in the production and use of FDCA is the development
of an effective
and selective dehydration process from biomass-derived sugars.
[0007] It is therefore desirable to develop methods for production of this
highly
important compound, as well as many other chemicals and metabolites, by
alternative means
that not only would substitute renewable for petroleum-based feedstocks, but
also use less
energy and capital-intensive technologies. In particular, the selective
control of sugar
dehydration could be a very powerful technology, leading to a wide range of
additional,
inexpensive building blocks.
Summary of the Invention
[0008] The present invention provides methods for producing a product of one
or more
enzymatic pathways. The pathways used in the methods of the invention involve
one or more
conversion steps such as, for example, an enzymatic conversion of guluronic
acid into D-
glucarate (Step 7); an enzymatic conversion of 5-ketogluconate (5-KGA) into L-
Iduronic acid
(Step 15); an enzymatic conversion of L-Iduronic acid into Idaric acid Step
7b); and an
enzymatic conversion of 5-ketogluconate into 4,6-dihydroxy 2,5-diketo
hexanoate (2,5-DDH)
(Step 16); an enzymatic conversion of 1,5-gluconolactone to gulurono-lactone
(Step 19). In
some embodiments the methods of the invention produce 2,5-furandicarboxylic
acid (FDCA)
as a product. The methods can include both enzymatic and chemical conversions
as steps.
Various pathways are also provided for converting glucose or fructose or
sucrose or galactose
into 5-dehdyro-4-deoxy-glucarate (DDG), and for converting the same sugars
into FDCA. The
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methods can also involve the use of engineered enzymes that perform reactions
with high
specificity and efficiency.
[0009] In a first aspect the invention provides a method for producing a
product of an
enzymatic or chemical pathway from a starting substrate. The pathway can
contain any one or
more of the following conversion steps: an enzymatic conversion of guluronic
acid into D-
glucarate (Step 7); an enzymatic conversion of 5-ketogluconate (5-KGA) into L-
Iduronic acid
(Step 15); an enzymatic conversion of L-Iduronic acid into Idaric acid (Step
7b); and an
enzymatic conversion of 5-ketocluconate into 4,6-dihydroxy 2,5-diketo
hexanoate (2,5-DDH)
(Step 16); an enzymatic conversion of 1,5-gluconolactone to gulurono-lactone
(Step 19).
[0010] In one embodiment the product of the enzymatic pathway is 5-dehydro-4-
deoxy-glucarate (DDG). In various embodiments the substrate of the method can
be glucose,
and the product can 5-dehydro-4-deoxy-glucarate (DDG). The method can involve
the steps of
the enzymatic conversion of D-glucose to 1,5-gluconolactone (Step 1); the
enzymatic
conversion of 1,5-gluconolactone to gulurono-lactone (Step 19); the enzymatic
conversion of
gulurono-lactone to guluronic acid (Step 1B); the enzymatic conversion of
guluronic acid to D-
glucarate (Step 7); and the enzymatic conversion of D-glucarate to 5-dehydro-4-
deoxy-
glucarate (DDG) (Step 8).
[0011] In another method of the invention the substrate is glucose and the
product is
DDG, and the method involves the steps of: the conversion of D-glucose to 1,5-
gluconolactone
(Step 1); the conversion of 1,5-gluconolactone to gluconic acid (Step la); the
conversion of
gluconic acid to 5-ketogluconate (5-KGA) (Step 14); the conversion of 5-
ketogluconate (5-
KGA) to L-Iduronic acid (Step 15); the conversion of L-Iduronic acid to Idaric
acid (Step 7b);
and the conversion of Idaric acid to DDG (Step 8a).
[0012] In another method of the invention the substrate is glucose and the
product is
DDG and the method involves the steps of the conversion of D-glucose to 1,5-
gluconolactone
(Step 1); the conversion of 1,5-gluconolactone to gluconic acid (Step la); the
conversion of
gluconic acid to 5-ketogluconate (5-KGA) (Step 14); the conversion of 5-
ketogluconate (5-
KGA) to 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 16); the conversion
of 4,6-
dihydroxy 2,5-diketo hexanoate (2,5-DDH) to 4-deoxy-5-threo-hexosulose uronate
(DTHU)
(Step 4); and the conversion of 4-deoxy-5-threo-hexosulose uronate (DTHU) to
DDG (Step 5).
[0013] In another method of the invention the substrate is glucose and the
product is
DDG, and the method involves the steps of: the conversion of D-glucose to 1,5-
gluconolactone
(Step 1); the conversion of 1,5-gluconolactone to gluconic acid (Step la); the
conversion of
gluconic acid to 5-ketogluconate (5-KGA) (Step 14); the conversion of 5-
ketogluconate (5-
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KGA) to L-Iduronic acid (Step 15); the conversion of L-Iduronic acid to 4-
deoxy-5-threo-
hexosulose uronate (DTHU) (Step 7B); and the conversion of 4-deoxy-5-threo-
hexosulose
uronate (DTHU) to DDG (Step 5).
[0014] Any of the methods disclosed herein can further involve the step of
converting
the DDG to 2,5-furan-dicarboxylic acid (FDCA). Converting the DDG to FDCA in
any of the
methods can involve contacting DDG with an inorganic acid to convert the DDG
to FDCA.
[0015] In another aspect the invention provides a method for synthesizing
derivatized
(esterified) FDCA. The method involves contacting DDG with an alcohol, an
inorganic acid at
a temperature in excess of 60 C to form derivatized FDCA. In different
embodiments the
alcohol is methanol, butanol or ethanol.
[0016] In another aspect the invention provides a method for synthesizing a
derivative of FDCA. The method involves contacting DDG with an alcohol, an
inorganic acid,
and a co-solvent to produce a derivative of DDG; optionally purifying the
derivative of DDG;
and contacting the derivative of DDG with an inorganic acid to produce a
derivative of FDCA.
The inorganic acid can be sulfuric acid and the alcohol can be ethanol or
butanol. In various
embodiments the co-solvent can be any of THF, acetone, acetonitrile, an ether,
butyl acetate,
an dioxane, chloroform, methylene chloride, 1,2-dichloroethane, a hexane,
toluene, and a
xylene.
[0017] In one embodiment in the derivative of DDG is di-ethyl DDG and the
derivative of FDCA is di-ethyl FDCA, and in another embodiment the derivative
of DDG is di-
butyl DDG and the derivative of FDCA is di-butyl FDCA.
[0018] In another aspect the invention provides a method for synthesizing
FDCA.
The method involves contacting DDG with an inorganic acid in a gas phase.
[0019] In another aspect the invention provides a method for synthesizing
FDCA.
The method involves contacting DDG with an inorganic acid at a temperature in
excess of 120
C.
[0020] In another aspect the invention provides a method for synthesizing
FDCA.
The method involves contacting DDG with an inorganic acid under anhydrous
reaction
conditions.
Description of the Drawings
[0021] FIG. 1 is a electrophoretic gel of crude lysates and purified enzymes
of proteins
474, 475, and 476.
[0022] FIGS. 2A-2H is a schematic illustration of the pathways of Routes 1, 2,
2A, 2C,
2D, 2E, 2F, respectively.
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[0023] FIGS. 3A-3c present a schematic illustration of the pathways of Routes
3, 4,
and 5, respectively.
[0024] FIG. 4 is an HPCL-MS analysis of the dehydration of gluconate with
gluconate
dehydratase to produce DHG by pSGI-359.
[0025] FIG. 5 is a graphical illustration of semicarbizide assay plots for
measuring the
activity of gluconate dehydratases.
[0026] FIGS. 6A-6B provide Lineweaver-Burk plots for the oxidation of
glucuronate
and iduronate with three enzymes of the invention.
[0027] FIG. 7A shows the results of an HPLC analysis of time points for the
isomerization of 5KGA and Iduronate using enzymes DTHU isomerases in the EC
5.3.1.17
family. Controls: dead enzyme is a control with heat inactivated enzyme. Med
Bl refers to
reactions without isomerase add/n. Time points, x axis 1=0.5 h; 2=1; 3=2 h;
4=16h. Figure 7b
shows an HPLC analysis of time points for the isomerization of 5KGA and
iduronate using
enzymes in the EC 5.3.1.17 family. Controls: dead enzyme is a control with
heat inactivated
enzyme; Med Bl: refers to reactions without isomerase add/n. Time points, X
axis: 1=0 h; 2= 1
h ; 3= 2 h ; 4=17 h.
[0028] FIG. 8 shows product formation for the isomerization of 5KGA and
iduronate
with enzymes in the EC 5.3.1.n1 family. The data were obtained from enzymatic
assays.
[0029] FIG. 9: HPLC analysis of the formation of 2,5-DDH and the reduction of
5KGA
concentration over time. Total ion counts for 2,5-DDH are shown.
[0030] FIG. 10 is a HPLC-MS chromatogram showing the production of guluronic
acid
lactone from 1,5-gluconolactone. An overlay of a trace of authentic guluronic
acid is shown.
[0031] FIG. 11 is a schematic illustration of the Scheme 6 reaction pathway.
[0032] FIGS. 12A-12B are LC-MS chromatograms showing 5-KGA and DDG
reaction products, respectively.
[0033] FIG. 13 is an LC-MS chromatogram showing FDCA and FDCA dibutyl ester
derivative reaction products.
[0034] FIG. 14A is a GC-MS analysis of a crude reaction sample of the diethyl-
FDCA
synthesis from the reaction of DDG with ethanol. Single peak corresponded to
diethyl-FDCA.
FIG. 14B is an MS fragmentation of the major product from the reaction of DDG
with ethanol.
[0035] FIG. 15A is a GC-MS analysis of a crude reaction sample of the diethyl-
FDCA
synthesis from the reaction of DDG with ethanol. Single peak corresponded to
diethyl-FDCA.
FIG. 15B is a MS fragmentation of the major product from the reaction of DDG
with ethanol.
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[0036] FIG. 16 is a schematic illustration of the synthesis of FDCA and its
derivatives
from DTHU.
[0037] FIG. 17 is a schematic illustration of Scheme 1. Cell free enzymatic
synthesis
of DDG from glucose. Enzymes are ST-1: glucose oxidase; ST-1A: hydrolysis-
chemical; ST-
14: gluconate dehydro genase (p S GI-504); ST-15 : 5 -dehydro-4-deoxy-D-
glucuronate isomerase
(DTHU IS, pSGI-434); ST-7B: Uronate dehydrogenase (UroDH, pSGI-476)); ST-8A
Glucarate dehydratase (GlucDH, pSGI-353); ST-A: NAD(P)H oxidase (NADH OX, pSGI-
431); ST-B: Catalase. Figure 17b shows the concentration of reaction
intermediates over the
first 3h as analyzed by HPLC. Formation of DDG is shown in both reactions.
Detailed Description of the Invention
[0038] The present invention provides methods for producing a product of an
enzymatic pathway. The methods can comprise the enzymatic conversion of a
substrate into a
product. By utilizing the enzymatic and chemical pathways of the invention it
is possible to
synthesize a wide variety of products in a highly efficient and economical
manner. One
product that can be produced by the methods and pathways of the invention is
2,5-furanyl
dicarboxylic acid (FDCA), which can be produced at commercial scales according
to the
invention. The methods can comprise one or more enzymatic and/or chemical
substrate-to-
product conversion steps disclosed herein. In some embodiments the enzymes
utilized perform
enzymatic conversion steps using activities unknown for the enzymes. These
novel activities
can therefore be employed in the invention to perform the conversion steps and
perform a
substrate to product conversion as part of a enzymatic and/or chemical
pathway. Any of the
products of any of the pathways disclosed herein (e.g., DDG, iduronic acid,
idaric acid,
glucaric acid, FDCA, etc.) can be produced on a commercial scale. i.e. in
quantities of at least
1 gram or at least 10 grams or at least 100 grams or at least 1 kg in a single
bioreactor or
reaction vessel, as disclosed herein.
[0039] The pathways of the invention are comprised of any one or more of the
steps
disclosed herein. It is understood that a step of a pathway of the invention
can involve the
forward reaction or the reverse reaction, i.e., the substrate A being
converted into product B,
while in the reverse reaction substrate B is converted into product A. In the
methods both the
forward and the reverse reactions are described as the step unless otherwise
noted.
[0040] The methods involve producing a product of a pathway, which can be an
enzymatic pathway. The methods involve one or more enzymatic and/or chemical
conversion
steps, which convert a substrate to a product. Steps that can be included in
the methods
include, for example, any one or more of: an enzymatic conversion of guluronic
acid into D-
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glucarate (Step 7); an enzymatic conversion of L-Iduronic acid to 4-deoxy-5-
threo-hexosulose
uronate (DTHU)(17); an enzymatic conversion of 5-ketogluconate (5-KGA) into L-
Iduronic
acid (Step 15); an enzymatic conversion of L-Iduronic acid into Idaric acid
Step 7B); and an
enzymatic conversion of 5-ketocluconate into 4,6-dihydroxy 2,5-diketo
hexanoate (2,5-DDH)
(Step 16); an enzymatic conversion of 1,5-gluconolactone to gulurono-lactone
(Step 19). Any
one or more of the aforementioned steps can be included in a method or pathway
of the
invention. An enzymatic step or pathway is a step or pathway that requires an
enzyme as a
catalyst in the reaction to make the step proceed. Chemical steps can be
performed without an
enzyme as a catalyst in the reaction. Any one or more of the steps recited in
the methods can
be an enzymatic step. In some embodiments every step of the pathway is an
enzymatic step,
while in other embodiments one or more steps in the pathway is a chemical
step.
[0041] In some embodiments any of the methods can include a step involving the
addition of the substrate of the reaction to a reaction mix containing the
enzyme that performs
the conversion. Thus the method of converting guluronic acid into D-glucarate
(step 7) can
involve the addition of guluronic acid as starting substrate to the reaction
mix; the enzymatic
conversion of L-iduronic acid to Idaric acid (7B) can involve the addition of
L-Iduronic acid as
starting substrate to the reaction mix; the enzymatic conversion of L-Iduronic
acid to 4-deoxy-
5-threo-hexosulose uronate (DTHU) (17) can involve the addition of L-iduronic
acid as
starting substrate to the reaction mix. Any of the methods can involve a step
of adding
glucose, fructose, galactose, sucrose, or mannose or another mono- or di-
saccharide to the
reaction mixture. Another step that can be included in any of the methods is a
step of purifying
from the reaction mixture a reaction product. Thus, a step of purifying
glucaric acid/D-
glucarate or L-Iduronic acid/iduronate, or Idaric acid, or 2,5-diketo
hexanedioic/DKHA can be
included in any of the methods described herein. Any of the methods disclose
can include a
step of isolating or purifying DDG or FDCA from the reaction mixture. And any
of the
methods can involve a step of adding an enzyme that performs any one or more
of the steps
described herein to the reaction mixture. A reaction mixture is a mixture of
at least one
substrate and at least one enzyme and involves the conversion of at least one
substrate into a
least one enzyme product. Any of the methods can involve a step of adding an
isolated
enzyme to a reaction mix, the enzyme performing a substrate to product
conversion step of a
pathway of the invention, and the isolated enzyme being at least 10% purified
or at least 20%
purified or at least 25% purified or at least 50% purified or at least 70%
purified or at least
80% purified or at least 90%, all w/w.
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[0042] Since many sugars can be converted into other sugars any of the methods
or
pathways of the invention can involve the use of glucose, sucrose, fructose or
galactose as the
starting substrate. Thus, in any pathway or reaction disclosed herein where
glucose is the
starting substrate it is understood that fructose or sucrose or galactose or
mannose or another
starting substrate can also be a starting substrate for that pathway or
reaction. In some
embodiments the sugar is converted into glucose which then enters the pathway
but in other
embodiments the pathway begins with fructose or sucrose or galactose or
mannose or another
mono- or di-saccharide.
[0043] The reactions of the invention can occur in a lysate of cells or a cell-
free lysate
that contains one or more enzymes that perform the enzymatic conversion, but
can also occur
in a reaction mixture containing components added by the user to form a
reaction mixture, or
can contain components purified from a cell lysate, or may be contained in a
whole cell
biocatalyst. The reaction can also occur in a mix made of purified components
that have been
combined, such as in a mix where the substrate and enzyme were combined to
form the
reaction mix. The reactions can occur in an in vitro reaction or can occur in
a recombinant
cell, and therefore the product(s) can be harvested by lysing the cells or by
collecting from the
culture medium. The reactions can occur in a laboratory container or reaction
vessel such as,
for example, a centrifuge tube, a test tube, a vial, a beaker, or a glass or
metal or plastic
container or reactor, a fermenter or fermentation vessel or bioreactor, an
algae pond, any of
which can be small scale or large scale. Any of the organisms described herein
can be utilized
as host cells to produce the product of a step or pathway of the invention.
The organisms can
also be used to produce one or more enzymes of the invention for use in a
method of the
invention. Various types of organisms can be used. Examples include: bacteria
of the family
Acetobacteraceae (e.g. bacteria of the genus Acetobacter, Acidiphilium,
Gluconobacter,
Gluconoacetobacter), or bacteria of the family Pseudomonadaceae (e.g., genus
Azotobacter,
Pseudomonas), or bacteria of the family Enterobacteriacea (e.g., of the genus
Escherichia (e.g.,
E. coli), Klebsiella). Yeast can also be used for these purposes such as yeast
of the genera
Saccharomyces, Ashbya, Kluveromyces, Lachancea, Zygosaccharomyces, Candida,
Pichia,
Arxula or Trichosporon or Blastobotrys. Cyanobacteria can also be used such as
those of the
genus Cyanothece (e.g. Cyanothece strains ATCC 51142, PCC 7424, PCC 7425, PCC
7822,
PCC 8801, PCC 8802), or Microcystis or Synechococcus (e.g., strains elongatus
PCC 7942,
PCC 7002, PCC 6301, CC9311, CC9605, CC9902, JA-2-3B'a(2-13), JA-3-3Ab, RCC307,
WH
7803, WH 8102) or Synechocystis, or Thermosynechococcus. Thus the present
invention
provides recombinant host cells comprising a recombinant nucleic acid of one
or more of SEQ
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ID NOs: 4-6, 20-32, 36-38, 47-54, 56, 62-66, 69-70, 72, and 79-84 or a codon-
optimized
sequence of any of SEQ ID NOs: 1-84. The host cells can also contain a vector
of the
invention described herein. A "codon optimized" sequence refers to changes in
the codons of a
sequence to those preferentially used in a particular organism so that the
encoded protein is
efficiently expressed in the organism carrying the sequence. The recombinant
nucleic acid
sequence can be comprised on a vector, as disclosed herein.
[0044] In various embodiments the methods of the invention are methods of
converting
glucose or fructose or sucrose or galactose to DDG, or glucose or fructose or
sucrose or
galactose to FDCA, or glucose or fructose or sucrose or galactose to DTHU or
DEHU, or for
converting DDG to FDCA. The methods can involve converting the starting
substrate in the
method into the product. The starting substrate is the chemical entity
considered to begin the
method and the product is the chemical entity considered to be the final end
product of the
method. Intermediates are those chemical entities that are created in the
method (whether
transiently or permanently) and that are present in the reaction pathway
between the starting
substrate and the product. In various embodiments the methods and pathways of
the invention
have about four or about five intermediates or 4-5 intermediates, or about 3
intermediates, or 3-
intermediates, or less than 6 or less than 7 or less than 8 or less than 9 or
less than 10 or less
than 15 or less than 20 intermediates, meaning these values not counting the
starting substrate
or the final end product.
[0045] The invention provides methods of producing FDCA and/or DDG, from
glucose
or fructose or sucrose or galactose that have high yields. The theoretical
yield is the amount of
product that would be formed if the reaction went to completion under ideal
conditions. In
different embodiments the methods of the invention produce DDG from glucose,
fructose, or
galactose with a theoretical yield of at least 50% molar, or at least 60%
molar or at least 70%
molar, or at least 80% molar, at least 90% molar or at least 95% molar or at
least 97% molar or
at least 98% molar or at least 99% molar, or a theoretical yield of 100%
molar. The methods
of the invention also can provide product with a carbon conservation of at
least 80% or at least
90% or at least 95% or at least 97% or at least 98% or at least 99% or 100%,
meaning that the
particular carbon atoms present in the initial substrate are present in the
end product of the
method at the recited percentage. In some embodiments the methods produce DDG
and/or
FDCA from glucose or fructose or sucrose or galactose via dehydration
reactions.
Example Synthesis Routes
[0046] The invention also provides specific pathways for synthesizing and
producing a
desired product. Any of the following described routes or pathways can begin
with glucose or
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fructose or sucrose or galactose or mannose and flow towards a desired
product. In some
embodiments D-glucose is the starting substrate and the direction of the
pathway towards any
intermediate or final product of the pathway is considered to be in the
downstream direction,
while the opposite direction towards glucose is considered the upstream
direction. It will be
realized that routes or pathways can flow in either the downstream or upstream
direction.
While glucose is used as an example starting substrate for pathways described
herein, it is also
understood that sucrose, fructose, galactose, or mannose or any intermediate
in any of the
pathways can also be the starting substrate in any method of the invention,
and DDG, DTHU,
FDCA, or any intermediate in any of the routes or pathways of the invention
can be the final
end product of a method of the invention. The disclosed methods therefore
include any one or
more steps disclosed in any of the routes or pathways of the invention for
converting any
starting substrate or intermediate into any end product or intermediate in the
disclosed routes or
pathways using one or more of the steps in the disclosed routes or pathways.
Thus, for
example the methods can be methods for converting glucose or fructose or
sucrose or galactose
or mannose to DDG, or to guluronic acid, or to galactarate, or to DTHU, or to
DEHU, or to
guluronic acid, or to iduronic acid, or to idaric acid, or to glucaric acid,
or for converting
galactarate to DDG, or for converting guluronic acid to D-glucarate, or for
converting 5-KGA
to L-Iduronic acid, or for converting L-Iduronic acid to Idaric acid, or for
converting 5-KGA to
2,5-DDH or DTHU, or for converting DHG to DEHU. In these embodiments the
methods
utilize the steps disclosed in the methods and pathways of the invention from
starting substrate
to the relevant end product. One or more of the steps can also be utilized in
methods flowing
in the "opposite" or upstream direction from the pathways disclosed herein.
[0047] Route 1 is illustrated in Figure 2a. Route 1 converts D-glucose (or any
intermediate in the pathway) into 5-dehydro-4-deoxy-glucarate (DDG) via an
enzymatic
pathway via a series of indicated steps. Route 1 converts D-glucose into DDG
via a pathway
having 1,5-gluconolactone, gluconic acid, 3-dehydro-gluconic acid (DHG), 4,6-
dihydroxy 2,5-
diketo hexanoate (2,5-DDH), and 4-deoxy-L-threo-hexosulose uronate (DTHU) as
intermediates and DDG as the final end product. For any of the pathways
additional
intermediates not shown can also be present. The steps are the enzymatic
conversion of D-
glucose to 1,5-gluconolactone (Step 1); the enzymatic conversion of 1,5-
gluconolactone to
gluconic acid (Step 1A); the enzymatic conversion of gluconic acid to 3-
dehydro-gluconic acid
(DHG) (Step 2); the enzymatic conversion of 3-dehydro-gluconic acid (DHG) to
4,6-dihydroxy
2,5-diketo hexanoate (2,5-DDH) (Step 3); the enzymatic conversion of 4,6-
dihydroxy 2,5-
diketo hexanoate (2,5-DDH) to 4-deoxy-L-threo-hexosulose uronate (DTHU) (Step
4); and the
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enzymatic conversion of 4-deoxy-L-threo-hexosulose uronate (DTHU) to 5-dehydro-
4-deoxy
glucarate (DDG) (Step 5). Route 1 also comprises sub-routes where the glucose
or any
intermediate in the pathway as a substrate is converted into any other
downstream intermediate
as final product, and each substrate to product sub-route is considered
disclosed as if each is set
forth herein in full.
[0048] Route 2 is illustrated in Figure 2b and converts D-glucose into DDG.
The steps
in the Route 2 pathway are the enzymatic conversion of D-glucose into 1,5-
gluconolactone
(Step 1); the enzymatic conversion of 1,5-gluconolactone to gluconic acid
(Step 1A); the
enzymatic conversion of gluconic acid to guluronic acid (Step 6); the
enzymatic conversion of
guluronic acid to D-glucarate (Step 7); the enzymatic conversion of D-
glucarate to DDG (Step
8). Route 2 also comprises sub-routes where glucose or any intermediate in the
pathway as
substrate is converted into any other downstream intermediate as final
product, and each sub-
route is considered disclosed as if each is set forth herein in full. For
example in some
embodiments the methods comprise steps for the conversion of glucose or
gluconic acid as
substrate into guluronic acid or D-glucarate as product using one or more of
the steps described
in Route 2.
[0049] Route 2A is illustrated in Figure 2c. The steps in Route 2A are the
enzymatic
conversion of D-glucose to 1,5-gluconolactone (Step 1); the enzymatic
conversion of 1,5-
gluconolactone to guluronic acid lactone (Step 19); the enzymatic conversion
of guluronic acid
lactone to guluronic acid (Step 1B); the enzymatic conversion of guluronic
acid to D-glucarate
(Step 7); the enzymatic conversion of D-glucarate to 5-dehydro-4-deoxy-
glucarate (DDG)
(Step 8). Route 2A also comprises sub-routes where glucose or any intermediate
in the
pathway as starting substrate is converted into any other downstream
intermediate as final end
product, and each sub-route is considered disclosed as if each is set forth
herein in full. For
example in some embodiments the methods comprise steps for the conversion of
glucose or
guluronic acid lactone as substrate into glucarate or DDG as product using one
or more of the
steps described in Route 2A.
[0050] Route 2B is illustrated in Figure 2d. The steps in Route 2B are the
enzymatic
conversion of D-glucose into gluconic acid (Steps 1 and 1A); the enzymatic
conversion of
gluconic acid into 5-ketogluconate (5-KGA) (Step 14); the enzymatic conversion
of 5-KGA
into L-Iduronic acid (Step 15); the enzymatic conversion of L-Iduronic acid
into Idaric acid
(Step 7B); the enzymatic conversion of Idaric acid into DDG (Step 8A). Route
2B also
comprises sub-routes where glucose or any intermediate in the pathway as
starting substrate is
converted into any other downstream intermediate as final end product, and
each sub-route is
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considered disclosed as if each is set forth herein in full. For example in
some embodiments
the methods comprise steps for the conversion of glucose or 5-KGA as substrate
into iduronic
acid or idaric acid as product using one or more of the steps described in
Route 2B.
[0051] Route 2C is illustrated in Figure 2e. The steps in Route 2C are the
enzymatic
conversion of D-glucose to gluconic acid (Steps 1 and 1A); the enzymatic
conversion of
gluconic acid to 5-ketogluconate (5-KGA) (Step 14); the enzymatic conversion
of 5-KGA to
4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) (Step 16); the enzymatic
conversion of 4,6-
dihydroxy 2,5-diketo hexanoate (2,5-DDH) to 4-deoxy-5-threo-hexosulose uronate
(DTHU)
(Step 4); the enzymatic conversion of DTHU to DDG (Step 5). Route 2C also
comprises sub-
routes where glucose or any intermediate in the pathway as starting substrate
is converted into
any other downstream intermediate as final end product, and each sub-route is
considered
disclosed as if each is set forth herein in full. For example in some
embodiments the methods
comprise steps for the conversion of glucose or gluconic acid as substrate
into 2,5-DDH or
DTHU using one or more steps described in Route 2C.
[0052] Route 2D is illustrated in Figure 2f The steps in Route 2D are the
enzymatic
conversion of D-glucose to gluconic acid (Steps 1 and 1A); the enzymatic
conversion of
gluconic acid to 5-ketogluconate (5-KGA) (Step 14); the enzymatic conversion
of 5-KGA to
Iduronic acid (Step 15); the enzymatic conversion of L-Iduronic acid to DTHU
(Step 17); the
enzymatic conversion of DTHU to DDG (Step 5). Route 2D also comprises sub-
routes where
glucose or any intermediate in the pathway as starting substrate is converted
into any other
downstream intermediate as final end product, and each sub-route is considered
disclosed as if
each is set forth herein in full. For example in some embodiments the methods
comprise steps
for the conversion of glucose or 5-KGA as substrate into L-iduronic acid or
DTHU using one
or more of the steps described in Route 2D.
[0053] Route 2E is illustrated in Figure 2g. The steps in Route 2D are the
enzymatic
conversion of D-glucose to 1,5-gluconolactone (Step 1); the enzymatic
conversion of 1,5-
gluconolactone to guluronic acid lactone (Step 19); the enzymatic conversion
of guluronic acid
lactone to guluronic acid (Step 1B); the enzymatic conversion of guluronic
acid to 4-deoxy-
erythro-hexosulose uronate (DEHU) (Step 17A); the enzymatic conversion of DEHU
to 3-
deoxy-D-erythro-2-hexulosaric acid (DDH) (Step 7A). Route 2E also comprises
sub-routes
where glucose or any intermediate in the pathway as starting substrate is
converted into any
other downstream intermediate as final end product, and each sub-route is
considered disclosed
as if each is set forth herein in full. For example in some embodiments the
methods comprise
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steps for the conversion of glucose as substrate into guluronic acid or DEHU
using one or
more of the steps described in Route 2E.
[0054] Route 2F is illustrated in Figure 2h. The steps in Route 2F are the
enzymatic
conversion of D-glucose to gluconic acid (Steps 1 and 1A); the enzymatic
conversion of
gluconic acid to guluronic acid (Step 6); the enzymatic conversion of
guluronic acid to 4-
deoxy-erythro-hexosulose uronate (DEHU) (Step 17A); the enzymatic conversion
of DEHU to
3-deoxy-D-erythro-2-hexulosaric acid (DDH) (Step 7A). Route 2F also comprises
sub-routes
where glucose or gluconic acid or any intermediate in the pathway as starting
substrate is
converted into guluronic acid or DDH or any other downstream intermediate as
final end
product using one or more of the steps of Route 2F, and each sub-route is
considered disclosed
as if each is set forth herein in full.
[0055] Route 3 is illustrated in Figure 3a. The steps in Route 3 are the
enzymatic
conversion of D-glucose to gluconic acid (Steps 1 and 1A); the enzymatic
conversion of
gluconic acid to 3-dehydro-gluconic acid (DHG) (Step 2); the enzymatic
conversion of DHG
to 4-deoxy-erythro-hexosulose uronate (DEHU) (Step 6A); the enzymatic
conversion of
DEHU to DDG (Step 7A). Route 3 also comprises sub-routes where glucose or
fructose or
sucrose or galactose or any intermediate in the pathway as starting substrate
is converted into
gluconic acid or DDH any other downstream intermediate of Route 3 as final end
product
using one or more of the steps of Route 3, and each sub-route is considered
disclosed as if each
is set forth herein in full.
[0056] Route 4 is illustrated in Figure 3b. The steps in Route 4 are the
enzymatic
conversion of D-glucose to a-D-gluco-hexodialdo-1,5-pyranose (Step 9); the
enzymatic
conversion of a-D-gluco-hexodialdo-1,5-pyranose to a-D-glucopyranuronic acid
(Step 10); the
enzymatic conversion of a-D-glucopyranuronic acid to D-glucaric acid 1,5-
lactone (Step 11);
the enzymatic conversion of D-glucaric acid 1,5-lactone to D-glucarate (Step
1C); the
enzymatic conversion of D-glucarate to DDG (Step 8). Route 4 also comprises
sub-routes
where glucose or any intermediate in the pathway as starting substrate is
converted into
glucarate or DDG or any other downstream intermediate as final end product
using one or
more of the steps of Route 4, and each sub-route is considered disclosed as if
each is set forth
herein in full.
[0057] Route 5 is illustrated in Figure 3c. The steps in Route 5 are the
enzymatic
conversion of D-galactose to D-galacto-hexodialdose (Step 9A); the enzymatic
conversion of
D-galacto-hexodialdose to galacturonate (Step 10A); the enzymatic conversion
of
galacturonate to galactarate (Step 11A); the enzymatic conversion of
galactarate to DDG (Step
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13). Route 5 also comprises sub-routes where galactose or any intermediate in
the pathway as
starting substrate is converted into any other downstream intermediate as
final product, and
each sub-route is considered disclosed as if each is set forth herein in full.
For example in
some embodiments the methods comprise steps for the conversion of galactose or
another
substrate into galacturonate or galactarate using the steps described in Route
5.
[0058] In various other embodiments the invention provides a method of
producing a
product of an enzymatic and/or chemical pathway from a starting substrate that
involves
performing Step 1, followed by Step 19, followed by Step 1B to produce a
guluronic acid
product. Optionally the pathway can continue with Step 7 to produce glucarate.
In another
embodiment the method involves performing Steps 1 and lA followed by Step 14,
followed by
Step 15 to produce Iduronic acid. Optionally the method can continue with Step
7B to produce
an Idaric acid product or with Step 17 to produce DTHU. In another embodiment
the method
involves performing Steps 1 and 1A, followed by Step 14 followed by Step 16 to
produce a
2,5-DDH product. In another embodiment the method involves performing Step 1
followed by
Step 19 to produce guluronic acid lactone.
The Enzymatic Steps
[0059] There are disclosed a wide variety of enzymes (and nucleic acids that
encode
the enzymes) that can perform the steps of the methods outlined herein. The
enzymes utilized
in the enzymatic steps of the invention can be proteins or polypeptides. In
addition to the
families and classes of enzymes disclosed herein for performing the steps of
the invention,
homologs having a sequence identity to any enzyme or nucleic acid or to any of
SEQ ID NOs
1-84, disclosed herein will also be useful in the invention. Enzymes and
nucleic acids that are
homologs of SEQ ID NOs: 1-84 have a sequence identity of at least 40% or at
least 50% or at
least 60% or at least 70% or at least 80% or at least 90% or at least 95% or
at least 97% or at
least 98% or at least 99% to any nucleic acid or enzyme of SEQ ID NO: 1-84, or
to a member
of an enzyme class disclosed herein. Percent sequence identity or homology
with respect to
amino acid or nucleotide sequences is defined herein as the percentage of
amino acid or
nucleotide residues in the candidate sequence that are identical with the
known polypeptides,
after aligning the sequences for maximum percent identity and introducing
gaps, if necessary,
to achieve the maximum percent identity or homology. Homology or identity at
the nucleotide
or amino acid sequence level may be determined using methods known in the art,
including but
not limited to BLAST (Basic Local Alignment Search Tool) analysis using the
algorithms
employed by the programs blastp, blastn, blastx, tblastn and tblastx (Altschul
(1997), Nucleic
Acids Res. 25, 3389-3402, and Karlin (1990), Proc. Natl. Acad. Sci. USA 87,
2264-2268),
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which are tailored for sequence similarity searching. Alternatively a
functional fragment of
any of the enzymes or nucleic acids encoding such enzymes or of any enzyme or
nucleic acid
of SEQ ID NOs 1-84 disclosed herein may also be used. The term "functional
fragment"
refers to a polypeptide that has an amino-terminal and/or carboxy-terminal
deletion and/or
internal deletion (which can be replaced to form a chimeric protein), where
the remaining
amino acid sequence has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
85%,
86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the corresponding positions in the reference sequence,
and/or that retains
about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
of
the activity of the full-length polypeptide. The EC numbers provided use the
enzyme
nomenclature of the Nomenclature Committee of the International Union of
Biochemistry and
Molecular Biology. In other embodiments the functional fragment retains the
requirement of
the presence of a co-factor necessary for the activity of a protein or protein
encoded by SEQ ID
NO:1-84.
[0060] Also disclosed is an expression vector having a sequence of SEQ ID NO:
4-6,
20-32, 36-38, 47-54, 56, 62-66, 69-70, 72, and 79-84. The vector can be a
bacterial, yeast, or
algal vector. Vectors designed for expression of a gene can also include a
promoter active in
the organism carrying the vector and operably linked to the sequence of the
invention. The
vector can contain a promoter or expression control sequence operatively
linked to a sequence
of SEQ ID NOs: 4-6, 20-32, 36-38, 47-54, 56, 62-66, 69-70, 72, and 79-84 or a
codon-
optimized sequence of any of them. A "promoter" refers to a nucleic acid
sequence capable of
binding RNA polymerase to initiate transcription of a gene in a 5' to 3'
("downstream")
direction. A sequence is "operably linked" to a promoter when the binding of
RNA
polymerase to the promoter is the proximate cause of said gene's
transcription.
[0061] Step 1 - Conversion (oxidation or dehydrogenation) of glucose to 1,5-
gluconolactone. This step can be performed with various enzymes, such as those
of the family
oxygen dependent glucose oxidases (EC 1.1.3.4) or NAD(P)-dependent glucose
dehydrogenases (EC 1.1.1.118, EC 1.1.1.119). Gluconobacter oxydans has been
shown to
efficiently oxidize glucose to gluconic acid and 5-ketogluconate (5-KGA) when
grown in a
fermentor. Enzymes of the family of soluble and membrane-bound PQQ-dependent
enzymes
(EC 1.1.99.35 and EC 1.1.5.2) found in Gluconobacter and other oxidative
bacteria can be
used. Quinoprotein glucose is another enzyme that is useful in performing this
step. The
specific enzyme selected will be dependent on the desired reaction conditions
and necessary
co-factors that will be present in the reaction, which are illustrated in
Table 1.
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[0062] Step lA ¨ Conversion (e.g., hydrolysis) of 1,5-gluconolactone to
gluconate.
This step can be performed chemically in aqueous media and the rate of
hydrolysis is
dependent on pH (Shimahara, K, Takahashi, T., Biochim. Biophys. Acta (1970),
201, 410).
Hydrolysis is faster in basic pH (e.g. pH 7.5) and slower in acid pH. Many
microorgranisms
also contain specific 1,5-glucono lactone hydrolases, and a few of them have
been cloned and
characterized (EC 3.1.1.17; Shinagawa, E Biosci. Biotechnol. Biochem. 2009,
73, 241-244).
[0063] Step 1B ¨ Conversion of Guluronic acid lactone to guluronic acid. The
chemical hydrolysis of guluronic acid lactone can be done by a spontaneous
reaction in
aqueous solutions. An enzyme capable of catalyzing this hydrolysis is
identified amongst the
large number of lactonases (EC 3.1.1. XX and more specifically 3.1.1.17,
3.1.1.25).
[0064] Step 2 ¨ Conversion of gluconic acid to 3-dehydro gluconic acid (DHG):
Several enzymes, such as gluconate dehydratases, can be used in the
dehydration of gluconic
acid to dehydro gluconic acid (DHG). Examples include those belonging to the
gluconate
dehydratase family (EC 4.2.1.39). A specific example of such a dehydratase has
been shown
to dehydrate gluconate (Kim, S. Lee, S.B. Biotechnol. Bioprocess Eng. (2008),
13, 436).
Particular examples of enzymes from this family and their cloning are shown in
Example 1.
[0065] Step 3: Conversion of 3-dehydro-gluconic acid (DHG) to 4,6-dihydroxy
2,5-
diketo hexanoate (2,5-DDH). Enzymes, 2-dehydro-3-deoxy-D-gluconate 5-
dehydrogenase (or
DHG dehydrogenases) (EC 1.1.1.127) for performing this conversion have been
described.
[0066] Step 4: Conversion of 4,6-dihydroxy 2,5-diketo hexanoate (2,5-DDH) to 4-
deoxy-L-threo-hexosulose uronate (DTHU). Enzymes of the family EC 5.3.1.12 can
be used
in this step, and Step 15 shows that five such enzymes were cloned and shown
to have activity
for the dehydration of 5-KGA. These enzymes will also show activity towards
2,5-DDH and
DTHU.
[0067] Step 5: Conversion of DTHU to 5-dehydro-4-deoxy-glucarate (DDG). DDG
can be produced from the chemical or enzymatic oxidation of DTHU, for example
with a mild
chemical catalyst capable of oxidizing aldehydes in the presence of alcohols.
Aldehyde
oxidases can be used to catalyze this oxidation. Oxidative bacteria such as
Acetobacter and
Gluconobacter (Hollmann et at Green Chem. 2011, 13, 226) will be useful in
screening.
Enzymes of the following families can perform this reaction: aldehyde oxidase
EC1.2.3.1,
aldehyde ferredoxin oxidoreductase (EC1.2.7.5), and in all the families of
EC1.2.1.-XX.
Enzymes of the family of uronate dehydrogenases (EC 1.1.1.203) (e.g. see Step
7) will also
have this activity. Other enzymes with both alcohol and aldehyde oxidation
activity can be
used, including enzymes in the alditol oxidase family (see Steps 19 and 6).
Other broad
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substrate oxidases include soluble and membrane bound PQQ-dependent
alcohol/aldehyde
oxidases. More specifically soluble periplasmic PQQ oxidases enzymes and their
homologs
belonging into Type I (EC 1.1.9.1) and II (EC 1.1.2.8) families as well as
membrane bound
PQQ oxidases belonging into EC 1.1.5.X families are useful. In other
embodiments aldehyde
dehydrogenases/oxidases that act on DTHU can be used.
[0068] Step 5 can also be performed using a dehydrogenase from acetic acid
bacteria
such Gluconobacter and Acetobacter and Gluconoacetobacter, and others. Whole
cell activity
is identified by screening microorganisms for the oxidation of DTHU. The
activity is
identified and one or more of the enzymes is cloned. Enzymes with uronate
dehydrogenase
activity described in Step-7 and 7B are also screened and found to have this
activity. A library
of soluble periplasmic and membrane bound PQQ-dependent enzymes is also cloned
and
several enzymes are found having this activity. Some of the enzymes found to
have the
activity are NAD(P)- or PQQ-dependent dehydrogenases, but others are FAD-
dependent
aldehyde dehydrogenases. SEQ ID NO: 71-72 are examples of NADP-dependent
dehydrogenases, and any one or a combination of them can be used to perform
Step 5. SEQ ID
NOs: 73-84 are examples of suitable PQQ-dependent dehydrogenases and any one
or any
combination of them can be used to perform Step 5.
[0069] Steps 6 and 6A: Conversion of gluconic acid to guluronic acid (6) and
conversion of 3-dehydro-gluconic acid (DHG) to 4-deoxy-5-erythro-hexosulose
uronate
(DEHU)(6A). The enzymes described in Step 5 are useful for these conversions.
Other useful
enzymes include NAD(P)-dependent dehydrogenases in the EC 1.1.1.)0( families
and more
specifically glucuronate dehydrogenase (EC 1.1.1.19), glucuronolactone
reductase (EC
1.1.1.20). In addition,a large number 02-dependent alcohol oxidases with broad
substate range
including sugars will be useful (EC 1.1.3.XX), including sorbitol/mannitol
oxidases (EC
1.1.3.40), hexose oxidases (EC 1.1.3.5), alcohol oxidases (EC 1.1.3.13) and
vanillin oxidase
(EC 1.1.3.38). PQQ-dependent enzymes and enzymes present in oxidative bacteria
can also be
used for these conversions.
[0070] Steps 7 and 7B: Conversion of guluronic acid to D-glucaric acid (7) and
conversion of L-Iduronic acid to Idaric acid (7B). These steps can be
accomplished with
enzymes of the family of uronate dehydrogenases (EC 1.1.1.203) or the
oxidases, as described
herein. Examples of uronate dehydrogenases include SEQ ID NO: 1-6, and any one
or any
combination of them can be used to perform Steps 7 and 7B.
[0071] Step 7A: Conversion of 4-deoxy-5-erythro-hexosulose uronate (DEHU) to 3-
deoxy-D-erythro-2-hexulosaric acid (DDH). The same enzymes described in Step 5
will be
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useful for performing this conversion. Similar to Step 5, for steps 7 and 7B
enzymes are
identified having the stated activity, which are NAD(P)- or PQQ-dependent
dehydrogenases,
but others are FAD-dependent aldehyde dehydrogenases. Examples of NADP-
dependent
gluconate-5-dehydrogenases include SEQ NO: 71-72 and examples of PQQ-dependent
dehydrogenases include SEQ ID NO: 73-84, and any one or any combination of
them can be
used to perform steps 7 and7B.
[0072] Steps 8 and 8A: Conversion of D-glucaric acid to 5-dehydro-4-deoxy-
glucarate
(DDG) (Step 8) and conversion of Idaric acid to DDG (Step 8A). Enzymes of the
family of
glucarate dehydratases (EC 4.2.1.40) can be used to perform these steps.
Enzymes of this
family have been cloned and have been shown to efficiently convert glucarate
to DDG. Two
D-glucarate dehydratases (EC 4.2.1.40) were cloned as shown in the Table of
cloned glucarate
dehydratases below. Both enzymes showed very high activity for the dehydration
of Glucarate
to DDG using the semicarbazide assay, as described in Step 2.
Cloned glucarate dehydratases
Organism pSGI (Vector) Gene 110 monomongonommi
E. coli 353 (pET28) POAES2 WT
Pseudomonas (SGI) 244 #8114 WT
[0073] Step 9 and 9A: Conversion of D-glucose to a-D-gluco-hexodialdo-1,5-
pyranose (9) and conversion of D-galactose to D-galacto-hexodialdose (9A).
Oxidases such as
those of the galactose oxidase family (EC 1.1.3.9) can be used in this step.
Mutant galactose
oxidases are also engineered to have activity on glucose and have been
described (Arnold, F.H.
et al ChemBioChem, 2002, 3(2), 781). Step 9A can be performed with enzymes of
the class
EC 1.1.3.9.
[0074] Step 10: Conversion of a-D-gluco-hexodialdo-1,5-pyranose to a-D-
glucopyranuronic acid (step 10) and D-galacto-hexodialdose to galacturonate
(10A). This step
can be performed using an enzyme of the family of aldehyde dehydrogenases.
Also an enzyme
identified from those of Step 5 will be useful for both of these conversions.
[0075] Step 11 and 11A: Conversion of a-D-glucopyranuronic acid to glucuronic
acid 1,5-lactone. Aldehyde dehydrogenases and oxidases as described in Step 5
will be useful
in performing this step. Uronate dehydrogenases described in Steps 7 and 7B
can also be
useful in performing this step. Step-11A is the conversion of galacturonate to
galactarate. The
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uronate dehydrogenase (EC 1.1.1.203), for example those described in Steps 7
and 7B, will be
useful in performing this step.
[0076] Step 12: Conversion of fructose to glucose. Glucose and fructose
isomerases
(EC 5.3.1.5) will be useful in performing this step.
[0077] Step 13: Conversion of galactarate to 5-dehydro-4-deoxy-D-glucarate
(DDG).
Enzymes of the family of galactarate dehydrogenases (EC 4.2.1.42) can be used
to perform this
step, and additional enzymes can be engineered for performing this step.
[0078] Step 14: Conversion of gluconate to 5-ketogluconate (5-KGA). A number
of
enzymes of the family of NAD(P)- dependent dehydrogenases (EC1.1.1.69) have
been cloned
and shown to have activity for the oxidation of gluconate or the reduction of
5KGA. For
example, the NADPH-dependent gluconate 5-dehydrogenase from Gluconobacter
(Expasy
P50199) was synthesized for optimal expression in E. coli as shown herein and
was cloned in
pET24 (pSGI-383). The enzyme was expressed and shown to have the required
activities.
Additional enzymes useful for performing this step include those of the family
of PQQ-
dependent enzymes present in Gluconobacter (Peters, B. et al. Appl. Microbiol
Biotechnol.,
(2013), 97, 6397), as well as the enzymes described in Step 6. Enzymes from
these families
can also be used to synthesize 5KGA from gluconate.
[0079] Step 15: Conversion of 5-KGA to L-Iduronic acid. This step can be
performed with various enzymes from different isomerase families, as further
described in
Example 4. Examples include isomerases of SEQ ID NOs: 7-19 or a homolog having
at least
70% sequence identity to an isomerase of SEQ ID NOs: 7-19; or by an isomerase
encoded by a
nucleic acid of SEQ ID NOs: 20-32 or a homolog of any of them.
[0080] Step 16: Conversion of 5-KGA to (45)-4,6-dihydroxy 2,5-diketo hexanoate
(2,5-DDH). This dehydration can be performed with enzymes in the gluconate
dehydratase
family (EC 4.2.3.39), such as those described in Example 5 or Step 17.
Examples of gluconate
dehydratases that can be used for Step 16 include SEQ ID NOs 33-35 (encoded by
SEQ ID
NOs: 36-38, and any one or any combination of them can be used to perform Step
16, or
homologs thereof
[0081] Step 17 and 17A: L-Iduronate to 4-deoxy-5-threo-hexosulose uronate
(DTHU) and Guluronate to 4-deoxy-erythro-5-hexosulose uronate (DEHU).
[0082] Enzymes of the family of dehydratases are identified that can be used
in the
performance of this step. Enzymes from the families of gluconate or glucarate
dehydratases
will have the desired activity for performing these steps. Furthermore, many
dehydratases of
the family (EC 4.2.1.X) will be useful in the performance of these steps. In
particular,
19
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enzymes that dehydrate 1,2-dyhydroxy acids to selectively produce 2-keto-acids
will be
useful, such as enzymes of the families: EC 4.2.1.6 (galactonate dehydratase)
, EC 4.2.1.8
(mannonate dehydratase), EC 4.2.1.25 (arabonate dehydratase), EC 4.2.1.39
(gluconate
dehydratase), EC 4.2.1.40 (glucarate dehydratase), EC 4.2.1.67 (fuconate
dehydratase), EC
4.2.1.82 (xylonate dehydratase), EC 4.2.1.90 (rhamnonate dehydratase) and
dihydroxy acid
dehydratases (4.2.1.9). Since known enzyme selectivity is the production of an
alpha-keto acid
the identified enzymes will produce DEHU and DTHU, respectively, as the
reaction
productsStep 19: Conversion of 1,5-gluconolactone to guluronic acid lactone.
This step can be
performed by enzymes of the family of alditol oxidases (EC 1.1.3.41) or the
enzymes
described in Step 6. Examples of alditol oxidases that can be used for Step 19
include SEQ ID
NOs 39-54 or a homolog of any of them, or by an alditol oxidase encoded by a
nucleic acid of
SEQ ID NOs: 47-54 or a homolog of any of them; and any one or any combination
of them can
be used to perform Step 19. Methods of Converting DDG to FDCA and of making
esterified
DDG and FDCAThe present invention also provides novel methods of converting
DDG to
FDCA and FDCA esters. Esters of FDCA include diethyl esters, dibutyl esters,
and other
esters. The methods involve converting DDG into a DDG ester by contacting DDG
with an
alcohol, an inorganic acid, and optionally a co-solvent to produce a
derivative of DDG. The
alcohol can be methanol, ethanol, propanol, isopropanol, butanol, isobutanol,
pentanol,
hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol,
tridecanol, tetradecanol,
pentadecanol, hexadecanol, heptadecanol, octadecanol, nonadecanol, eicosanol,
dimethyl
sulfoxide, dimethylformamide, polyethylene glycol, methyl isobutyl ketone, or
any Cl -C20
alcohol. The inorganic acid can be sulfuric acid, phosphoric acid, perchloric
acid, nitric acid,
hydrochloric acid, hydrofluoric acid, hydroboromic acid and hydriodic acid.
The co-solvent
can be any of or any mixture of THF, acetone, acetonitrile, an ether, butyl
acetate, an dioxane,
chloroform, methylene chloride, 1,2-dichloroethane, a hexane, toluene, and a
xylene. Any
combination of the alcohols, inorganic acids, and co-solvents can be utilized
in the reactions.
The esterified DDG can then be converted into esterified FDCA, for example by
contacting it
with an acid catalyst.
DDG Purification
[0083] DDG purification for dehydration or esterification was performed by
acidifying the DDG, e.g., by lowering the pH of the reaction with the addition
of conc HC1 to
pH ¨2.5. At this pH proteins and any residual glucarate precipitate are
removed by filtration
and the mixture is lyophilized to give a white powder consisting of DDG and
the reaction salts.
The mixture can be lyophilized at neutral pH after the enzymes have been
removed by
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filtration. Without further purification the DDG can then be dehydrated to
give 2,5-FDCA, or
be esterified to dibutyl-DDG (or di-ethyl DDG) prior to dehydration. One or
more steps of
purifying or esterifying DDG can be added to any of the methods and pathways
disclosed
herein that produce DDG. Other methods for purifying DDG from the aqueous
mixture can
also be used. These include separations using membranes or ion exchange resins
that capture
salts or DDG etc.
[0084] The invention therefore provides a method of purifying DDG that
involves
acidifying DDG in a solution, filtering the solution through a filter
membrane, and removing
water from the solution (e.g., by lyophilization ro spray drying). The
solution with the DDG
can be acidified to a pH of 2.5-3.5 or pH of 3.0-4.0 or pH of 3.5-4.5 or pH of
4.0-5.0 or pH of
4.5-5.5 or pH of 5.0-6.0 or pH of 5.5-6.5 or pH of 6.0-7.0 or pH of 6.5-7.5 or
pH of 7.0-8.0 or
pH of 7.5-8.5 or pH of about 8. The amount of water removed can be greater
than 80% or
greater than 85% or greater than 87% of the water or greater than 90% of the
water or greater
than 95% of the water or greater than 97% or greater than 98% or greater than
99% of the
water from the solvent comprising the DDG. Yields of greater than 25% or 30%
or 35% or
40% or 45% molar can be obtained. In one embodiment the method does not
involve a step of
ion exchange chromatography.
Methods for synthesizing FDCA and FDCA Derivatives
[0085] The invention also provides various methods of synthesizing FDCA. One
method for synthesizing FDCA involves contacting DDG with an alcohol, an
inorganic acid at
a high temperature to form FDCA. The alcohol can be any alcohol (e.g., any of
those
described above), and examples include (but are not limited to) methanol,
ethanol, propanol,
and butanol. Diols can also be used. The high temperature can be a temperature
greater than
70 C or greater than 80 C or greater than 90 C or greater than 100 C or
greater than 110 C
or greater than 120 C or greater than 130 C or greater than 140 C or
greater than 150 C to
form FDCA. Reaction yields of greater than 20% or greater than 30% or greater
than 35% or
greater than 40% can be achieved.
[0086] The invention also provides methods for synthesizing derivatives of
FDCA.
The methods involve contacting a derivative of DDG with an inorganic acid to
produce a
derivative of FDCA. The inorganic acid can be, for example, sulfuric acid, or
any inorganic
acid such as those described above. Optionally, the derivative of DDG can be
purified prior to
contacting it with the second inorganic acid. Non-limiting examples of
derivatives of DDG or
FDCA include, but are not limited to, methyl DDG, ethyl DDG, propyl DDG, butyl
DDG,
isobutyl DDG, di-methyl DDG, di-ethyl DDG, di-propyl DDG, di-butyl DDG. The
derivative
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of FDCA produced can be, but is not limited to, methyl FDCA, ethyl FDCA,
propyl FDCA,
butyl FDCA, di-methyl FDCA, di-ethyl FDCA, di-propyl FDCA, di-butyl FDCA, and
isobutyl
FDCA. The derivate of FDCA produced corresponds to the derivative of DDG used
in the
method. The derivative of FDCA can then be de-esterified to produce FDCA. The
method can
also be conducted in the gas phase, e.g., using the parameters described
below.
[0087] Another method for synthesizing FDCA or derivatives of FDCA involves
contacting DDG or derivatives of DDG (any described herein) with an inorganic
acid in a gas
phase, which can be done with a short residence time, e.g., of less than 10
seconds or less than
8 seconds, or less than 6 seconds or less than 5 seconds or less than 4
seconds or less than 3
seconds or less than 2 seconds or less than 1 second. The residence time
refers to the time that
the sample is present in the reaction zone of the high temperature flow
through reactor. The
method can also be conducted at high temperatures, for example at temperatures
greater than
150 C, greater than 200 C, greater than 250 C, greater than 300 C or
greater than 350 C.
Yields of greater than 25% or greater than 30% or greater than 40% or greater
than 45% or
greater than 50% molar are obtainable. Another method for synthesizing FDCA
involves
contacting DDG with an inorganic acid at a temperature in excess of 80 C or
90 C or 100 C
or 110 C or 120 C. Another method for synthesizing FDCA involves contacting
DDG with
an inorganic acid under anhydrous reaction conditions. In various embodiments
the anhydrous
conditions can be established by lyophilizing the DDG in any method of
synthesizing FDCA
disclosed herein so that the DDG contains less than 10% or less than 9% or
less than 8% or
less than 7% or less than 6% or less than 5% or less than 4% or less than 3%
water or less than
2% water, by weight.
[0088] The methods of the invention for synthesizing FDCA and its derivatives
as
described herein provide a significantly higher yield than has been available.
In different
embodiments molar yields of FDCA (v. DDG) can be obtained of greater than 10%
or greater
than 15% or greater than 20% or greater than 25% or greater than 30% or
greater than 35% or
greater than 40% or greater than 45% or greater than 50% or greater than 60%
or greater than
65% or from about 40% to about70%, or from about 45% to about 65%, or from
about 50% to
about 60%.
EXAMPLES
Example 1 ¨ Step 2, Gluconic Acid to 3-dehydro-gluconic acid (DHG)
[0089] Enzymes with natural activity for the dehydration of gluconate are
useful in
the invention (EC 4.2.1.39). Three enzymes from this family were cloned as
shown in Table 1.
Enzyme pSGI-365 was cloned and shown to be a dehydratase with broad substrate
range
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having strong activity for the dehydration of gluconate (Kim, S. Lee, S.B.
Biotechnol.
Bioprocess Eng. 2008, 13, 436).
Table 1: Enzymes used in this experiment and identity homology. All expressed
in P.
fluorescens
FOOPOilithiMitE .itiSGIF(V.kettik.)MMMcatettvm000007W17SYNMEijitkggtittHiAtnM
Achromobacter 365 (pRANGER) E3HJU7 Syn Plluorescens
Achromobacter 359 (pRANGER) #0385 wt Plluorescens
Acinetobacter 360 (pRANGER) #0336 wt Plluorescens
FggggggggggggggggggggggggggMA5WAdht.61tftib= .365E3tETTITO
-11S,1q140-viAolootot000tiomomm 78 79
1/$0143S9iA0Ittontabotitr(SG1)mgi 95
[0090] Proteins 359, 360, and 365 (SEQ ID NOs 33-35, respectively) showed 2-5
mole/min per mg of crude enzyme lysate activity for the synthesis of
dehydration of
gluconate (gel not shown). pSGI-359 was isolated by precipitation with
ammonium sulfate
and re-dissolving in buffer and assayed by the semicarbazide assay. Activities
of 46.2 U/mL
or 5.3 U/mg (1 unit=gmole/min) for the dehydration of gluconate were
calculated from
semicarbazide assay plots. Reaction buffer (93 mL) containing Kpi 10 mM pH 8.0
with 2 mM
MgC12 and 3.5 gr (0.016 mole) of sodium gluconate was mixed with 7 mL of the
previous
gluconate dehydratase solution. The reaction was incubated at 45 C for 16 h
before one
aliquot was analyzed by HPLC-MS (Figure 4). As shown in Figure 4 one new major
product
with the molecular weight of DHG was produced. The product was also shown to
have
activity with DHG dehydratases.
[0091] All proteins were cloned on the pRANGERTM (Lucigen, Middleton, WI)
expression vector and were expressed in a Pseudomonas fluorecens strain.
pRANGERTM is a
broad host commercially available plasmid vector containing the pBBR1
replicon, Kanamycin
resistance and an pBAD promoter for inducible expression of genes. For the
enzyme assay a
modification of the semicarbazide assay for the quantification of alpha keto
acid was used to
calculate the activity of each enzyme (Kim, S.; Lee, S.B. Biochem J. 2005,
387, 271). SEQ ID
NOs: 30-32 and 33-35 show the amino acid and nucleotide sequences,
respectively, of the
gluconate dehydratases #0385, #0336, and E3HJU7.
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Example 2 ¨ Step 3 - 3-dehydro-gluconic acid (DHG) to (45)-4,6-dihydroxy 2,5-
diketo
hexanoate (2,5-DDH)
[0092] Enzymes of the family (EC 1.1.1.127) can be used to perform this step.
Two
examples are 2-dehydro-3-deoxy-D-gluconate 5-dehydrogenase and DHG
dehydrogenases.
Five enzymes from this family were cloned as shown in Table 2 below. pRANGERTM
vector
was used in every case.
Table 2: Cloned of DHG oxidoreductase (or 2-dehydro-3-deoxy-D-gluconate 5-
dehydrogenase)
i',-:mgooismmagMMMM5AJSGuivti.ttptyMMGto#4DMMOitVT/$YIVZMEKttittt$Ii)tattittgq
Agrobacterium sp (SGI) 374 #9041 WT P.
fluorescens
Agrobacterium tumefaciens
375 #8939 WT P.
fluorescens
(SGI)
E. coli 376 P37769 WT P.
fluorescens
Sphingomonas (SGI) 395 #5112 WT P.
fluorescens
Hoeflea phototrophica
396 #7103 WT P.
fluorescens
(SGI)
[0093] The product prepared from the dehydration of gluconate in Step 2 was
used as
substrate for assaying the lysates of Table 2. As shown in the following Table
3, enzymes
were identified showing activity for the oxidation of DHG in assays measuring
NADH
formation (absorbance increase at 340 nm).
Table 3: Activity calculations for oxidation of DHG to 2,5-DDH using DHG
oxidoreductase.
A unit = mole/min of NADH
giiiiiiiiiMininininwmgmoggagtfAng-04WitiNLIMIGYENZ
maggEm
MggggggggnEvit-q5m-p114.--411knatiRtGympli%5M
L=mmmmmmw:,:mmmmm-,:mmmm,mmmmmmmnm:,:mmmm
pSGI_395 0.012 0.070 (0.02) 0.120
pSGI_396 0.033 0.139 (0.018) 0.418
pSGI_374 0.007 0.043 (0.012) 0.091
pSGI 376 0.007 0.121 (0.01) 1.610
[0094] Further verification of the formation of 2,5-DDH by these enzymes was
shown in Step 16 where the reduction of 2,5-DDH (made from the dehydration of
5KGA) with
pSGI-395 at acidic pH was shown.
Example 3 ¨ Steps 7 and 7B - Conversion of guluronic acid to D-glucaric acid
(7) and
conversion of L-Iduronic acid to Idaric acid (7B).
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[0095] To demonstrate Steps 7 and 7B the following study was performed.
Uronate
dehydrogenases (EC 1.1.1.203) are enzymes that oxidize glucuronic and
galacturonic acid.
Three enzymes with sequence similarity to the known uronate dehydrogenase
(Expasy:
Q7CRQ0; Prather, K.J, et al., J. Bacteriol. 2009, 191, 1565) were cloned from
bacterial strains
as shown in Tables 4 & 5.
Table 4 ¨ Cloned Uronate Dehydrogenases
0t0p4MginiQggaggi iir$posslottna
Agrobacterium #474 #8807 BL21DE3
Rhizobium #475 #8958 BL21DE3
Ps eudomonas #476 #1770 BL21DE3
Table 5 ¨ Sequence Identity
wcwiA
474 Agrobactenum 73 49 90
M41SLfthiiiibiiiiifMMMMMA 51 74
F-4176Pkudornonasaomm 50
[0096] Each protein was expressed with a His tag from pET28 and was purified
prior
to their screening. Protein gels of the crude lysates and purified enzymes are
shown in the gel
of Fig. 1. After purification all enzymes were tested for activity against
glucuronate, as well as
against guluronate and iduronate. Kinetic measurements at different substrate
concentrations
were performed and the calculated activities and Km values for each enzyme are
shown in
Table 6. All enzymes showed good activity for glucuronate, and also for L-
iduronate and
guluronate.
Table 6: Activity and Km value for purified uronate dehydrogenases.
Vmax (tM/minJmg), and Kin (mM)
x00.01
mmm:nmGitieltitOltitteNM=MidttrottAtemo monommmom
iiEggggggnM OggggggggggggggrgggggggggggA agCOR9Pjy):M
474 128.2 ; 0.37 0.96 ; 29.8 0.017
475 47.4 ; 0.22 0.59 ; 42.1 0.016
476 90.9 ; 0.34 1.36 ; 29.6 0.014
[0097] Each plasmid shown in Table 4 was transformed in BL21DE3 E. coli cells.
Clarified lysates were mixed with equal volume of (25 mL) of equilibration
buffer and purified
on an Ni NTA column. Activity of each purified enzyme was measured in by
mixing 0.050
mL of various dilutions of each purified enzyme with 0.95 mL of reaction
buffer (100 mM
TrisHC1, pH 8.0, 50 mM NaC1, 0.75 mM NAD+). The reaction progress was measured
by
monitoring of the formation of NADH at 340 nm. Figures 6a and 6b provide
Lineweaver-Burk
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WO 2015/143381 PCT/US2015/021848
plots for the oxidation of glucuronate and iduronate, with all three enzymes
shown in Figure 6.
Clear positive slopes were obtained with all enzymes giving the activities
shown in the table
above. Protein sequences of the uronate dehydrogenases are shown as SEQ ID
NOs: 1-3 and
the genes as SEQ ID NO: 4-6.
[0098] Pyrroloquinoline (PQQ) dependent aldehyde dehydrogenases also showed
good
activity for the oxidation of both guluronate and iduronate. These are soluble
periplasmic
enzymes that were expressed in the E. coli cytosol after their periplasmic
target sequence was
removed. The activities of crude lysates in units (gmole/min) per milligram of
total lysate
protein are shown in the following Table 6A. The actual activity of each
enzyme is at least 2-
5x higher if purified (see expression in Figure 3).
Table 6A: Activities of PQQ-dependent dehydrogenases with iduronate and
guluronate (Unit=
gmole/min)
Enzyme Iduronate U/mg Guluronate U/mg
P75804 (SEQ ID NO: 73) 8.7 3.2
9522 (SEQ ID NO: 74) 7.3 6.1
6926 (SEQ ID NO: 75) 9.2 4.1
7510 (SEQ ID NO: 76) 7.3 3.7
7215 (SEQ ID NO: 77) 14.2 8.3
8386 (SEQ ID NO: 78) 4.3 1.5
[0099] The activities shown on Table 6A were measured using an artificial
electron
acceptor DCPIP (2,6-dichloroindophenol) according to the following protocol:
In 0.95 mL of
20 mM Triethanol amine (pH 8.0) containing 0.2 mM DCPIP, 0.2 mM PMS (phnazine
ethosulffate) and substrate (10-40 mM), 0.050 mL of enzyme (as crude lysate or
10-100x
diluted with buffer) is added and the reaction progress is followed by the
change of DCPIP
absorbance at 600 nm. Because in their natural state these enzymes are
transferring electrons to
other proteins or cofactors in the membrane electron transport chain, the in
vitro activity is
measured using artificial electron acceptors with DCPIP being the most common.
[00100] The enzymes on Table 6A were active against a number of other
aldehydes
including butyraldehyde, butyraldehyde and glycerol (but not glucose).
Therefore, these
enzymes will oxidize the aldehyde group of iduronate and guluronate to give
iduronic and
glucaric acid respectively. In order to confirm this selectivity, two of these
enzymes, #403 and
#412, were expressed in the periplasm of E. coli by fusing them with the
periplasmic target
sequence of #403 (a native E. coli enzyme). Both proteins were expressed in
the periplasm but
in lower levels compared to the cytosol . The previous recombinant cells
oxidized
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benzaldehyde to benzoic acid in good yields and in lower yields produced
glucaric and idaric
acid from guluronate and iduronate.
Example 4 - Step-15: Conversion of 5-ketogluconate (5-KGA) to L-Iduronic acid
(15) or
guluronic acid (15A).
[00101] This example illustrates the identification of an enzyme capable of
isomerizing
5-KGA to iduronic acid (Step 15) or guluronic acid (Step 15A). Thirteen
enzymes from three
different isomerase families were cloned as shown in Table 7, while their %
sequence identity
is shown in Table 8.
Table 7: Isomerases cloned
Ugggggggn Eggggggggggggggggggn EgggggggggggggnArettetype&b.MMEM=::*
Expasy
Rhizobium 433 #8938 WT
5.3.1.17 E. coli 434 Q46938 (Expasy) WT
5.3.1.17 Rhizobium 435 #3891 WT
5.3.1.17 Pannonibacter 436 #7102 WT
5.3.1.n1 Lactobacillus 458 A5YBJ4 (Expasy) SYN
5.3.1.n1 Acidophilum 440 F0J748 (Expasy) SYN
5.3.1.n1 Bacillus 437 #9209 WT
5.3.1.n1 Ochrobactrum 438 #9732 WT
5.3.1.n1 Halomonas 439 #7403 WT
5.3.1.12 Sphingobacteria 478 #1874 WT
5.3.1.12 Thermotoga 479 Q9WXR9 SYN
5.3.1.12 Bacillus 480 Q9KFI6 SYN
5.3.1.12 Bacillus 481 034808 SYN
Table 8: % Identities of isomerases
433 5.3.1.17 65 44 43 16 13 18 11 14 6 11 11 7
436 5.3.1.17 45 46 18 14 15 12 13 5 10 11
7
-:43-4V 5.3.1.17 46 17 10 15 10 13 6 10 12 7
-:43 5.3.1.17 18 16 18 14 16 9 11 13 7
5.3.1.n1 37 57 41 44 6 7 9 5
5.3.1.n1 40 67 50 6 6 6 5
437 5.3.1.n1 46 51 8 7 10 6
438 5.3.1.n1 52 5 5 6 4
439 5.3.1.n1 6 7 8 5
481 5.3.1.12 7 36 54
5.3.1.12 7 7
479 5.3.1.12 37
5.3.1.12
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[00102] As shown in Table 8, enzymes with medium homology (underlined) within
each family were selected for cloning. The data demonstrated that enzymes from
all families
showed activity for the isomerization of 5-KGA giving L-iduronate as the main
product. Two
enzymes from the 5.3.1.17 family (433 & 434) were also used in the example
showing the
formation of DDG from 5-ketogluconate (5KGA).
[00103] Activity for the isomerization of 5KGA and iduronate using enzymes
from
Table 7 was measured using an enzymatic method that detected the formation of
products by
their activity against two different enzymes. For example, isomerization of
5KGA was
detected by measuring the activity of the product iduronate using uronate
dehydrogenase
(pSGI-476). Isomerization of iduronate was detected by measuring the activity
5KGA
reductase (pSGI-383, EC 1.1.1.69) of the product 5KGA. Presence of the
products was also
detected by GC-MS.
[00104] Enzymes from all families showed varying activity for the
isomerization of
5KGA and iduronate. Two enzymes from EC 5.3.1.12 were used in a cell free
reaction to
isomerize 5KGA and ultimately produce DDG as described in the example. The
enzymes
were purified and showed a single band by gel electrophoresis. The purified
isomerases were
used in reactions using lysate and buffer containing 5KGA or Iduronate.
Product formation
was demonstrating using both HPLC and the previously described enzymatic
methods. Results
for 17h of incubation using both HPLC and enzyme assays are shown in Figure
7a. All
enzymes showed good activity for the isomerization of both 5KGA and iduronate.
Yields for
iduronate isomerization by pSGI433, pSGI 434, pSGI 435, and p SGI 436 were
56%, 48%
42%, (436 not measured), respectively when measured enzymatically and 78.8%,
78.5%,
73.3% and 76.6%, respectively when measured by HPLC assay. Yields after 16h
for 5KGA
isomerization by the same enzymes were 18%, 17%, and 19% respectively (436 not
measured)
when measured by enzymatic assay, and 16.6%, 17.8%, 16.3%, and 16.9%,
respectively, when
measured by HPLC assay.
EC 5.3.1.12 enzymes
[00105] Enzymes from the EC 5.3.1.12 family (glucuronate isomerases) were also
purified by gel electrophoresis, isolated, and used to prepare reactions by
mixing with buffer
(50 mM HEPES, 1 mM ZnC12, pH 8.0) that contained 5 mM of 5KGA or Iduronate.
The
reactions were incubated at 30 C and analyzed for product formation using
both HPLC and
enzymatic methods. Results are shown in Figure 7b.
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5.3.1.17 Enzymes
[00106] Enzymes pSGI-478 and pSGI-479 (5-dehydro-4-deoxy-D-glucuronate
isomerases) showed isomerization activity for both 5KGA and iduronate. This
activity was
also confirmed with the enzymatic assays as above. Yields for isomerization of
iduronate by
pSGI-478 and -479 were 50% and 37%, respectively, when measured enzymatically,
and 20%
and 18% when measured by HPLC. Yields for 5KGA isomerization were 23% and 26%,
respectively, when measured enzymatically, and 24% and 16%, respectively when
measured
by HPLC. Results are shown in Figure 7a.
5.3.1.n1 Enzymes
[00107] Enzymes in this family were purified by gel electrophoresis. Product
formation was measured using enzymatic assays as described above and the
results are shown
in Figure 8. All enzymes cloned in this family were shown to have activity for
the
isomerization of 5KGA and iduronate.
[00108] In each case plasmids were transformed in BL21DE3 and proteins
purified on
a Ni NTA column.
Example 5: Step 16 ¨ 5-keto-gluconate (5KGA) to (4S)-4,6-dihydroxy 2,5-diketo
hexanoate (2,5-DDH)
[00109] The three gluconate dehydratases described in Step 2 (Example 1) were
expressed as described in Example 1, along with a purified glucarate
dehydratase from Step 8.
Enzymatic reactions for activity were performed and HPLC-MS analysis showed
the formation
of 2,5-DDH (Figure 9), which was also confirmed by the fact that formation of
the new
product was accompanied by the reduction of 5-KGA only in the samples
containing gluconate
dehydratases, as well as by enzymatic assays with DHG dehydratase (pSGI-395).
Good slopes
at 340 nm indicating large enzyme activity were obtained when NADH, pSGI-395
lysate and
aliquots of the previous reactions were mixed (data not shown). This result in
combination
with the HPLC analysis prove that the gluconate dehydratases examined
dehydrate 5KGA to
2,5-DDH.
Example 6: Step 19 ¨ Conversion of 1,5-gluconolactone to guluronic acid 6-
lactone.
[00110] 1,5-gluconolactone oxidation is a side activity of enzymes from the
alditol
oxidases (EC 1.1.3.41) family. These enzymes oxidize various alditols such as
sorbitol,
xylitol, glycerol and others. Enzymes were identified having activity for the
oxidation of 1,5-
gluconolacone, as shown in Table 6 below.
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Table 6. Alditol oxidases with activity on 1,5-gluconolactone.
mogggggggggggggggggggg MgggggggOggggggggggg4.61iitiititilattbiiemmummumuniii
SorbitolReaction Setup
Enzyme Enzyme 5ourc
signisigiiiigmognmog
monomm mmonomonomommogn Enzyme Substrate
Ngggggggn Eggggggggggggggggggggn MMMMMMgggggggnMggganom-MMNTdltmg mg/mM
ri
AO#13 Terriglobuds roseus 0.23 0.02 5.3 15 / 85
7%
AO#22 Granulicella mallensis 0.27 0.015 7.6 15 / 85
9%
AO#28 Streptomyces acidiscabies 1.30 0.010 15 15 / 85
8%
AO#36 Actinomycetales (SGI) 1.83 0.102 25 90 / 35
46%
AO#51 Frankia sp 0.59 0.019 NT NT
NT
AO#57 Propionibacteriacaeoe (SGI) 1.47 0.051 40
70 / 57 6%
AO#76 Streptomyces sp. 1.45 0.045 8.2 15 / 85
23%
AO#251* Paenibacillus sp. 0.47 0.003 24 15 8.5 -
2%
*crude lysate
[00111] Reactions were prepared using lysates of all the purified enzymes
shown on
Table 6. Reactions were prepared in 50 mM K-phosphate buffer, pH 7.0 with 0.5
mg/mL
catalase and incubated at 30 C. A new product was observed by HPLC-MS
analysis showing
the same retention time as guluronate after comparison with authentic
standards (Figure 10).
This was confirmed by GC-MS, where the product also had the same MS
fingerprint as
guluronate. It is therefore clear that all the alditol oxidases described in
the Table oxidize the
6-0H of 1,5-gluconolactone to produce the guluronic acid lactone. All alditol
oxidases were
cloned in pET28a with a HisTag and were expressed in BL21DE3 and purified on a
Ni NTA
column.
Example 7 ¨ Synthesis of FDCA and Other Intermediates
[00112] Purified DDG mono potassium salt was used for the dehydration to 2,5-
FDCA. Sulfuric acid was added to DDG and the reaction stirred at 60 C. The in
situ yield
was calculated (by HPLC-MS) to be ¨24% and ¨27%.
[00113] The reaction solutions were combined and then diluted by pouring into
ice (to
neutralize the heat). Approximately equivalent volume of THF was added, and
the solution
transferred to a separation funnel. Sodium chloride salt was added until
separation was
achieved. The solution was agitated between additions for best possible
dissolution. The
aqueous layer was removed, and the THF layer washed 3x more with sat. NaCL
solution.
Sodium sulfate was added and the solution left sitting overnight. Two layers
formed again
overnight. The aqueous layer was discarded and then silica gel was added to
the solution. It
was then concentrated down to solids via rotovap. The solids were loaded into
a silica flash
column and then separated via chromatographically. The fraction was
concentrated and dried.
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The isolated yield was 173.9mg. Corrected yield: 24.9%. 1H and 13C NMR and
HPLC-MS
analysis confirmed the product
Dehydration of DDG Dibuty1-2,5-FDCA in BuOH/H2SO4
[00114] Dehydration of un-derivitized lyophilized DDG containing the
dehydration
salts in BuOH was done using a Dean-Stark apparatus. Under these conditions,
DDG was
added to BuOH, and then H2504 was added and the reaction heated at 140 C.
After stirring
for 4 h HPLC-MS analysis shows the disappearance of DDG and the formation of
dibuty1-2,5-
FDCA. The in situ yield was calculated (by HPLC-MS) to be 36.5%.
[00115] The mixture was extracted with water, 1% NaOH, and again with water.
Then the organic layer was concentrated to a final mass of 37.21g. A portion
of this mass
(3.4423g) was removed and 0.34 g of dibutyl-2,5-FDCA was purified using HPLC.
Extrapolating the yield of the isolated product to the total amount of
compound isolated from
the reaction (37.21g) and taking into account the amount of salts present in
the original DDG
(-60% pure by weight) the reaction yield was calculated to be 42%. 1H and 13C
NMR and
HPLC-MS analysis confirmed the product
Synthesis of dibutyl DDG
[00116] In another aspect the invention provides a method for synthesizing a
derivative of DDG. The method involves contacting DDG with an alcohol, an
inorganic acid,
and optionally a co-solvent to produce a derivative of DDG. Optionally the
derivative of DDG
can be purified. The reaction can have a yield of the derivative of DDG of at
least 10% molar
yield or at least 15% molar yield or at least 20% molar yield or at least 25%
or at least 30% or
at least 35% molar yield or at least 40% molar yield. The inorganic acid can
be sulfuric acid
and the alcohol can be methanol, ethanol, propanol, butanol, isobutanol, or
any C 1 -C20
alcohol. In various embodiments the co-solvent can be any of THF, acetone,
acetonitrile, an
ether, butyl acetate, an dioxane, chloroform, methylene chloride, 1,2-
dichloroethane, a hexane,
toluene, and a xylene. When the alcohol is ethanol the DDG derivative will be
DDG mono-
ethyl ester and/or DDG diethyl ester. When the alcohol is butanol the DDG
derivative will be
DDG mono-butyl ester and/or DDG dibutyl ester.
[00117] DDG mono-potassium salt was used for derivatization according to the
following protocol. In a 1L Morton type indented reaction vessel equipped with
a mechanical
stirrer and heating mantle was charged with 60:40 DDG:KC1 (31.2 mmol), BuOH,
and
heptane. In a separate vial, sulfuric acid was added to water, and allowed to
cool after
dissolution. The solution was then added to the flask. The solution was kept
at 30 C.
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[00118] The precipitate was filtered off concentrated. The remaining gel was
dissolved in Et0Ac, and then TLC plates were spotted with the solutions and
the plates were
sprayed with a phosphomolybdic acid mixture, and then heated to at least 150 C
on a hot plate
to identify the DDG-DBE fraction. Isolated yield: 4.62 g (15.2 mmol, 47%
yield), > 98%
purity. 1H and 13C NMR and HPLC-MS analysis confirmed the product.
[00119] Different solvents can be used in the synthesis of DDG esters, such as
mixtures of BuOH (5%-95% v/v) with co-solvents such as THF, acetone,
acetonitrile, ethers
(dibutyl, ditheyl etc), esters such as Butyl-acetate, 1,6-dioxane, chloroform,
methylene
chloride, 1,2-dichloroethane, hexanes, toluene, and xylenes may be used as
cosolvents.
Reaction catalysts such as acids (sulfuric, hydrochloric, polyphosphoric or
immobilized acids
such as DOWEX) or bases (pyridine, ethyl-amine, diethyl-amine, boron
trifluoride) or other
catalysts commonly used for the esterification of carboxylic acids.
Dehydration of dibutyl-DDG to dibutyl-FDCA in n-BuOH/H2SO4
[00120] A stock solution of DDG-DBE (di-butyl ester) was made in butanol and
transferred to a clean, dry 100mL round-bottomed flask equipped with a stir
bar. To the flask,
25mL of conc. sulfuric acid was added. The flask was sealed and then stirred
at 60 C for 2hrs.
The in situ yield was calculated to be ¨56%. The reaction solution was
concentrated and the
residue was dissolved in MTBE and transferred to a separation funnel, and then
washed with
water. The recovered organic layer was concentrated and then separated via
HPLC for an
isolated yield: 250.7 mg (-90% purity) and 35% isolated yield (corrected for
purity). 1C and
13C NMR and HPLC-MS analysis confirmed the product.
Example 8 ¨ Cell free synthesis of DDG and FDCA and derivatives from 5-KGA
(Route 2A)
[00121] This example illustrates the enzymatic conversion of 5KGA to DDG using
purified enzymes according to Scheme 6 (a sub-Scheme of 2B), and also
illustrates the DDG
produced being dehydrated to FDCA using chemical steps. The Scheme involves
the steps of
isomerization of 5KGA (Step 15) and the subsequent oxidation to idaric acid
(Step 7B). DDG
was also dehydrated under differing chemical conditions to FDCA. The last step
(Step-8A)
was performed using glucarate dehydratase from E. coli.
[00122] Scheme 6 is illustrated in Figure 11. The scheme was performed using a
cell
free enzymatic synthesis of DDG from 5-KGA. The Scheme involves the
performance of
steps 15, 7B and 8A (see Fig. 2d). Two additional proteins were used to
complete the reaction
path, the first being NADH-oxidase (Step A) that is recycling the NAD+
cofactor in the
presence of oxygen, and catalase (Step B) that decomposes the peroxide
produced from the
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WO 2015/143381 PCT/US2015/021848
action of NADH oxidase. The enzymes are shown in the following Table 7. All
enzymes
contained a HisTag and were purified using an Ni-NTA column. Yields for this
synthesis of
[00123] DDG were calculated to be at least 88-97%.
Table 7:
STEP Enzyme EC Orqanl$m
pSGI-433
15 (DTHU_IS) 5.3.1.17 Rhizobium (SGI)
pSGI-434
15 (DTHU_IS) 5.3.1.17 E. colt
7B pSGI-476 (UroDH) 1.1.1.203 Pseudomonas (SGI)
8A pSGI-353 (GlucDH) 4.2.1.40 E. colt
pSGI-431
A (NADH_OX) 1.6.3.1 Thermus thermophidus
Catalase 1.11.1.6 Corynbacterium
[00124] 500 mL of liquid culture was purified for each isomerase for the
reaction.
Besides the enzymes shown on Table 7, each reaction contained 50 mM TrisHC1
(pH 8.0), 50
mM NaC1, 1 mM ZnC12 and 2 mM MgC12, 1 mM MnC12 and 1 mM NAD+. Reactions were
analyzed by HPLC after 16 h of incubation and Figure 12 presents the
chromatograms.
[00125] For dehydration to FDCA, the reaction mixtures of both samples were
combined and lyophilized into a white powder, which was split into two samples
and each
dissolved in AcOH with 0.25M H2SO4 or in 4.5 mL BuOH with 0.25M H2SO4. Both
reactions
were heated in sealed vials for 2-4 h at 120 C. Reaction products are shown
in Figure 13.
[00126] Samples 1 and 2 represent authentic standard and the 3h time point
from the
reaction in AcOH/ H2504, respectively. Spiking of sample 2 with sample 1 gave
a single peak
further verifying the FDCA product. Samples 1 and 3 (Figure 13) represent
authentic standard
and the 4h time point from the reaction in BuOH/ H2504, respectively. The
formation of
FDCA from the enzymatic reactions further confirms the presence of DDG in
these samples.
Example 9 ¨ Synthesis of DDG from Glucose and Gluconate
[00127] This example shows the enzymatic conversion of glucose and gluconate
to
DDG. The reaction was conducted with purified enzymes, and crude lysates as a
catalyst.
Enzymes and substrates were combined in a bio-reactor as shown in the Table
below:
kignignin
ummaa gaggmwm anggg maamaa2wgggaggn ognmaaoiinnuaaaaagnmaaaam mgmo
i
Rxn-1 Glucose 2 mg 7 mL 50 mL2 7.5 mL1 1 mL3
4 mL4 2 mg
600 mg
Rxn-2 Gluconate - 7 mL 50 mL 7.5 mL 1 mL 4 mL 2 mg
700 mg
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1. Lysate from 500 mL liquid culture of recombinant E. coli with plasmid
2. Lysate from 2L liquid culture of BL21DE3/pSGI-434
3. Purified enzyme, ¨30 Units of activity (or 3 mg of purified GlucD)
4. Lysate from 250 mL of culture
[00128] The reaction was incubated at 35 oC and dissolved oxygen and pH were
kept
at 20% and 8 respectively. Time points were analyzed by HPLC-MS and the
results are shown
in Figure 17b.
Extracted chromatograms verified the DDG mass (not shown) and
corresponding MS fragmentation. The results clearly showed production of DDG
during
incubation of the enzymes with either glucose or gluconate.
Example 10 - Construction of expression cassettes for recombinant glucarate
dehydratases
[00129] The following example describes the creation of recombinant nucleic
acid
constructs that contained coding sequence of a D-glucarate dehydratase
activity (GDH, EC
4.2.1.40) for heterologous expression in E. coli cells.
[00130] Genes encoding D-Glucarate dehydratase from E. coli (Expasy: POAES2;),
Acinetobacter ADP1 (Expasy: POAES2), as well as a proprietary Pseudomonas
bacterial strain
(#8114) were PCR-amplified from genomic DNA.
[00131] Each of the PCR-amplified genes was subsequently cloned into the
bacterial
transformation vector pET24a(+), in which the expression of each of the GDH
genes was
placed under control of a T7 promoter. The nucleotide sequences of each of the
PCR-
amplified inserts were also verified by sequencing confirmation.
Example 11 - E. coli strains expressing recombinant glucarate dehydratases.
[00132]
Each of the expression vectors constructed as described in Example 9
was introduced into NovaBlue(DE3) E. coli by heat shock-mediated
transformation. Putative
transformants were selected on LB agar supplemented with Kanamycin (50 ug/m1).
Appropriate PCR primers were used in colony-PCR assays to confirm positive
clones that
contained each of the expression vectors.
[00133]
For each expression vector, a bacterial colony was picked from
transformation plates and allowed to grow at 30 C in liquid LB media
supplemented with
Kanamycin (50 ug/m1) for two days. The culture was then transferred into vials
containing
15% glycerol and stored at -80 C as a frozen pure culture.
Example 12 - Demonstration of in vitro synthesis of DDG by using cell lysate
of recombinant
E. coli cells expressing a GDH enzyme
[00134] This Example describes how in vitro synthesis of DDG
intermediate was
achieved using recombinant glucarate dehydratase (GDH) enzymes produced in E.
coli cells.
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[00135] Preparation of cell lysates: Recombinant bacterial strains
constructed as
described previously in Example 2 were grown individually in 3 mL of liquid LB
media
supplemented with Kanamycin (50 g/ml) at 30 C on a rotating shaker with
rotation speed
pre-set at 250 rpm for 1 day. This preculture was used to inoculate 100 mL of
TB media
containing Kanamycin (5Oug/m1), followed by incubation at 30 C on a rotating
shaker pre-set
at 250 rpm for 2-3 hour until early log phase (OD600-0.5-0.6) before isopropyl
D-1
thiogalactopyranoside (IPTG; 0.25 mM final concentration) was added to induce
protein
expression. Cells were allowed to grow for another 18 hours at 30 C before
they were
harvested by centrifugation, resuspended in 15 mL of lysis buffer (10 mM
phosphate buffer,
pH 7.8, 2 mM MgC12) and were lysed by sonication. The production of
recombinant enzymes
in E. coli cells was quantified using standard pre-cast SDS-PAGE gels system
(BioRad), and
specific activity was measured according to a procedure described by Gulick et
at.
(Biochemistry 39, 4590-4602, 2000). Crude cell lysates or purified enzymes
(using the
HisTag) were then tested for the ability to convert gram amounts of glucarate
to DDG as
described in greater detail below.
Enzymatic Dehydration of Glucarate
[00136] A large scale oxidation of glucarate using glucarate
dehydratase was
prepared. 350 mL of water 25 g of glucaric acid sodium salt (0.1 mole) and 4.5
gr of KOH
(0.8 mole) were mixed in an Erlenmyer flask. Residual solid glucarate was
dissolved by the
slow addition of 5M KOH solution (-3 mL) and the pH was adjusted to 7.4. In
this solution
100 mg of purified glucarate dehydratase and 2 mM MgC12 were added, and the
mixture was
placed in an orbital shaker at 30 C for 20 h. The next day the precipitate is
removed by
filtration. The pH of the reaction was essentially unchanged. Analysis of the
reaction revealed
the presence of only DDG in the solution, indicating >95% yield.
Purification of DDG product from enzymatic reactions:
[00137] DDG produced via enzymatic dehydration was purified by using either of
the
two following techniques. The enzymatic dehydration reactions were acidified
to pH-3.0 with
6M HC1, filtered to eliminate precipitate, and subsequently lyophilized to
produce a white
powder consisting of DDG and salts. The same DDG purity (but lower amount of
salts) can be
obtained if the reaction was filtered through a 10 KDa membrane to remove
proteins and then
lyophilized. Without any further purification both previous lyophilized
powders can be
dehydrated to FDCA (or its esters) or can be esterified to dibutyl DDG as
shown in other
examples of this application.
CA 02943348 2016-09-20
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[00138] Results of HPLC-MS analyses indicated that DDG product
constituted
at least 95% of the total products in the samples obtained from either of the
two purification
techniques.
Example 13 - Demonstration of in vitro synthesis of FDCA from DDG in one-step
chemical
reaction
[00139] Applicants have discovered that the synthesis of FDCA (i.e. the free
acid
form) could be achieved by a chemical conversion of DDG to FDCA in the
presence of H2SO4=
The reaction was performed as follows. Approximately 20 mg of DDG acid (crude
lyophilized
powder with salts previously purified as described in Example 3) and 0.25 M of
H2SO4 were
added into an air tight sealed tube containing 1 mL of water and 1 mL of DMSO.
The DDG
was found completely dissolved in this solution. The reaction was stirred at
105 C for 18
hours. Results of an HPLC-MS analysis performed on a crude reaction sample
indicated the
formation of FDCA free acid (FDCA: 2,5-furan dicarboxylic acid) as the major
product, as
well as insignificant amounts of some other unidentified byproducts. As a
control in HPLC-
MS analysis, a commercial FDCA was analyzed in the same conditions.
Example 14 - Demonstration of in vitro synthesis of FDCA-esters (dimethyl-,
diethyl-,
dibutyl-, and isopropyl- esters)
Synthesis of diethyl-2,5 FDCA from purified DDG:
[00140] In an air tight sealed tube, 18 mL of Et0H, 0.2 gram (1 mmole) of DDG
acid,
previously purified as described in Example 11, and 0.25 M of H2504 were
added. The DDG
acid was not completely dissolved in this solution. The reaction was gently
stirred at 105 C for
18 hours. Results of a GC-MS analysis of a crude reaction sample indicated
that the formation
of diethyl-FDCA the major product. As a control, an authentic FDCA was
chemically
synthesized, esterified to diethyl-FDCA and analyzed in the same conditions.
Example 15 - Synthesis of dibuty1-2,5 FDCA from purified DDG
[00141] In an air tight sealed tube, 18 mL of n-BuOH, 0.2 gram (1 mmole) of
DDG
acid, previously purified as described in Example 11, and 0.25 M of H2504 were
added. The
DDG acid was not completely dissolved in this solution. The reaction was
gently stirred at
105 C for 18 hours. As shown in FIGURE 15, results of the GC-MS analysis of a
reaction
sample indicated that diethyl-FDCA (FDCA: 2,5-furan dicarboxylic acid) was
formed as the
major product. As a control, an authentic FDCA was chemically synthesized,
esterified to
diethyl-FDCA, and analyzed in the same conditions.
Example 16 - Synthesis of dibuty1-2,5 FDCA from crude DDG (unpurified):
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[00142] 0.2 gram (1 mmole) of crude DDG acid, which was an unpurified
lyophilized
powder obtained directly from the enzymatic dehydration of glucarate as
described in Example
11, was added into an air tight sealed tube contanning 18 mL of n-BuOH,
followed by addition
of 0.25 M of H2SO4. The crude DDG acid was not completely dissolved in this
solution. The
reaction was gently stirred at 105 C for 18 hours. Results of a GC-MS analysis
of a crude
reaction sample indicated that diethyl-FDCA (FDCA: 2,5-furan dicarboxylic
acid) was formed
as the major product. The GC-MS result indicated that the present of
contaminant salts in
crude/unpurified lyophilized powder did not significantly affect the reaction
outcome. As a
control, an authentic FDCA was chemically synthesized, esterified to diethyl-
FDCA, and
analyzed in the same conditions.
Example 17 - In vitro production of FDCA and/or esters using immobilized acids
[00143] In industrial practices, immobilized acids offer many advantages for
performing dehydrations since they can typically operate in several types of
solvent (aqueous,
organic or mixed, etc.). In addition, they can be easily recycled and be re-
used. Following
some examples of the synthesis of esters of FDCA using immobilized
AMBERLYST015
(Rohm and Haas, Philadelphia, PA) and DOWEX050 WX8 (Dow Chemical Co, Midland,
MI).
Synthesis of dibutyl-FDCA from crude DDG by using DOWEX050 WX8
[00144] In an air tight sealed tube, 2 mL of n-Butanol, 20 mg of crude DDG
acid
(unpurified lyophilized powder containing salts) and 200 mg of DOWEX050 WX8
were
combined. The DDG was not completely dissolved in this solution. The reaction
was gently
stirred at 105 C for 18 hours. Results of the GC-MS analysis of a crude
reaction sample
indicated that diethyl-FDCA (FDCA: 2,5-furan dicarboxylic acid) was formed as
the major
product. This GC-MS result indicated that the present of contaminant salts
(phosphate and
NaC1) in crude/unpurified lyophilized powder did not significantly affect the
reaction outcome.
As a control, an authentic FDCA was chemically synthesized esterified to
diethyl-FDCA and
analyzed in the same conditions.
Synthesis of dibutyl-FDCA from crude DDG by using AMBERLYST015
[00145] In an air tight sealed tube, 2 mL of n-Butanol, 20 mg of crude DDG
acid (crude
lyophilized powder with salts) and 200 mg of AMBERLYST015 (Rohm and Haas,
Philadelphia, PA) were combined. The DDG was not completely dissolved in this
solution.
The reaction was gently stirred at 105 C for 18 hours. Results of the GC-MS
analysis of a
crude reaction sample indicated that diethyl-FDCA (FDCA: 2,5-furan
dicarboxylic acid) was
formed as the major product. This GC-MS result indicated that the present of
contaminant
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WO 2015/143381 PCT/US2015/021848
salts (phosphate and NaC1) in crude/unpurified lyophilized powder did not
significantly affect
the reaction outcome. As a control, an authentic FDCA was chemically
synthesized esterified
to diethyl-FDCA and analyzed in the same conditions.
Synthesis of ethyl-FDCA from crude DDG by using AMBERLYST015
[00146] In an air tight sealed tube, 2 mL of ethanol, 20 mg of crude DDG acid
(unpurified lyophilized powder containing salts) and 200 mg of AMBERLYST015
(Rohm and
Haas, Philadelphia, PA) were combined. The DDG was not completely dissolved in
this
solution. The reaction was gently stirred at 105 C for 18 hours. Results of
the GC-MS analysis
of a crude reaction sample indicated that diethyl-FDCA (FDCA: 2,5-furan
dicarboxylic acid)
was formed as the major product. This GC-MS result indicated that the present
of contaminant
salts (phosphate and NaC1) in crude/unpurified lyophilized powder did not
significantly affect
the reaction outcome. As a control, a commercial FDCA was chemically
esterified to diethyl-
FDCA and analyzed in the same conditions.
Synthesis of diethyl-FDCA from crude DDG by using DOWEX050 WX8
[00147] In an air tight sealed tube, 2 mL of ethanol, 20 mg of crude DDG acid
(unpurified lyophilized powder containing salts) and 200 mg of DOWEX050 WX8
were
combined. The DDG was not completely dissolved in this solution. The reaction
was gently
stirred at 105 C for 18 hours. Results of the GC-MS analysis of a crude
reaction sample
indicated that diethyl-FDCA (FDCA: 2,5-furan dicarboxylic acid) was formed as
the major
product. This GC-MS result indicated that the present of contaminant salts
(phosphate and
NaC1) in crude/unpurified lyophilized powder did not significantly affect the
reaction outcome.
As a control, a commercial FDCA was chemically esterified to diethyl-FDCA and
analyzed in
the same conditions.
Example 18 - Production of FDCA derivatives
[00148] The synthesis of a number of high-value FDCA derivatives is described
in
Figure 16 in which dehydration of DTHU produces furfural-5-carboxylic acid,
i.e. FCA, which
is then chemically or enzymatically oxidized to FDCA, be reduced to FCH, or be
transaminated (using chemical reductive amination or transaminase) to amino
acid-AFC.
Example 19 ¨ Production of di-butyl FDCA in a gas phase reaction
[00149] In this example the inlet of the GC was used as a high temperature
reactor to
catalyze the dehydration of di-butyl DDG to di-butyl FDCA. The resulting
products were
chromatographically separated detected by mass spectrometry. A solution of di-
butyl DDG
(10 mM) and sulfuric acid (100 mM) in butanol was placed in a GC vial. The
vial was injected
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WO 2015/143381 PCT/US2015/021848
into a GC and FDCA Dibutyl ester was observed. The reaction occurred in the
300 C inlet
(residence time = 4 seconds). The average yield of 6 injections was 54%.
GC Settings: Direct liquid inject / MS detector
Inlet: 300 C, total flow 29.51 ml/min, split ratio 10:1, split flow 24.1
ml/min, Septum
Purge flow 3 mL/min.
GC liner: 4 mm, glass wool (P/N 5183-4647)
Column Flow: 2.41 ml/min He constant pressure control
Oven Program: At 40 C hold for 2 min, then ramp 25 C/min to 275 C, then
ramp
40 C/min to 325 C, hold for 2 min.
Column: HP-5M5, Agilent Technologies, 30m x 0.25mm x 0.25um.
Total Runtime: 14.65 minutes
MSD Transfer line: 290 C
MS Source: 250 C
MS Quad: 150 C
Retention times:
2,3-FDCA Dibutyl ester: 9.3 min
2,5-FDCA Dibutyl ester: 9.7 min
[00150] All publications and patent applications mentioned in this
specification are
herein incorporated by reference to the same extent as if each individual
publication or patent
application was specifically and individually indicated to be incorporated by
reference.
[00151] No admission is made that any reference constitutes prior art. The
discussion
of the references states what their authors assert, and the applicants reserve
the right to
challenge the accuracy and pertinence of the cited documents. It will be
clearly understood
that although a number of prior art publications are referred to herein, this
reference does not
constitute an admission that any of these documents forms part of the common
general
knowledge in the art.
[00152] It should also be understood that the foregoing examples are offered
to
illustrate, but not limit, the invention.
39
