U.S. patent application number 16/428627 was filed with the patent office on 2020-03-26 for methods and organisms for the growth-coupled production of 1,4-butanediol.
The applicant listed for this patent is Genomatica, Inc.. Invention is credited to Anthony P. Burgard, Mark J. Burk, Stephen J. Van Dien.
Application Number | 20200095616 16/428627 |
Document ID | / |
Family ID | 39768484 |
Filed Date | 2020-03-26 |
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United States Patent
Application |
20200095616 |
Kind Code |
A1 |
Burgard; Anthony P. ; et
al. |
March 26, 2020 |
METHODS AND ORGANISMS FOR THE GROWTH-COUPLED PRODUCTION OF
1,4-BUTANEDIOL
Abstract
The invention provides a non-naturally occurring microorganism
comprising one or more gene disruptions, the one or more gene
disruptions occurring in genes encoding an enzyme obligatory to
coupling 1,4-butanediol production to growth of the microorganism
when the gene disruption reduces an activity of the enzyme, whereby
theone or more gene disruptions confers stable growth-coupled
production of 1,4-butanediol onto the non-naturally occurring
microorganism. The microorganism can further comprise a gene
encoding an enzyme in a 1,4-butanediol (BDO) biosynthetic pathway.
The invention additionally relates to methods of using
microorganisms to produce BDO.
Inventors: |
Burgard; Anthony P.;
(Elizabeth, PA) ; Van Dien; Stephen J.;
(Encinitas, CA) ; Burk; Mark J.; (San Diego,
CA) |
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Applicant: |
Name |
City |
State |
Country |
Type |
Genomatica, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
39768484 |
Appl. No.: |
16/428627 |
Filed: |
May 31, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15424723 |
Feb 3, 2017 |
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16428627 |
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13914422 |
Jun 10, 2013 |
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15424723 |
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13065303 |
Mar 18, 2011 |
8470582 |
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13914422 |
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11891602 |
Aug 10, 2007 |
7947483 |
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13065303 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01D 3/002 20130101;
C12N 9/0006 20130101; C12P 7/18 20130101 |
International
Class: |
C12P 7/18 20060101
C12P007/18; B01D 3/00 20060101 B01D003/00; C12N 9/04 20060101
C12N009/04 |
Claims
1. A non-naturally occurring microorganism comprising one or more
gene disruptions, said one or more gene disruptions occurring in
genes encoding an enzyme obligatory to coupling 1,4-butanediol
production to growth of said microorganism when said gene
disruption reduces an activity of said enzyme, whereby said one or
more gene disruptions confers stable growth-coupled production of
1,4-butanediol onto said non-naturally occurring microorganism.
2. The non-naturally occurring microorganism of claim 1, wherein
said one or more gene disruptions comprise a metabolic modification
listed in Table 6 or 7.
3. The non-naturally occurring microorganism of claim 1, wherein
said one or more gene disruptions comprise a deletion of said one
or more genes.
4. The non-naturally occurring microorganism of claim 1, wherein
said non-naturally occurring microorganism is selected from the
group of microorganisms having a metabolic modification listed in
Table 6 or 7.
5. A non-naturally occurring microorganism comprising a set of
metabolic modifications obligatory to coupling 1,4-butanediol
production to growth of said microorganism, said set of metabolic
modifications comprising disruption of one or more genes, or an
ortholog thereof, wherein said set of metabolic modifications
comprises disruption of adhE and ldhA, wherein said microorganism
exhibits stable growth-coupled production of 1,4-butanediol.
6. The non-naturally occurring microorganism of claim 5, wherein
said set of metabolic modifications further comprise disruption of
mdh; adhE, ldhA, pflAB, mdh, and aspA; or adhE, ldhA, pflAB, mdh,
and aspA.
7. The non-naturally occurring microorganism of claim 6, wherein
said set of metabolic modifications further comprise disruption of
one or more genes selected from the set of genes comprising mqo,
aspA, sfcA, maeB, pntAB, and gdhA; or pykA, pykF, dhaKLM, deoC,
edd, yiaE, ycdW, prpC, and gsk.
8. The non-naturally occurring microorganism of claim 7, wherein
said set of metabolic modifications comprise disruption of sfcA and
maeB; or pykA, pykF, dhaKLM, deoC, edd, yiaE and ycdW.
9. The non-naturally occurring microorganism of claim 8, wherein
said set of metabolic modifications further comprise disruption of
prpC and gsk.
10. The non-naturally occurring microorganism of claim 6, wherein
said set of metabolic modifications further comprise disruption of
pflAB.
11. The non-naturally occurring microorganism of claim 7, wherein
said set of metabolic modifications further comprise disruption of
pflAB.
12. The non-naturally occurring microorganism of claim 8, wherein
said set of metabolic modifications further comprise disruption of
pflAB.
13. The non-naturally occurring microorganism of claim 9, wherein
said set of metabolic modifications further comprise disruption of
pflAB.
14. The non-naturally occurring microorganism of claim 5, wherein
said microorganism further comprises a 1,4-butanediol (BDO)
biosynthetic pathway, said pathway comprising at least one
exogenous nucleic acid encoding 4-hydroxybutanoate dehydrogenase,
CoA-independent succinic semialdehyde dehydrogenase, succinyl-CoA
synthetase, CoA-dependent succinic semialdehyde dehydrogenase,
4-hydroxybutyrate:CoA transferase, glutamate:succinic semialdehyde
transaminase, glutamate decarboxylase, CoA-independent aldehyde
dehydrogenase, CoA-dependent aldehyde dehydrogenase or alcohol
dehydrogenase, wherein said exogenous nucleic acid is expressed in
sufficient amounts to produce 1,4-butanediol (BDO).
15. A method of producing a non-naturally occurring microorganism
having stable growth-coupled production of 1,4-butanediol,
comprising: (a) identifying in silico a set of metabolic
modifications requiring 1,4-butanediol production during
exponential growth, and (b) genetically modifying a microorganism
to contain said set of metabolic modifications requiring
1,4-butanediol production.
16-23. (canceled)
24. A method of producing 1,4-butanediol coupled to the growth of a
microorganism, comprising: (a) culturing under exponential growth
phase in a sufficient amount of nutrients and media the
non-naturally occurring microorganism of claim 8; and (b) isolating
1,4-butanediol produced from said non-naturally occurring
microorganism.
25-31. (canceled)
32. The method of claim 24, wherein said non-naturally occurring
microorganism further comprises a 1,4-butanediol (BDO) biosynthetic
pathway, said pathway comprising at least one exogenous nucleic
acid encoding 4-hydroxybutanoate dehydrogenase, CoA-independent
succinic semialdehyde dehydrogenase, succinyl-CoA synthetase,
CoA-dependent succinic semialdehyde dehydrogenase,
4-hydroxybutyrate:CoA transferase, glutamate:succinic semialdehyde
transaminase, glutamate decarboxylase, CoA-independent aldehyde
dehydrogenase, CoA-dependent aldehyde dehydrogenase or alcohol
dehydrogenase, wherein said exogenous nucleic acid is expressed in
sufficient amounts to produce 1,4-butanediol (BDO).
Description
[0001] This application is a continuation of application Ser. No.
15/424,723, filed Feb. 3, 2017, which is a continuation of
application Ser. No. 13/914,422, filed Jun. 10, 2013, which is a
continuation of application Ser. No. 13/065,303, filed Mar. 18,
2011, now U.S. Pat. No. 8,470,582, which is is a continuation of
application Ser. No. 11/891,602, filed Aug. 10, 2007, now U.S. Pat.
No. 7,947,483, the entire contents of each of which are
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates generally to in silico design
of organisms, and more specifically to organisms having
1,4-butanediol biosynthetic capability.
[0003] 1,4-Butanediol (BDO) is a four carbon dialcohol that
currently is manufactured exclusively through various petrochemical
routes. BDO is part of a large volume family of solvents and
polymer intermediates that includes gamma-butyrolactone (GBL),
tetrahydrofuran (THF), pyrrolidone, N-methylpyrrolidone (NMP), and
N-vinyl-pyrrolidone. The overall market opportunity for this family
exceeds $4.0 B.
[0004] Approximately 2.5B lb BDO is produced globally per year with
4-5% annual growth and a recent selling price ranging from
$1.00-1.20/lb. The demand for BDO stems largely from its use as an
intermediate for polybutylene terephthalate (PBT) plastic resins,
polyurethane thermoplastics and co-polyester ethers. BDO also
serves as a primary precursor to THF, which is employed as an
intermediate for poly(tetramethylene glycol) PTMEG copolymers
required for lycra and spandex production. Approximately 0.7 B lb
of THF is produced globally per year with an annual growth rate
over 6%. A significant percentage of growth (>30%) for both BDO
and THF is occurring in Asia (China and India). GBL currently is a
smaller volume (0.4 B lb/year) product which has numerous
applications as a solvent, as an additive for inks, paints, and
dyes, as well as the primary precursor to pyrrolidone derivatives
such as NMP.
[0005] Conventional processes for the synthesis of BDO use
petrochemical feedstocks for their starting materials. For example,
acetylene is reacted with 2 molecules of formaldehyde in the Reppe
synthesis reaction (Kroschwitz and Grant, Encyclopedia of Chem.
Tech., John Wiley and Sons, Inc., New York (1999)), followed by
catalytic hydrogenation to form 1,4-butanediol. It has been
estimated that 90% of the acetylene produced in the U.S. is
consumed for butanediol production. Alternatively, it can be formed
by esterification and catalytic hydrogenation of maleic anhydride,
which is derived from butane. Downstream, butanediol can be further
transformed; for example, by oxidation to .gamma.-butyrolactone,
which can be further converted to pyrrolidone and
N-methyl-pyrrolidone, or hydrogenolysis to tetrahydrofuran (FIG.
1). These compounds have varied uses as polymer intermediates,
solvents, and additives, and have a combined market of nearly 2
billion lb/year.
[0006] The conventional hydrocarbon feedstock-based approach
utilizes methane to produce formaldehyde. Thus, a large percentage
of the commercial production of BDO relies on methane as a starting
material. The production of acetylene also relies on
petroleum-based starting material (see FIG. 1). Therefore, the
costs of BDO production fluctuate with the price of petroleum and
natural gas.
[0007] It is desirable to develop a method for production of these
chemicals by alternative means that not only substitute renewable
for petroleum-based feedstocks, and also use less energy- and
capital-intensive processes. The Department of Energy has proposed
1,4-diacids, and particularly succinic acid, as key
biologically-produced intermediates for the manufacture of the
butanediol family of products (DOE Report, "Top Value-Added
Chemicals from Biomass", 2004). However, succinic acid is costly to
isolate and purify and requires high temperatures and pressures for
catalytic reduction to butanediol.
[0008] Thus, there exists a need for alternative means for
effectively producing commercial quantities of 1,4-butanediol and
its chemical precursors. The present invention satisfies this need
and provides related advantages as well.
SUMMARY OF INVENTION
[0009] The invention provides a non-naturally occurring
microorganism comprising one or more gene disruptions, the one or
more gene disruptions occurring in genes encoding an enzyme
obligatory to coupling 1,4-butanediol production to growth of the
microorganism when the gene disruption reduces an activity of the
enzyme, whereby theone or more gene disruptions confers stable
growth-coupled production of 1,4-butanediol onto the non-naturally
occurring microorganism. The microorganism can further comprise a
gene encoding an enzyme in a 1,4-butanediol (BDO) biosynthetic
pathway. The invention additionally relates to methods of using
microorganisms to produce BDO.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The patent or application file contains at least one drawing
executed in color. Copies of this patent or patent application
publication with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0011] FIG. 1 is a schematic diagram showing an entry point of
4-hydroxybutanoic acid (4-HB) into the product pipeline of the
1,4-butanediol (BDO) family of chemicals, and comparison with
chemical synthesis routes from petrochemical feedstocks. Solid
black arrows show chemical synthesis routes; dashed blue arrows
show a biosynthetic route to 4-HB and subsequent conversion steps
to BDO family chemicals.
[0012] FIG. 2 is a schematic diagram showing biochemical pathways
to 4-hydroxybutyurate (4-HB) and .gamma.-butyrolactone (GBL)
production. Enzymes catalyzing the 4-HB biosynthetic reactions are:
(1) CoA-independent succinic semialdehyde dehydrogenase; (2)
succinyl-CoA synthetase; (3) CoA-dependent succinic semialdehyde
dehydrogenase; (4) glutamate:succinic semialdehyde transaminase;
(5) glutamate decarboxylase; (6) 4-hydroxybutanoate dehydrogenase.
Conversion (7) corresponds to a spontaneous, non-enzymatic reaction
which converts 4-HB to GBL.
[0013] FIG. 3 is a schematic diagram showing the chemical synthesis
of 1,4-butanediol (BDO) and downstream products
.gamma.-butyrolactone (GBL), tetrahydrofuran (THF) and several
pyrrolidones.
[0014] FIG. 4 is a schematic process flow diagram of bioprocesses
for the production of .gamma.-butyrolactone. Panel (a) illustrates
fed-batch fermentation with batch separation and panel (b)
illustrates fed-batch fermentation with continuous separation.
[0015] FIG. 5 shows the hypothetical production envelopes of an
OptKnock-designed strain contrasted against a typical
non-growth-coupled production strain. Note that the potential
evolutionary trajectories of the OptKnock strain are fundamentally
different in that they lead to a high producing phenotype.
[0016] FIG. 6 shows biochemical pathways to 1,4-butanediol. 1)
CoA-independent succinic semialdehyde dehydrogenase; 2)
succinyl-CoA synthetase; 3) CoA-dependent succinic semialdehyde
dehydrogenase; 4) glutamate:succinate semialdehyde transaminase; 5)
glutamate decarboxylase; 6) 4-hydroxybutanoate dehydrogenase; 7)
4-hydroxybutyryl CoA:acetyl-CoA transferase; 8) aldehyde
dehydrogenase; 9) alcohol dehydrogenase.
[0017] FIGS. 7A and 7B show the anaerobic growth rate versus BDO
yield solution boundaries for an E. coli strain possessing the BDO
production pathways shown and OptKnock predicted knockouts in FIG.
5 assuming in FIG. 7A PEP carboxykinase irreversibility and in FIG.
7B PEP carboxykinase reversibility. A basis glucose uptake rate of
20 mmol/gDW/hr is assumed along with a non-growth associated ATP
maintenance requirement of 7.6 mmol/gDW/hr.
[0018] FIG. 8 shows a pictorial representation of E. coli central
metabolism.
[0019] FIG. 9 shows the anaerobic growth rate versus BDO yield
solution boundaries for an E. coli strain possessing the BDO
production pathways shown and OptKnock predicted knockouts in FIG.
5 assuming PEP carboxykinase reversibility. A basis glucose uptake
rate of 20 mmol/gDW/hr is assumed along with a non-growth
associated ATP maintenance requirement of 7.6 mmol/gDW/hr.
DETAILED DESCRIPTION OF THE INVENTION
[0020] This invention is directed to the design and production of
cells and organisms having biosynthetic production capabilities for
4-hydroxybutanoic acid (4-HB), .gamma.-butyrolactone and
1,4-butanediol. In one embodiment, the invention utilizes in silico
stoichiometric models of Escherichia coli metabolism that identify
metabolic designs for biosynthetic production of 4-hydroxybutanoic
acid (4-HB) and 1,4-butanediol (BDO). The results described herein
indicate that metabolic pathways can be designed and recombinantly
engineered to achieve the biosynthesis of 4-HB and downstream
products such as 1,4-butanediol in Escherichia coli and other cells
or organisms. Biosynthetic production of 4-HB, for example, for the
in silico designs can be confirmed by construction of strains
having the designed metabolic genotype. These metabolically
engineered cells or organisms also can be subjected to adaptive
evolution to further augment 4-HB biosynthesis, including under
conditions approaching theoretical maximum growth.
[0021] The invention is further directed to metabolic engineering
strategies for attaining high yields of 1,4-butanediol (BDO) in
Escherichia coli (see Examples V-VII). As disclosed herein, a
genome-scale stoichiometric model of E. coli metabolism was
employed using the bilevel optimization framework OptKnock to
identify in silico strategies with multiple knockouts. The
deletions are placed such that the redundancy in the network is
reduced with the ultimate effect of coupling growth to the
production of BDO in the network. The growth-coupled BDO production
characteristic of the designed strains make them genetically stable
and amenable to continuous bioprocesses. Strain design strategies
were identified assuming the addition of non-native reaction
capabilities into E. coli leading to a metabolic pathway from
succinate semialdehyde to BDO. Out of the hundreds of strategies
identified by OptKnock, one design emerged as satisfying multiple
criteria. This design, utilizing the removal of adhE, ldhA, mdh,
aspA, and pflAB, 1) led to a high predicted BDO yield at maximum
growth, 2) required a reasonable number of knockouts, 3) had no
detrimental effect on the maximum theoretical BDO yield, 4) brought
about a tight coupling of BDO production with cell growth, and 5)
was robust with respect to the assumed irreversibility or
reversibility of PEP carboxykinase. Also disclosed herein are
methods for the experimental testing of the strain designs and
their evolution towards the theoretical maximum growth.
[0022] In certain embodiments, the 4-HB biosynthesis
characteristics of the designed strains make them genetically
stable and particularly useful in continuous bioprocesses. Separate
strain design strategies were identified with incorporation of
different non-native or heterologous reaction capabilities into E.
coli leading to 4-HB and 1,4-butanediol producing metabolic
pathways from either CoA-independent succinic semialdehyde
dehydrogenase, succinyl-CoA synthetase and CoA-dependent succinic
semialdehyde dehydrogenase, or glutamate:succinic semialdehyde
transaminase. In silico metabolic designs were identified that
resulted in the biosynthesis of 4-HB in both E. coli and yeast
species from each of these metabolic pathways. The 1,4-butanediol
intermediate .gamma.-butyrolactone can be generated in culture by
spontaneous cyclization under conditions at pH<7.5, particularly
under acidic conditions, such as below pH 5.5, for example,
pH<7, pH<6.5, pH<6, and particularly at pH<5.5 or
lower.
[0023] Strains identified via the computational component of the
platform can be put into actual production by genetically
engineering any of the predicted metabolic alterations which lead
to the biosynthetic production of 4-HB, 1,4-butanediol or other
intermediate and/or downstream products. In yet a further
embodiment, strains exhibiting biosynthetic production of these
compounds can be further subjected to adaptive evolution to further
augment product biosynthesis. The levels of product biosynthesis
yield following adaptive evolution also can be predicted by the
computational component of the system.
[0024] As used herein, the term "non-naturally occurring" when used
in reference to a microbial organism or microorganism of the
invention is intended to mean that the microbial organism has at
least one genetic alteration not normally found in a naturally
occurring strain of the referenced species, including wild-type
strains of the referenced species. Genetic alterations include, for
example, modifications introducing expressible nucleic acids
encoding metabolic polypeptides, other nucleic acid additions,
nucleic acid deletions and/or other functional disruption of the
microbial genetic material. Such modification include, for example,
coding regions and functional fragments thereof, for heterologous,
homologous or both heterologous and homologous polypeptides for the
referenced species. Additional modifications include, for example,
non-coding regulatory regions in which the modifications alter
expression of a gene or operon. Exemplary metabolic polypeptides
include enzymes within a 4-HB biosynthetic pathway and enzymes
within a biosynthetic pathway for a BDO family of compounds.
[0025] A metabolic modification refers to a biochemical reaction
that is altered from its naturally occurring state. Therefore,
non-naturally occurring microorganisms having genetic modifications
to nucleic acids encoding metabolic polypeptides or, functional
fragments thereof. Exemplary metabolic modifications are described
further below for both E. coli and yeast microbial organisms.
[0026] As used herein, the term "isolated" when used in reference
to a microbial organism is intended to mean an organism that is
substantially free of at least one component as the referenced
microbial organism is found in nature. The term includes a
microbial organism that is removed from some or all components as
it is found in its natural environment. The term also includes a
microbial organism that is removed from some or all components as
the microbial organism is found in non-naturally occurring
environments. Therefore, an isolated microbial organism is partly
or completely separated from other substances as it is found in
nature or as it is grown, stored or subsisted in non-naturally
occurring environments. Specific examples of isolated microbial
organisms include partially pure microbes, substantially pure
microbes and microbes cultured in a medium that is non-naturally
occurring.
[0027] As used herein, the terms "microbial," "microbial organism"
or "microorganism" is intended to mean any organism that exists as
a microscopic cell that is included within the domains of archaea,
bacteria or eukarya. Therefore, the term is intended to encompass
prokaryotic or eukaryotic cells or organisms having a microscopic
size and includes bacteria, archaea and eubacteria of all species
as well as eukaryotic microorganisms such as yeast and fungi. The
term also includes cell cultures of any species that can be
cultured for the production of a biochemical.
[0028] As used herein, the term "4-hydroxybutanoic acid" is
intended to mean a 4-hydroxy derivative of butyric acid having the
chemical formula C.sub.4H.sub.8O.sub.3 and a molecular mass of
104.11 g/mol (126.09 g/mol for its sodium salt). The chemical
compound 4-hydroxybutanoic acid also is known in the art as 4-HB,
4-hydroxybutyrate, gamma-hydroxybutyric acid or GHB. The term as it
is used herein is intended to include any of the compound's various
salt forms and include, for example, 4-hydroxybutanoate and
4-hydroxybutyrate. Specific examples of salt forms for 4-HB include
sodium 4-HB and potassium 4-HB. Therefore, the terms
4-hydroxybutanoic acid, 4-HB, 4-hydroxybutyrate,
4-hydroxybutanoate, gamma-hydroxybutyric acid and GHB as well as
other art recognized names are used synonymously herein.
[0029] As used herein, the term "monomeric" when used in reference
to 4-HB is intended to mean 4-HB in a non-polymeric or
underivatized form. Specific examples of polymeric 4-HB include
poly-4-hydroxybutanoic acid and copolymers of, for example, 4-HB
and 3-HB. A specific example of a derivatized form of 4-HB is
4-HB-CoA. Other polymeric 4-HB forms and other derivatized forms of
4-HB also are known in the art.
[0030] As used herein, the term ".gamma.-butyrolactone" is intended
to mean a lactone having the chemical formula C.sub.4H.sub.6O.sub.2
and a molecular mass of 86.089 g/mol. The chemical compound
.gamma.-butyrolactone also is know in the art as GBL,
butyrolactone, 1,4-lactone, 4-butyrolactone, 4-hydroxybutyric acid
lactone, and gamma-hydroxybutyric acid lactone. The term as it is
used herein is intended to include any of the compound's various
salt forms.
[0031] As used herein, the term "1-4 butanediol" is intended to
mean an alcohol derivative of the alkane butane, carrying two
hydroxyl groups which has the chemical formula
C.sub.4H.sub.10O.sub.2 and a molecular mass of 90.12 g/mol. The
chemical compound 1-4 butanediol also is known in the art as BDO
and is a chemical intermediate or precursor for a family of
compounds referred to herein as BDO family of compounds, some of
which are exemplified in FIG. 1.
[0032] As used herein, the term "tetrahydrofuran" is intended to
mean a heterocyclic organic compound corresponding to the fully
hydrogenated analog of the aromatic compound furan which has the
chemical formula C.sub.4H.sub.8O and a molecular mass of 72.11
g/mol. The chemical compound tetrahydrofuran also is known in the
art as THF, tetrahydrofuran, 1,4-epoxybutane, butylene oxide,
cyclotetramethylene oxide, oxacyclopentane, diethylene oxide,
oxolane, furanidine, hydrofuran, tetra-methylene oxide. The term as
it is used herein is intended to include any of the compound's
various salt forms.
[0033] As used herein, the term "CoA" or "coenzyme A" is intended
to mean an organic cofactor or prosthetic group (nonprotein portion
of an enzyme) whose presence is required for the activity of many
enzymes (the apoenzyme) to form an active enzyme system. Coenzyme A
functions in certain condensing enzymes, acts in acetyl or other
acyl group transfer and in fatty acid synthesis and oxidation,
pyruvate oxidation and in other acetylation.
[0034] As used herein, the term "substantially anaerobic" when used
in reference to a culture or growth condition is intended to mean
that the amount of oxygen is less than about 10% of saturation for
dissolved oxygen in liquid media. The term also is intended to
include sealed chambers of liquid or solid medium maintained with
an atmosphere of less than about 1% oxygen.
[0035] The non-naturally occurring microbal organisms of the
invention can contain stable genetic alterations, which refers to
microorganisms that can be cultured for greater than five
generations without loss of the alteration. Generally, stable
genetic alterations include modifications that persist greater than
10 generations, particularly stable modifications will persist more
than about 25 generations, and more particularly, stable genetic
modifications will be greater than 50 generations, including
indefinitely.
[0036] Those skilled in the art will understand that the genetic
alterations, including metabolic modifications exemplified herein
are described with reference to E. coli and yeast genes and their
corresponding metabolic reactions. However, given the complete
genome sequencing of a wide variety of organisms and the high level
of skill in the area of genomics, those skilled in the art will
readily be able to apply the teachings and guidance provided herein
to essentially all other organisms. For example, the E. coli
metabolic alterations exemplified herein can readily be applied to
other species by incorporating the same or analogous encoding
nucleic acid from species other than the referenced species. Such
genetic alterations include, for example, genetic alterations of
species homologs, in general, and in particular, orthologs,
paralogs or nonorthologous gene displacements.
[0037] An ortholog is a gene or genes that are related by vertical
descent and are responsible for substantially the same or identical
functions in different organisms. For example, mouse epoxide
hydrolase and human epoxide hydrolase can be considered orthologs
for the biological function of hydrolysis of epoxides. Genes are
related by vertical descent when, for example, they share sequence
similarity of sufficient amount to indicate they are homologous, or
related by evolution from a common ancestor. Genes can also be
considered orthologs if they share three-dimensional structure but
not necessarily sequence similarity, of a sufficient amount to
indicate that they have evolved from a common ancestor to the
extent that the primary sequence similarity is not identifiable.
Genes that are orthologous can encode proteins with sequence
similarity of about 25% to 100% amino acid sequence identity. Genes
encoding proteins sharing an amino acid similarity less that 25%
can also be considered to have arisen by vertical descent if their
three-dimensional structure also shows similarities. Members of the
serine protease family of enzymes, including tissue plasminogen
activator and elastase, are considered to have arisen by vertical
descent from a common ancestor.
[0038] Orthologs include genes or their encoded gene products that
through, for example, evolution, have diverged in structure or
overall activity. For example, where one species encodes a gene
product exhibiting two functions and where such functions have been
separated into distinct genes in a second species, the three genes
and their corresponding products are considered to be orthologs.
For the growth-coupled production of a biochemical product, those
skilled in the art will understand that the orthologous gene
harboring the metabolic activity to be disrupted is to be chosen
for construction of the non-naturally occurring microorganism. An
example of orthologs exhibiting separable activities is where
distinct activities have been separated into distinct gene products
between two or more species or within a single species. A specific
example is the separation of elastase proteolysis and plasminogen
proteolysis, two types of serine protease activity, into distinct
molecules as plasminogen activator and elastase. A second example
is the separation of mycoplasma 5'-3' exonuclease and Drosophila
DNA polymerase III activity. The DNA polymerase from the first
species can be considered an ortholog to either or both of the
exonuclease or the polymerase from the second species and vice
versa.
[0039] In contrast, paralogs are homologs related by, for example,
duplication followed by evolutionary divergence and have similar or
common, but not identical functions. Paralogs can originate or
derive from, for example, the same species or from a different
species. For example, microsomal epoxide hydrolase (epoxide
hydrolase I) and soluble epoxide hydrolase (epoxide hydrolase II)
can be considered paralogs because they represent two distinct
enzymes, co-evolved from a common ancestor, that catalyze distinct
reactions and have distinct functions in the same species. Paralogs
are proteins from the same species with significant sequence
similarity to each other suggesting that they are homologous, or
related through co-evolution from a common ancestor. Groups of
paralogous protein families include HipA homologs, luciferase
genes, peptidases, and others.
[0040] A nonorthologous gene displacement is a nonorthologous gene
from one species that can substitute for a referenced gene function
in a different species. Substitution includes, for example, being
able to perform substantially the same or a similar function in the
species of origin compared to the referenced function in the
different species. Although generally, a nonorthologous gene
displacement will be identifiable as structurally related to a
known gene encoding the referenced function, less structurally
related but functionally similar genes and their corresponding gene
products nevertheless will still fall within the meaning of the
term as it is used herein. Functional similarity requires, for
example, at least some structural similarity in the active site or
binding region of a nonorthologous gene compared to a gene encoding
the function sought to be substituted. Therefore, a nonorthologous
gene includes, for example, a paralog or an unrelated gene.
[0041] Therefore, in identifying and constructing the non-naturally
occurring microbial organisms of the invention having 4-HB, GBL
and/or BDO biosynthetic capability, those skilled in the art will
understand with applying the teaching and guidance provided herein
to a particular species that the identification of metabolic
modifications can include identification and inclusion or
inactivation of orthologs. To the extent that paralogs and/or
nonorthologous gene displacements are present in the referenced
microorganism that encode an enzyme catalyzing a similar or
substantially similar metabolic reaction, those skilled in the art
also can utilize these evolutionally related genes.
[0042] Orthologs, paralogs and nonorthologous gene displacements
can be determined by methods well known to those skilled in the
art. For example, inspection of nucleic acid or amino acid
sequences for two polypeptides will reveal sequence identity and
similarities between the compared sequences. Based on such
similarities, one skilled in the art can determine if the
similarity is sufficiently high to indicate the proteins are
related through evolution from a common ancestor. Algorithms well
known to those skilled in the art, such as Align, BLAST, Clustal W
and others compare and determine a raw sequence similarity or
identity, and also determine the presence or significance of gaps
in the sequence which can be assigned a weight or score. Such
algorithms also are known in the art and are similarly applicable
for determining nucleotide sequence similarity or identity.
Parameters for sufficient similarity to determine relatedness are
computed based on well known methods for calculating statistical
similarity, or the chance of finding a similar match in a random
polypeptide, and the significance of the match determined. A
computer comparison of two or more sequences can, if desired, also
be optimized visually by those skilled in the art. Related gene
products or proteins can be expected to have a high similarity, for
example, 25% to 100% sequence identity. Proteins that are unrelated
can have an identity which is essentially the same as would be
expected to occur by chance, if a database of sufficient size is
scanned (about 5%). Sequences between 5% and 24% may or may not
represent sufficient homology to conclude that the compared
sequences are related. Additional statistical analysis to determine
the significance of such matches given the size of the data set can
be carried out to determine the relevance of these sequences.
[0043] Exemplary parameters for determining relatedness of two or
more sequences using the BLAST algorithm, for example, can be as
set forth below. Briefly, amino acid sequence alignments can be
performed using BLASTP version 2.0.8 (Jan. 5, 1999) and the
following parameters: Matrix: 0 BLOSUM62; gap open: 11; gap
extension: 1; x_dropoff: 50; expect: 10.0; wordsize: 3; filter: on.
Nucleic acid sequence alignments can be performed using BLASTN
version 2.0.6 (Sep. 16, 1998) and the following parameters: Match:
1; mismatch: -2; gap open: 5; gap extension: 2; x_dropoff: 50;
expect: 10.0; wordsize: 11; filter: off. Those skilled in the art
will know what modifications can be made to the above parameters to
either increase or decrease the stringency of the comparison, for
example, and determine the relatedness of two or more
sequences.
[0044] The invention provides a non-naturally occurring microbial
biocatalyst including a microbial organism having a
4-hydroxybutanoic acid (4-HB) biosynthetic pathway that includes at
least one exogenous nucleic acid encoding 4-hydroxybutanoate
dehydrogenase, CoA-independent succinic semialdehyde dehydrogenase,
succinyl-CoA synthetase, CoA-dependent succinic semialdehyde
dehydrogenase, glutamate:succinic semialdehyde transaminase or
glutamate decarboxylase, wherein the exogenous nucleic acid is
expressed in sufficient amounts to produce monomeric
4-hydroxybutanoic acid (4-HB). Succinyl-CoA synthetase is also
referred to as succinyl-CoA synthase or succinyl CoA ligase.
[0045] The non-naturally occurring microbial biocatalysts of the
invention include microbial organisms that employ combinations of
metabolic reactions for biosynthetically producing the compounds of
the invention. Exemplary compounds produced by the non-naturally
occurring microorganisms include, for example, 4-hydroxybutanoic
acid, 1,4-butanediol and .gamma.-butyrolactone. The relationships
of these exemplary compounds with respect to chemical synthesis or
biosynthesis are exemplified in FIG. 1.
[0046] In one embodiment, a non-naturally occurring microbial
organism is engineered to produce 4-HB. This compound is one useful
entry point into the 1,4-butanediol family of compounds. The
biochemical reactions for formation of 4-HB from succinate, from
succinate through succinyl-CoA or from .alpha.-ketoglutarate are
shown in FIG. 2.
[0047] The invention is described herein with general reference to
the metabolic reaction, reactant or product thereof, or with
specific reference to one or more nucleic acids or genes encoding
an enzyme associated with or catalyzing the referenced metabolic
reaction, reactant or product. Unless otherwise expressly stated
herein, those skilled in the art will understand that reference to
a reaction also constitutes reference to the reactants and products
of the reaction. Similarly, unless otherwise expressly stated
herein, reference to a reactant or product also references the
reaction and that reference to any of these metabolic constitutes
also references the gene or genes encoding the enzymes that
catalyze the referenced reaction, reactant or product. Likewise,
given the well known fields of metabolic biochemistry, enzymology
and genomics, reference herein to a gene or encoding nucleic acid
also constitutes a reference to the corresponding encoded enzyme
and the reaction it catalyzes as well as the reactants and products
of the reaction.
[0048] The production of 4-HB via biosynthetic modes using the
microbial organisms of the invention is particularly useful because
it results in monomeric 4-HB. The non-naturally occurring microbial
organisms of the invention and their biosynthesis of 4-HB and BDO
family compounds also is particularly useful because the 4-HB
product (1) is secreted; (2) is devoid of any derivatizations such
as Coenzyme A; (3) avoids thermodynamic changes during
biosynthesis, and (4) allows for the spontaneous chemical
conversion of 4-HB to .gamma.-butyrolactone (GBL) in acidic pH
medium. This latter characteristic is exemplified as step 7 in FIG.
2 and also is particularly useful for efficient chemical synthesis
or biosynthesis of BDO family compounds such as 1,4-butanediol
and/or tetrahydrofuran (THF), for example.
[0049] Microbial organisms generally lack the capacity to
synthesize 4-HB and therefore, any of the compounds shown in FIG. 1
are known to be within the 1,4-butanediol family of compounds or
known by those in the art to be within the 1,4-butanediol family of
compounds. Moreover, organisms having all of the requisite
metabolic enzymatic capabilities are not known to produce 4-HB from
the enzymes described and biochemical pathways exemplified herein.
Rather, with the possible exception of a few anaerobic
microorganisms described further below, the microorganisms having
the enzymatic capability use 4-HB as a substrate to produce, for
example, succinate. In contrast, the non-naturally occurring
microbial organisms of the invention generate 4-HB as a product. As
described above, the biosynthesis of 4-HB in its monomeric form is
not only particularly useful in chemical synthesis of BDO family of
compounds, it also allows for the further biosynthesis of BDO
family compounds and avoids altogether chemical synthesis
procedures.
[0050] The non-naturally occurring microbial organisms of the
invention that can produce monomeric 4-HB are produced by ensuring
that a host microbial organism includes functional capabilities for
the complete biochemical synthesis of at least one 4-HB
biosynthetic pathway of the invention. Ensuring at least one
requisite 4-HB biosynthetic pathway confers 4-HB biosynthesis
capability onto the host microbial organism.
[0051] Three requisite 4-HB biosynthetic pathways are exemplified
herein and shown for purposes of illustration in FIG. 2. One
requisite 4-HB biosynthetic pathway includes the biosynthesis of
4-HB from succinate. The enzymes participating in this 4-HB pathway
include CoA-independent succinic semialdehyde dehydrogenase and
4-hydroxybutanoate dehydrogenase. Another requisite 4-HB
biosynthetic pathway includes the biosynthesis from succinate
through succinyl-CoA. The enzymes participating in this 4-HB
pathway include succinyl-CoA synthetase, CoA-dependent succinic
semialdehyde dehydrogenase and 4-hydroxybutanoate dehydrogenase. A
third requisite 4-HB biosynthetic pathway includes the biosynthesis
of 4-HB from .alpha.-ketoglutarate. The enzymes participating in
this 4-HB biosynthetic pathway include glutamate:succinic
semialdehyde transaminase, glutamate decarboxylase and
4-hydroxybutanoate dehydrogenase. Each of these 4-HB biosynthetic
pathways, their substrates, reactants and products are described
further below in the Examples.
[0052] The non-naturally occurring microbial organisms of the
invention can be produced by introducing expressible nucleic acids
encoding one or more of the enzymes participating in one or more
4-HB biosynthetic pathways. Depending on the host microbial
organism chosen for biosynthesis, nucleic acids for some or all of
a particular 4-HB biosynthetic pathway can be expressed. For
example, if a chosen host is deficient in both enzymes in the
succinate to 4-HB pathway (the succinate pathway) and this pathway
is selected for 4-HB biosynthesis, then expressible nucleic acids
for both CoA-independent succinic semialdehyde dehydrogenase and
4-hydroxybutanoate dehydrogenase are introduced into the host for
subsequent exogenous expression. Alternatively, if the chosen host
is deficient in 4-hydroxybutanoate dehydrogenase then an encoding
nucleic acid is needed for this enzyme to achieve 4-HB
biosynthesis.
[0053] In like fashion, where 4-HB biosynthesis is selected to
occur through the succinate to succinyl-CoA pathway (the
succinyl-CoA pathway), encoding nucleic acids for host deficiencies
in the enzymes succinyl-CoA synthetase, CoA-dependent succinic
semialdehyde dehydrogenase and/or 4-hydroxybutanoate dehydrogenase
are to be exogenously expressed in the recipient host. Selection of
4-HB biosynthesis through the .alpha.-ketoglutarate to succinic
semialdehyde pathway (the .alpha.-ketoglutarate pathway) will
utilize exogenous expression for host deficiencies in one or more
of the enzymes for glutamate:succinic semialdehyde transaminase,
glutamate decarboxylase and/or 4-hydroxybutanoate
dehydrogenase.
[0054] Depending on the 4-HB biosynthetic pathway constituents of a
selected host microbial organism, the non-naturally occurring
microbial 4-HB biocatalysts of the invention will include at least
one exogenously expressed 4-HB pathway-encoding nucleic acid and up
to all six 4-HB pathway encoding nucleic acids. For example, 4-HB
biosynthesis can be established from all three pathways in a host
deficient in 4-hydroxybutanoate dehydrogenase through exogenous
expression of a 4-hydroxybutanoate dehydrogenase encoding nucleic
acid. In contrast, 4-HB biosynthesis can be established from all
three pathways in a host deficient in all six enzymes through
exogenous expression of all six of CoA-independent succinic
semialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependent
succinic semialdehyde dehydrogenase, glutamate:succinic
semialdehyde transaminase, glutamate decarboxylase and
4-hydroxybutanoate dehydrogenase.
[0055] Given the teachings and guidance provided herein, those
skilled in the art will understand that the number of encoding
nucleic acids to introduce in an expressible form will parallel the
4-HB pathway deficiencies of the selected host microbial organism.
Therefore, a non-naturally occurring microbial organism of the
invention can have one, two, three, four, five or six encoding
nucleic acids encoding the above enzymes constituting the 4-HB
biosynthetic pathways. In some embodiments, the non-naturally
occurring microbial organisms also can include other genetic
modifications that facilitate or optimize 4-HB biosynthesis or that
confer other useful functions onto the host microbial organism. One
such other functionality can include, for example, augmentation of
the synthesis of one or more of the 4-HB pathway precursors such as
succinate, succinyl-CoA and/or .alpha.-ketoglutarate.
[0056] In some embodiments, a non-naturally occurring microbial
organism of the invention is generated from a host that contains
the enzymatic capability to synthesize 4-HB. In this specific
embodiment it can be useful to increase the synthesis or
accumulation of a 4-HB pathway product to, for example, drive 4-HB
pathway reactions toward 4-HB production. Increased synthesis or
accumulation can be accomplished by, for example, overexpression of
nucleic acids encoding one or more of the above-described 4-HB
pathway enzymes. Over expression of the 4-HB pathway enzyme or
enzymes can occur, for example, through exogenous expression of the
endogenous gene or genes, or through exogenous expression of the
heterologous gene or genes. Therefore, naturally occurring
organisms can be readily generated to be non-naturally 4-HB
producing microbial organisms of the invention through
overexpression of one, two, three, four, five or all six nucleic
acids encoding 4-HB biosynthetic pathway enzymes. In addition, a
non-naturally occurring organism can be generated by mutagenesis of
an endogenous gene that results in an increase in activity of an
enzyme in the 4-HB biosynthetic pathway.
[0057] In particularly useful embodiments, exogenous expression of
the encoding nucleic acids is employed. Exogenous expression
confers the ability to custom tailor the expression and/or
regulatory elements to the host and application to achieve a
desired expression level that is controlled by the user. However,
endogenous expression also can be utilized in other embodiments
such as by removing a negative regulatory effector or induction of
the gene's promoter when linked to an inducible promoter or other
regulatory element. Thus, an endogenous gene having a naturally
occurring inducible promoter can be up-regulated by providing the
appropriate inducing agent, or the regulatory region of an
endogenous gene can be engineered to incorporate an inducible
regulatory element, thereby allowing the regulation of increased
expression of an endogenous gene at a desired time. Similarly, an
inducible promoter can be included as a regulatory element for an
exogenous gene introduced into a non-naturally occurring microbial
organism (see Example II).
[0058] "Exogenous" as it is used herein is intended to mean that
the referenced molecule or the referenced activity is introduced
into the host microbial organism. Therefore, the term as it is used
in reference to expression of an encoding nucleic acid refers to
introduction of the encoding nucleic acid in an expressible form
into the microbial organism. When used in reference to a
biosynthetic activity, the term refers to an activity that is
introduced into the host reference organism. The source can be, for
example, a homologous or heterologous encoding nucleic acids that
expresses the referenced activity following introduction into the
host microbial organism. Therefore, the term "endogenous" refers to
a referenced molecule or activity that is present in the host.
Similarly, the term when used in reference to expression of an
encoding nucleic acid refers to expression of an encoding nucleic
acid contained within the microbial organism. The term
"heterologous" refers to a molecule or activity derived from a
source other than the referenced species whereas "homologous"
refers to a molecule or activity derived from the host microbial
organism. Accordingly, exogenous expression of an encoding nucleic
acid of the invention can utilize either or both a heterologous or
homologous encoding nucleic acid.
[0059] Sources of encoding nucleic acids for a 4-HB pathway enzyme
can include, for example, any species where the encoded gene
product is capable of catalyzing the referenced reaction. Such
species include both prokaryotic and eukaryotic organisms
including, but not limited to, bacteria, including archaea and
eubacteria, and eukaryotes, including yeast, plant, insect, animal,
and mammal, including human. For example, the microbial organisms
having 4-HB biosynthetic production are exemplified herein with
reference to E. coli and yeast hosts. However, with the complete
genome sequence available for now more than 550 species (with more
than half of these available on public databases such as the NCBI),
including 395 microorganism genomes and a variety of yeast, fungi,
plant, and mammalian genomes, the identification of genes encoding
the requisite 4-HB biosynthetic activity for one or more genes in
related or distant species, including for example, homologues,
orthologs, paralogs and nonorthologous gene displacements of known
genes, and the interchange of genetic alterations between organisms
is routine and well known in the art. Accordingly, the metabolic
alterations enabling biosynthesis of 4-HB and other compounds of
the invention described herein with reference to a particular
organism such as E. coli or yeast can be readily applied to other
microorganisms, including prokaryotic and eukaryotic organisms
alike. Given the teachings and guidance provided herein, those
skilled in the art will know that a metabolic alteration
exemplified in one organism can be applied equally to other
organisms.
[0060] In some instances, such as when an alternative 4-HB
biosynthetic pathway exists in an unrelated species, 4-HB
biosynthesis can be conferred onto the host species by, for
example, exogenous expression of a paralog or paralogs from the
unrelated species that catalyzes a similar, yet non-identical
metabolic reaction to replace the referenced reaction. Because
certain differences among metabolic networks exist between
different organisms, those skilled in the art will understand that
the actual genes usage between different organisms may differ.
However, given the teachings and guidance provided herein, those
skilled in the art also will understand that the teachings and
methods of the invention can be applied to all microbial organisms
using the cognate metabolic alterations to those exemplified herein
to construct a microbial organism in a species of interest that
will synthesize monomeric 4-HB.
[0061] Host microbial organisms can be selected from, and the
non-naturally occurring microbial organisms generated in, for
example, bacteria, yeast, fungus or any of a variety of other
microorganisms applicable to fermentation processes. Exemplary
bacteria include species selected from E. coli, Klebsiella oxytoca,
Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes,
Mannheimia succiniciproducens, Rhizobium etli, Bacillus subtilis,
Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas
mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces
coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens,
and Pseudomonas putida. Exemplary yeasts or fungi include species
selected from Saccharomyces cerevisiae, Schizosaccharomyces pombe,
Kluyveromyces lactis, Kluyveromyces marxianus, Aspergillus terreus,
Aspergillus niger and Pichia pastoris.
[0062] Methods for constructing and testing the expression levels
of a non-naturally occurring 4-1-1B-producing host can be
performed, for example, by recombinant and detection methods well
known in the art. Such methods can be found described in, for
example, Sambrook et al., Molecular Cloning: A Laboratory Manual,
Third Ed., Cold Spring Harbor Laboratory, New York (2001); Ausubel
et al., Current Protocols in Molecular Biology, John Wiley and
Sons, Baltimore, Md. (1999). 4-HB and GBL can be separated by, for
example, HPLC using a Spherisorb 5 ODS1 column and a mobile phase
of 70% 10 mM phosphate buffer (pH=7) and 30% methanol, and detected
using a UV detector at 215 nm (Hennessy et al. J. Forensic Sci.
46(6):1-9 (2004)). BDO is detected by gas chromatography or by HPLC
and refractive index detector using an Aminex HPX-87H column and a
mobile phase of 0.5 mM sulfuric acid (Gonzalez-Pajuelo et al., Met.
Eng. 7:329-336 (2005)).
[0063] The non-naturally occurring microbial organisms of the
invention are constructed using methods well known in the art as
exemplified above to exogenously express at least one nucleic acid
encoding a 4-HB pathway enzyme in sufficient amounts to produce
monomeric 4-HB. Exemplary levels of expression for 4-HB enzymes in
each pathway are described further below in the Examples. Following
the teachings and guidance provided herein, the non-naturally
occurring microbial organisms of the invention can achieve
biosynthesis of monomeric 4-HB resulting in intracellular
concentrations between about 0.1-25 mM or more. Generally, the
intracellular concentration of monomeric 4-HB is between about 3-20
mM, particularly between about 5-15 mM and more particularly
between about 8-12 mM, including about 10 mM or more. Intracellular
concentrations between and above each of these exemplary ranges
also can be achieved from the non-naturally occurring microbial
organisms of the invention.
[0064] As described further below, one exemplary growth condition
for achieving biosynthesis of 4-HB includes anaerobic culture or
fermentation conditions. In certain embodiments, the non-naturally
occurring microbial organisms of the invention can be sustained,
cultured or fermented under anaerobic or substantially anaerobic
conditions. Briefly, anaerobic conditions refers to an environment
devoid of oxygen. Substantially anaerobic conditions include, for
example, a culture, batch fermentation or continuous fermentation
such that the dissolved oxygen concentration in the medium remains
between 0 and 10% of saturation. Substantially anaerobic conditions
also includes growing or resting cells in liquid medium or on solid
agar inside a sealed chamber maintained with an atmosphere of less
than 1% oxygen. The percent of oxygen can be maintained by, for
example, sparging the culture with an N.sub.2/CO.sub.2 mixture or
other suitable non-oxygen gas or gases.
[0065] The invention also provides a non-naturally occurring
microbial biocatalyst including a microbial organism having
4-hydroxybutanoic acid (4-HB) and 1,4-butanediol (BDO) biosynthetic
pathways that include at least one exogenous nucleic acid encoding
4-hydroxybutanoate dehydrogenase, CoA-independent succinic
semialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependent
succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA
transferase, glutamate:succinic semialdehyde transaminase,
glutamate decarboxylase, CoA-independent aldehyde dehydrogenase,
CoA-dependent aldehyde dehydrogenase or alcohol dehydrogenase,
wherein the exogenous nucleic acid is expressed in sufficient
amounts to produce 1,4-butanediol (BDO).
[0066] Non-naturally occurring microbial organisms also can be
generated which biosynthesize BDO. Following the teachings and
guidance provided previously for the construction of microbial
organisms that synthesize 4-HB, additional BDO pathways can be
incorporated into the 4-HB producing microbial organisms to
generate organisms that also synthesize BDO and other BDO family
compounds. The chemical synthesis of BDO and its downstream
products are illustrated in FIG. 3. The non-naturally occurring
microbial organisms of the invention capable of BDO biosynthesis
circumvent these chemical synthesis using 4-HB as an entry point as
illustrated in FIG. 2. As described further below, the 4-HB
producers can be used to chemically convert 4-HB to GBL and then to
BDO or THF, for example. Alternatively, the 4-HB producers can be
further modified to include biosynthetic capabilities for
conversion of 4-HB and/or GBL to BDO.
[0067] The additional BDO pathways to introduce into 4-HB producers
include, for example, the exogenous expression in a host deficient
background or the overexpression of a CoA-independent aldehyde
dehydrogenase, CoA-dependent aldehyde dehydrogenase or an alcohol
dehydrogenase. In the absence of endogenous acyl-CoA synthetase
capable of modifying 4-HB, the non-naturally occurring BDO
producing microbial organisms can further include an exogenous
acyl-CoA synthetase selective for 4-HB. Exemplary alcohol and
aldehyde dehydrogenases that can be used for these in vivo
conversions from 4-HB to BDO are listed below in Table 1.
TABLE-US-00001 TABLE 1 Alcohol and Aldehyde Dehydrogenases for
Conversion of 4-HB to BDO. ALCOHOL DEHYDROGENASES ec:1.1.1.1
alcohol dehydrogenase ec:1.1.1.2 alcohol dehydrogenase (NADP+)
ec:1.1.1.4 (R,R)-butanediol dehydrogenase ec:1.1.1.5 acetoin
dehydrogenase ec:1.1.1.6 glycerol dehydrogenase ec:1.1.1.7
propanediol-phosphate dehydrogenase ec:1.1.1.8 glycerol-3-phosphate
dehydrogenase (NAD+) ec:1.1.1.11 D-arabinitol 4-dehydrogenase
ec:1.1.1.12 L-arabinitol 4-dehydrogenase ec:1.1.1.13 L-arabinitol
2-dehydrogenase ec:1.1.1.14 L-iditol 2-dehydrogenase ec:1.1.1.15
D-iditol 2-dehydrogenase ec:1.1.1.16 galactitol 2-dehydrogenase
ec:1.1.1.17 mannitol-1-phosphate 5-dehydrogenase ec:1.1.1.18
inositol 2-dehydrogenase ec:1.1.1.21 aldehyde reductase ec:1.1.1.23
histidinol dehydrogenase ec:1.1.1.26 glyoxylate reductase
ec:1.1.1.27 L-lactate dehydrogenase ec:1.1.1.28 D-lactate
dehydrogenase ec:1.1.1.29 glycerate dehydrogenase ec:1.1.1.30
3-hydroxybutyrate dehydrogenase ec:1.1.1.31 3-hydroxyisobutyrate
dehydrogenase ec:1.1.1.35 3-hydroxyacyl-CoA dehydrogenase
ec:1.1.1.36 acetoacetyl-CoA reductase ec:1.1.1.37 malate
dehydrogenase ec:1.1.1.38 malate dehydrogenase
(oxaloacetate-decarboxylating) ec:1.1.1.39 malate dehydrogenase
(decarboxylating) ec:1.1.1.40 malate dehydrogenase
(oxaloacetate-decarboxylating) (NADP+) ec:1.1.1.41 isocitrate
dehydrogenase (NAD+) ec:1.1.1.42 isocitrate dehydrogenase (NADP+)
ec:1.1.1.54 allyl-alcohol dehydrogenase ec:1.1.1.55 lactaldehyde
reductase (NADPH) ec:1.1.1.56 ribitol 2-dehydrogenase ec:1.1.1.59
3-hydroxypropionate dehydrogenase ec:1.1.1.60
2-hydroxy-3-oxopropionate reductase ec:1.1.1.61 4-hydroxybutyrate
dehydrogenase ec:1.1.1.66 omega-hydroxydecanoate dehydrogenase
ec:1.1.1.67 mannitol 2-dehydrogenase ec:1.1.1.71 alcohol
dehydrogenase [NAD(P)+] ec:1.1.1.72 glycerol dehydrogenase (NADP+)
ec:1.1.1.73 octanol dehydrogenase ec:1.1.1.75 (R)-aminopropanol
dehydrogenase ec:1.1.1.76 (S,S)-butanediol dehydrogenase
ec:1.1.1.77 lactaldehyde reductase ec:1.1.1.78 methylglyoxal
reductase (NADH-dependent) ec:1.1.1.79 glyoxylate reductase (NADP+)
ec:1.1.1.80 isopropanol dehydrogenase (NADP+) ec:1.1.1.81
hydroxypyruvate reductase ec:1.1.1.82 malate dehydrogenase (NADP+)
ec:1.1.1.83 D-malate dehydrogenase (decarboxylating) ec:1.1.1.84
dimethylmalate dehydrogenase ec:1.1.1.85 3-isopropylmalate
dehydrogenase ec:1.1.1.86 ketol-acid reductoisomerase ec:1.1.1.87
homoisocitrate dehydrogenase ec:1.1.1.88 hydroxymethylglutaryl-CoA
reductase ec:1.1.1.90 aryl-alcohol dehydrogenase ec:1.1.1.91
aryl-alcohol dehydrogenase (NADP+) ec:1.1.1.92 oxaloglycolate
reductase (decarboxylating) ec:1.1.1.94 glycerol-3-phosphate
dehydrogenase [NAD(P)+] ec:1.1.1.95 phosphoglycerate dehydrogenase
ec:1.1.1.97 3-hydroxybenzyl-alcohol dehydrogenase ec:1.1.1.101
acylglycerone-phosphate reductase ec:1.1.1.103 L-threonine
3-dehydrogenase ec:1.1.1.104 4-oxoproline reductase ec:1.1.1.105
retinol dehydrogenase ec:1.1.1.110 indolelactate dehydrogenase
ec:1.1.1.112 indanol dehydrogenase ec:1.1.1.113 L-xylose
1-dehydrogenase ec:1.1.1.129 L-threonate 3-dehydrogenase
ec:1.1.1.137 ribitol-5-phosphate 2-dehydrogenase ec:1.1.1.138
mannitol 2-dehydrogenase (NADP+) ec:1.1.1.140 sorbitol-6-phosphate
2-dehydrogenase ec:1.1.1.142 D-pinitol dehydrogenase ec:1.1.1.143
sequoyitol dehydrogenase ec:1.1.1.144 perillyl-alcohol
dehydrogenase ec:1.1.1.156 glycerol 2-dehydrogenase (NADP+)
ec:1.1.1.157 3-hydroxybutyryl-CoA dehydrogenase ec:1.1.1.163
cyclopentanol dehydrogenase ec:1.1.1.164 hexadecanol dehydrogenase
ec:1.1.1.165 2-alkyn-1-ol dehydrogenase ec:1.1.1.166
hydroxycyclohexanecarboxylate dehydrogenase ec:1.1.1.167
hydroxymalonate dehydrogenase ec:1.1.1.174 cyclohexane-1,2-diol
dehydrogenase ec:1.1.1.177 glycerol-3-phosphate 1-dehydrogenase
(NADP+) ec:1.1.1.178 3-hydroxy-2-methylbutyryl-CoA dehydrogenase
ec:1.1.1.185 L-glycol dehydrogenase ec:1.1.1.190
indole-3-acetaldehyde reductase (NADH) ec:1.1.1.191
indole-3-acetaldehyde reductase (NADPH) ec:1.1.1.192
long-chain-alcohol dehydrogenase ec:1.1.1.194 coniferyl-alcohol
dehydrogenase ec:1.1.1.195 cinnamyl-alcohol dehydrogenase
ec:1.1.1.198 (+)-borneol dehydrogenase ec:1.1.1.202 1,3-propanediol
dehydrogenase ec:1.1.1.207 (-)-menthol dehydrogenase ec:1.1.1.208
(+)-neomenthol dehydrogenase ec:1.1.1.216 farnesol dehydrogenase
ec:1.1.1.217 benzyl-2-methyl-hydroxybutyrate dehydrogenase
ec:1.1.1.222 (R)-4-hydroxyphenyllactate dehydrogenase ec:1.1.1.223
isopiperitenol dehydrogenase ec:1.1.1.226
4-hydroxycyclohexanecarboxylate dehydrogenase ec:1.1.1.229 diethyl
2-methyl-3-oxosuccinate reductase ec:1.1.1.237
hydroxyphenylpyruvate reductase ec:1.1.1.244 methanol dehydrogenase
ec:1.1.1.245 cyclohexanol dehydrogenase ec:1.1.1.250 D-arabinitol
2-dehydrogenase ec:1.1.1.251 galactitol 1-phosphate 5-dehydrogenase
ec:1.1.1.255 mannitol dehydrogenase ec:1.1.1.256 fluoren-9-ol
dehydrogenase ec:1.1.1.257 4-(hydroxymethyl)benzenesulfonate
dehydrogenase ec:1.1.1.258 6-hydroxyhexanoate dehydrogenase
ec:1.1.1.259 3-hydroxypimeloyl-CoA dehydrogenase ec:1.1.1.261
glycerol-1-phosphate dehydrogenase [NAD(P)+] ec:1.1.1.265
3-methylbutanal reductase ec:1.1.1.283 methylglyoxal reductase
(NADPH-dependent) ec:1.1.1.286 isocitrate-homoisocitrate
dehydrogenase ec:1.1.1.287 D-arabinitol dehydrogenase (NADP+)
butanol dehydrogenase ALDEHYDE DEHYDROGENASES ec:1.2.1.2 formate
dehydrogenase ec:1.2.1.3 aldehyde dehydrogenase (NAD+) ec:1.2.1.4
aldehyde dehydrogenase (NADP+) ec:1.2.1.5 aldehyde dehydrogenase
[NAD(P)+] ec:1.2.1.7 benzaldehyde dehydrogenase (NADP+) ec:1.2.1.8
betaine-aldehyde dehydrogenase ec:1.2.1.9
glyceraldehyde-3-phosphate dehydrogenase (NADP+) ec:1.2.1.10
acetaldehyde dehydrogenase (acetylating) ec:1.2.1.11
aspartate-semialdehyde dehydrogenase ec:1.2.1.12
glyceraldehyde-3-phosphate dehydrogenase (phosphorylating)
ec:1.2.1.13 glyceraldehyde-3-phosphate dehydrogenase (NADP+)
(phosphorylating) ec:1.2.1.15 malonate-semialdehyde dehydrogenase
ec:1.2.1.16 succinate-semialdehyde dehydrogenase [NAD(P)+]
ec:1.2.1.17 glyoxylate dehydrogenase (acylating) ec:1.2.1.18
malonate-semialdehyde dehydrogenase (acetylating) ec:1.2.1.19
aminobutyraldehyde dehydrogenase ec:1.2.1.20 glutarate-semialdehyde
dehydrogenase ec:1.2.1.21 glycolaldehyde dehydrogenase ec:1.2.1.22
lactaldehyde dehydrogenase ec:1.2.1.23 2-oxoaldehyde dehydrogenase
(NAD+) ec:1.2.1.24 succinate-semialdehyde dehydrogenase ec:1.2.1.25
2-oxoisovalerate dehydrogenase (acylating) ec:1.2.1.26
2,5-dioxovalerate dehydrogenase ec:1.2.1.27
methylmalonate-semialdehyde dehydrogenase (acylating) ec:1.2.1.28
benzaldehyde dehydrogenase (NAD+) ec:1.2.1.29 aryl-aldehyde
dehydrogenase ec:1.2.1.30 aryl-aldehyde dehydrogenase (NADP+)
ec:1.2.1.31 L-aminoadipate-semialdehyde dehydrogenase ec:1.2.1.32
aminomuconate-semialdehyde dehydrogenase ec:1.2.1.36 retinal
dehydrogenase ec:1.2.1.39 phenylacetaldehyde dehydrogenase
ec:1.2.1.41 glutamate-5-semialdehyde dehydrogenase ec:1.2.1.42
hexadecanal dehydrogenase (acylating) ec:1.2.1.43 formate
dehydrogenase (NADP+) ec:1.2.1.45
4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase
ec:1.2.1.46 formaldehyde dehydrogenase ec:1.2.1.47
4-trimethylammoniobutyraldehyde dehydrogenase ec:1.2.1.48
long-chain-aldehyde dehydrogenase ec:1.2.1.49 2-oxoaldehyde
dehydrogenase (NADP+) ec:1.2.1.51 pyruvate dehydrogenase (NADP+)
ec:1.2.1.52 oxoglutarate dehydrogenase (NADP+) ec:1.2.1.53
4-hydroxyphenylacetaldehyde dehydrogenase ec:1.2.1.57 butanal
dehydrogenase ec:1.2.1.58 phenylglyoxylate dehydrogenase
(acylating) ec:1.2.1.59 glyceraldehyde-3-phosphate dehydrogenase
(NAD(P)+) (phosphorylating) ec:1.2.1.62 4-formylbenzenesulfonate
dehydrogenase ec:1.2.1.63 6-oxohexanoate dehydrogenase ec:1.2.1.64
4-hydroxybenzaldehyde dehydrogenase ec:1.2.1.65 salicylaldehyde
dehydrogenase ec:1.2.1.66 mycothiol-dependent formaldehyde
dehydrogenase ec:1.2.1.67 vanillin dehydrogenase ec:1.2.1.68
coniferyl-aldehyde dehydrogenase ec:1.2.1.69 fluoroacetaldehyde
dehydrogenase ec:1.2.1.71 succinylglutamate-semialdehyde
dehydrogenase
[0068] Therefore, in addition to any of the various modifications
exemplified previously for establishing 4-HB biosynthesis in a
selected host, the BDO producing microbial organisms can include
any of the previous combinations and permutations of 4-HB pathway
metabolic modifications as well as any combination of expression
for CoA-independent aldehyde dehydrogenase, CoA-dependent aldehyde
dehydrogenase or an alcohol dehydrogenase to generate biosynthetic
pathways for BDO. Therefore, the BDO producers of the invention can
have exogenous expression of, for example, one, two, three, four,
five, six, seven, eight, nine or all 10 enzymes corresponding to
any of the six 4-HB pathway and/or any of the 4 BDO pathway
enzymes.
[0069] Design and construction of the genetically modified
microbial organisms is carried out using methods well known in the
art to achieve sufficient amounts of expression to produce BDO. In
particular, the non-naturally occurring microbial organisms of the
invention can achieve biosynthesis of BDO resulting in
intracellular concentrations between about 0.1-25 mM or more.
Generally, the intracellular concentration of BDO is between about
3-20 mM, particularly between about 5-15 mM and more particularly
between about 8-12 mM, including about 10 mM or more. Intracellular
concentrations between and above each of these exemplary ranges
also can be achieved from the non-naturally occurring microbial
organisms of the invention. As with the 4-HB producers, the BDO
producers also can be sustained, cultured or fermented under
anaerobic conditions.
[0070] The invention further provides a method for the production
of 4-HB. The method includes culturing a non-naturally occurring
microbial organism having a 4-hydroxybutanoic acid (4-HB)
biosynthetic pathway comprising at least one exogenous nucleic acid
encoding 4-hydroxybutanoate dehydrogenase, CoA-independent succinic
semialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependent
succinic semialdehyde dehydrogenase, glutamate:succinic
semialdehyde transaminase or glutamate decarboxylase under
substantially anaerobic conditions for a sufficient period of time
to produce monomeric 4-hydroxybutanoic acid (4-HB). The method can
additionally include chemical conversion of 4-HB to GBL and to BDO
or THF, for example.
[0071] It is understood that, in methods of the invention, any of
the one or more exogenous nucleic acids can be combined in a
non-naturally occurring microbial organism of the invention so long
as the desired product is produced, for example, 4-HB, BDO, THF or
GBL. For example, a non-naturally occurring microbial organism
having a 4-HB biosynthetic pathway can comprise at least two
exogenous nucleic acids encoding desired enzymes, such as the
combination of 4-hydroxybutanoate dehydrogenase and CoA-independent
succinic semialdehyde dehydrogenase; 4-hydroxybutanoate
dehydrogenase and CoA-dependent succinic semialdehyde
dehydrogenase; CoA-dependent succinic semialdehyde dehydrogenase
and succinyl-CoA synthetase; succinyl-CoA synthetase and glutamate
decarboxylase, and the like. Thus, it is understood that any
combination of two or more enzymes of a biosynthetic pathway can be
included in a non-naturally occurring microbial organism of the
invention. Similarly, it is understood that any combination of
three or more enzymes of a biosynthetic pathway can be included in
a non-naturally occurring microbial organism of the invention, for
example, 4-hydroxybutanoate dehydrogenase, CoA-independent succinic
semialdehyde dehydrogenase and succinyl-CoA synthetase;
4-hydroxybutanoate dehydrogenase, CoA-dependent succinic
semialdehyde dehydrogenase and glutamate:succinic semialdehyde
transaminase, and so forth, as desired, so long as the combination
of enzymes of the desired biosynthetic pathway results in
production of the corresponding desired product.
[0072] Any of the non-naturally occurring microbial organisms
described previously can be cultured to produce the biosynthetic
products of the invention. For example, the 4-HB producers can be
cultured for the biosynthetic production of 4-HB. The 4-HB can be
isolated or be treated as described below to generate GBL, THF
and/or BDO. Similarly, the BDO producers can be cultured for the
biosynthetic production of BDO. The BDO can be isolated or
subjected to further treatments for the chemical synthesis of BDO
family compounds such as those downstream compounds exemplified in
FIG. 3.
[0073] In some embodiments, culture conditions include anaerobic or
substantially anaerobic growth or maintenance conditions. Exemplary
anaerobic conditions have been described previously and are well
known in the art. Exemplary anaerobic conditions for fermentation
processes are described below in the Examples. Any of these
conditions can be employed with the non-naturally occurring
microbial organisms as well as other anaerobic conditions well
known in the art. Under such anaerobic conditions, the 4-HB and BDO
producers can synthesize monomeric 4-HB and BDO, respectively, at
intracellular concentrations of 5-10 mM or more as well as all
other concentrations exemplified previously.
[0074] A number of downstream compounds also can be generated for
the 4-HB and BDO producing non-naturally occurring microbial
organisms of the invention. With respect to the 4-HB producing
microbial organisms of the invention, monomeric 4-HB and GBL exist
in equilibrium in the culture medium. The conversion of 4-HB to GBL
can be efficiently accomplished by, for example, culturing the
microbial organisms in acid pH medium. A pH less than or equal to
7.5, in particular at or below pH 5.5, spontaneously converts 4-HB
to GBL as illustrated in FIG. 1.
[0075] The resultant GBL can be separated from 4-HB and other
components in the culture using a variety of methods well known in
the art. Such separation methods include, for example, the
extraction procedures exemplified in the Examples as well as
methods which include continuous liquid-liquid extraction,
pervaporation, membrane filtration, membrane separation, reverse
osmosis, electrodialysis, distillation, crystallization,
centrifugation, extractive filtration, ion exchange chromatography,
size exclusion chromatography, adsorption chromatography, and
ultrafiltration. All of the above methods are well known in the
art. Separated GBL can be further purified by, for example,
distillation.
[0076] Another down stream compound that can be produced from the
4-HB producing non-naturally occurring microbial organisms of the
invention includes, for example, BDO. This compound can be
synthesized by, for example, chemical hydrogenation of GBL.
Chemical hydrogenation reactions are well known in the art. One
exemplary procedure includes the chemical reduction of 4-HB and/or
GBL or a mixture of these two components deriving from the culture
using a heterogeneous or homogeneous hydrogenation catalyst
together with hydrogen, or a hydride-based reducing agent used
stoichiometrically or catalytically, to produce 1,4-butanediol.
[0077] Other procedures well known in the art are equally
applicable for the above chemical reaction and include, for
example, WO No. 82/03854 (Bradley, et al.), which describes the
hydrogenolysis of gamma-butyrolactone in the vapor phase over a
copper oxide and zinc oxide catalyst. British Pat. No. 1,230,276,
which describes the hydrogenation of gamma-butyrolactone using a
copper oxide-chromium oxide catalyst. The hydrogenation is carried
out in the liquid phase. Batch reactions also are exemplified
having high total reactor pressures. Reactant and product partial
pressures in the reactors are well above the respective dew points.
British Pat. No. 1,314,126, which describes the hydrogenation of
gamma-butyrolactone in the liquid phase over a
nickel-cobalt-thorium oxide catalyst. Batch reactions are
exemplified as having high total pressures and component partial
pressures well above respective component dew points. British Pat.
No. 1,344,557, which describes the hydrogenation of
gamma-butyrolactone in the liquid phase over a copper
oxide-chromium oxide catalyst. A vapor phase or vapor-containing
mixed phase is indicated as suitable in some instances. A
continuous flow tubular reactor is exemplified using high total
reactor pressures. British Pat. No. 1,512,751, which describes the
hydrogenation of gamma-butyrolactone to 1,4-butanediol in the
liquid phase over a copper oxide-chromium oxide catalyst. Batch
reactions are exemplified with high total reactor pressures and,
where determinable, reactant and product partial pressures well
above the respective dew points. U.S. Pat. No. 4,301,077, which
describes the hydrogenation to 1,4-butanediol of
gamma-butyrolactone over a Ru--Ni--Co--Zn catalyst. The reaction
can be conducted in the liquid or gas phase or in a mixed
liquid-gas phase. Exemplified are continuous flow liquid phase
reactions at high total reactor pressures and relatively low
reactor productivities. U.S. Pat. No. 4,048,196, which describes
the production of 1,4-butanediol by the liquid phase hydrogenation
of gamma-butyrolactone over a copper oxide-zinc oxide catalyst.
Further exemplified is a continuous flow tubular reactor operating
at high total reactor pressures and high reactant and product
partial pressures. And U.S. Pat. No. 4,652,685, which describes the
hydrogenation of lactones to glycols.
[0078] A further downstream compound that can be produced form the
4-HB producing microbial organisms of the invention includes, for
example, THF. This compound can be synthesized by, for example,
chemical hydrogenation of GBL. One exemplary procedure well known
in the art applicable for the conversion of GBL to THF includes,
for example, chemical reduction of 4-HB and/or GBL or a mixture of
these two components deriving from the culture using a
heterogeneous or homogeneous hydrogenation catalyst together with
hydrogen, or a hydride-based reducing agent used stoichiometrically
or catalytically, to produce tetrahydrofuran. Other procedures well
know in the art are equally applicable for the above chemical
reaction and include, for example, U.S. Pat. No. 6,686,310, which
describes high surface area sol-gel route prepared hydrogenation
catalysts. Processes for the reduction of gamma butyrolactone to
tetrahydrofuran and 1,4-butanediol also are described.
[0079] The culture conditions can include, for example, liquid
culture procedures as well as fermentation and other large scale
culture procedures. As described further below in the Examples,
particularly useful yields of the biosynthetic products of the
invention can be obtained under anaerobic or substantially
anaerobic culture conditions.
[0080] The invention further provides a method of manufacturing
4-HB. The method includes fermenting a non-naturally occurring
microbial organism having a 4-hydroxybutanoic acid (4-HB)
biosynthetic pathway comprising at least one exogenous nucleic acid
encoding 4-hydroxybutanoate dehydrogenase, CoA-independent succinic
semialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependent
succinic semialdehyde dehydrogenase, glutamate:succinic
semialdehyde transaminase or glutamate decarboxylase under
substantially anaerobic conditions for a sufficient period of time
to produce monomeric 4-hydroxybutanoic acid (4-HB), the process
comprising fed-batch fermentation and batch separation; fed-batch
fermentation and continuous separation, or continuous fermentation
and continuous separation.
[0081] The culture and chemical hydrogenations described above also
can be scaled up and grown continuously for manufacturing of 4-HB,
GBL, BDO and/or THF. Exemplary growth procedures include, for
example, fed-batch fermentation and batch separation; fed-batch
fermentation and continuous separation, or continuous fermentation
and continuous separation. All of these processes are well known in
the art. Employing the 4-HB producers allows for simultaneous 4-HB
biosynthesis and chemical conversion to GBL, BDO and/or THF by
employing the above hydrogenation procedures simultaneous with
continuous cultures methods such as fermentation. Other
hydrogenation procedures also are well known in the art and can be
equally applied to the methods of the invention.
[0082] Fermentation procedures are particularly useful for the
biosynthetic production of commercial quantities of 4-HB and/or
BDO. Generally, and as with non-continuous culture procedures, the
continuous and/or near-continuous production of 4-HB or BDO will
include culturing a non-naturally occurring 4-HB or BDO producing
organism of the invention in sufficient neutrients and medium to
sustain and/or nearly sustain growth in an exponential phase.
Continuous culture under such conditions can be include, for
example, 1 day, 2, 3, 4, 5, 6 or 7 days or more. Additionally,
continuous culture can include 1 week, 2, 3, 4 or 5 or more weeks
and up to several months. Alternatively, organisms of the invention
can be cultured for hours, if suitable for a particular
application. It is to be understood that the continuous and/or
near-continuous culture conditions also can include all time
intervals in between these exemplary periods.
[0083] Fermentation procedures are well known in the art. Briefly,
fermentation for the biosynthetic production of 4-HB, BDO or other
4-HB derived products of the invention can be utilized in, for
example, fed-batch fermentation and batch separation; fed-batch
fermentation and continuous separation, or continuous fermentation
and continuous separation. Examples of batch and continuous
fermentation procedures well known in the art are exemplified
further below in the Examples.
[0084] In addition, to the above fermentation procedures using the
4-HB or BDO producers of the invention for continuous production of
substantial quantities of monomeric 4-HB and BDO, respectively, the
4-HB producers also can be, for example, simultaneously subjected
to chemical synthesis procedures as described previously for the
chemical conversion of monomeric 4-HB to, for example, GBL, BDO
and/or THF. The BDO producers can similarly be, for example,
simultaneously subjected to chemical synthesis procedures as
described previously for the chemical conversion of BDO to, for
example, THF, GBL, pyrrolidones and/or other BDO family compounds.
In addition, the products of the 4-HB and BDO producers can be
separated from the fermentation culture and sequentially subjected
to chemical conversion, as disclosed herein.
[0085] Briefly, hydrogenation of GBL in the fermentation broth can
be performed as described by Frost et al., Biotechnology Progress
18: 201-211 (2002). Another procedure for hydrogenation during
fermentation include, for example, the methods described in, for
example, U.S. Pat. No. 5,478,952. This method is further
exemplified in the Examples below.
[0086] Therefore, the invention additionally provides a method of
manufacturing .gamma.-butyrolactone (GBL), tetrahydrofuran (THF) or
1,4-butanediol (BDO). The method includes fermenting a
non-naturally occurring microbial organism having 4-hydroxybutanoic
acid (4-FIB) and 1,4-butanediol (BDO) biosynthetic pathways, the
pathways comprise at least one exogenous nucleic acid encoding
4-hydroxybutanoate dehydrogenase, CoA-independent succinic
semialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependent
succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA
transferase, glutamate:succinic semialdehyde transaminase,
glutamate decarboxylase, CoA-independent 1,4-butanediol
semialdehyde dehydrogenase, CoA-dependent 1,4-butanediol
semialdehyde dehydrogenase, 1,4-butanediol alcohol dehydrogenase,
either CoA-dependent or independent, under substantially anaerobic
conditions for a sufficient period of time to produce
1,4-butanediol (BDO), GBL or THF, the fermenting comprising
fed-batch fermentation and batch separation; fed-batch fermentation
and continuous separation, or continuous fermentation and
continuous separation.
[0087] In addition to the biosynthesis of 4-HB, BDO and other
products of the invention as described herein, the non-naturally
occurring microbial organisms and methods of the invention also can
be utilized in various combinations with each other and with other
microbial organisms and methods well known in the art to achieve
product biosynthesis by other routes. For example, one alternative
to produce BDO other than use of the 4-HB producers and chemical
steps or other than use of the BDO producer directly is through
addition of another microbial organism capable of converting 4-HB
or a 4-HB product exemplified herein to BDO.
[0088] One such procedure includes, for example, the fermentation
of a 4-HB producing microbial organism of the invention to produce
4-HB, as described above and below. The 4-HB can then be used as a
substrate for a second microbial organism that converts 4-HB to,
for example, BDO, GBL and/or THF. The 4-HB can be added directly to
another culture of the second organism or the original culture of
4-HB producers can be depleted of these microbial organisms by, for
example, cell separation, and then subsequent addition of the
second organism to the fermentation broth can utilized to produce
the final product without intermediate purification steps. One
exemplary second organism having the capacity to biochemically
utilize 4-HB as a substrate for conversion to BDO, for example, is
Clostridium acetobutylicum (see, for example, Jewell et al.,
Current Microbiology, 13:215-19 (1986)).
[0089] In other embodiments, the non-naturally occurring microbial
organisms and methods of the invention can be assembled in a wide
variety of subpathways to achieve biosynthesis of, for example,
4-HB and/or BDO as described. In these embodiments, biosynthetic
pathways for a desired product of the invention can be segregated
into different microbial organisms and the different microbial
organisms can be co-cultured to produce the final product. In such
a biosynthetic scheme, the product of one microbial organism is the
substrate for a second microbial organism until the final product
is synthesized. For example, the biosynthesis of BDO can be
accomplished as described previously by constructing a microbial
organism that contains biosynthetic pathways for conversion of a
substrate such as endogenous succinate through 4-HB to the final
product BDO. Alternatively, BDO also can be biosynthetically
produced from microbial organisms through co-culture or
co-fermentation using two organisms in the same vessel. A first
microbial organism being a 4-HB producer with genes to produce 4-HB
from succinic acid, and a second microbial organism being a BDO
producer with genes to convert 4-HB to BDO.
[0090] Given the teachings and guidance provided herein, those
skilled in the art will understand that a wide variety of
combinations and permutations exist for the non-naturally occurring
microbial organisms and methods of the invention together with
other microbial organisms, with the co-culture of other
non-naturally occurring microbial organisms having subpathways and
with combinations of other chemical and/or biochemical procedures
well known in the art to produce 4-HB, BDO, GBL and THF products of
the invention.
[0091] One computational method for identifying and designing
metabolic alterations favoring biosynthesis of a product is the
OptKnock computational framework, Burgard et al., Biotechnol
Bioeng, 84: 647-57 (2003). OptKnock is a metabolic modeling and
simulation program that suggests gene deletion strategies that
result in genetically stable microorganisms which overproduce the
target product. Specifically, the framework examines the complete
metabolic and/or biochemical network of a microorganism in order to
suggest genetic manipulations that force the desired biochemical to
become an obligatory byproduct of cell growth. By coupling
biochemical production with cell growth through strategically
placed gene deletions or other functional gene disruption, the
growth selection pressures imposed on the engineered strains after
long periods of time in a bioreactor lead to improvements in
performance as a result of the compulsory growth-coupled
biochemical production. Lastly, when gene deletions are constructed
there is a negligible possibility of the designed strains reverting
to their wild-type states because the genes selected by OptKnock
are to be completely removed from the genome. Therefore, this
computational methodology can be used to either identify
alternative pathways that lead to biosynthesis of 4-HB and/or BDO
or used in connection with the non-naturally occurring microbial
organisms for further optimization of 4-HB and/or BDO
biosynthesis.
[0092] Briefly, OptKnock is a term used herein to refer to a
computational method and system for modeling cellular metabolism.
The OptKnock program relates to a framework of models and methods
that incorporate particular constraints into flux balance analysis
(FBA) models. These constraints include, for example, qualitative
kinetic information, qualitative regulatory information, and/or DNA
microarray experimental data. OptKnock also computes solutions to
various metabolic problems by, for example, tightening the flux
boundaries derived through flux balance models and subsequently
probing the performance limits of metabolic networks in the
presence of gene additions or deletions. OptKnock computational
framework allows the construction of model formulations that enable
an effective query of the performance limits of metabolic networks
and provides methods for solving the resulting mixed-integer linear
programming problems. The metabolic modeling and simulation methods
referred to herein as OptKnock are described in, for example, U.S.
patent application Ser. No. 10/043,440, filed Jan. 10, 2002, and in
International Patent No. PCT/US02/00660, filed Jan. 10, 2002.
[0093] Another computational method for identifying and designing
metabolic alterations favoring biosynthetic production of a product
is metabolic modeling and simulation system termed SimPheny.RTM..
This computational method and system is described in, for example,
U.S. patent application Ser. No. 10/173,547, filed Jun. 14, 2002,
and in International Patent Application No. PCT/US03/18838, filed
Jun. 13, 2003.
[0094] SimPheny.RTM. is a computational system that can be used to
produce a network model in silico and to simulate the flux of mass,
energy or charge through the chemical reactions of a biological
system to define a solution space that contains any and all
possible functionalities of the chemical reactions in the system,
thereby determining a range of allowed activities for the
biological system. This approach is referred to as
constraints-based modeling because the solution space is defined by
constraints such as the known stoichiometry of the included
reactions as well as reaction thermodynamic and capacity
constraints associated with maximum fluxes through reactions. The
space defined by these constraints can be interrogated to determine
the phenotypic capabilities and behavior of the biological system
or of its biochemical components. Analysis methods such as convex
analysis, linear programming and the calculation of extreme
pathways as described, for example, in Schilling et al., J. Theor.
Biol. 203:229-248 (2000); Schilling et al., Biotech. Bioeng.
71:286-306 (2000) and Schilling et al., Biotech. Prog. 15:288-295
(1999), can be used to determine such phenotypic capabilities. As
described in the Examples below, this computation methodology was
used to identify and analyze the feasible as well as the optimal
4-HB biosynthetic pathways in 4-HB non-producing microbial
organisms.
[0095] As described above, one constraints-based method used in the
computational programs applicable to the invention is flux balance
analysis. Flux balance analysis is based on flux balancing in a
steady state condition and can be performed as described in, for
example, Varma and Palsson, Biotech. Bioeng. 12:994-998 (1994).
Flux balance approaches have been applied to reaction networks to
simulate or predict systemic properties of, for example, adipocyte
metabolism as described in Fell and Small, J. Biochem. 138:781-786
(1986), acetate secretion from E. coli under ATP maximization
conditions as described in Majewski and Domach, Biotech. Bioeng.
35:732-738 (1990) or ethanol secretion by yeast as described in
Vanrolleghem et al., Biotech. Prog. 12:434-448 (1996).
Additionally, this approach can be used to predict or simulate the
growth of E. coli on a variety of single-carbon sources as well as
the metabolism of H. influenzae as described in Edwards and
Palsson, Proc. Natl. Acad. Sci. USA 97:5528-5533 (2000), Edwards
and Palsson, J. Biol. Chem. 274:17410-17416 (1999) and Edwards et
al., Nature Biotech. 19:125-130 (2001).
[0096] Once the solution space has been defined, it can be analyzed
to determine possible solutions under various conditions. This
computational approach is consistent with biological realities
because biological systems are flexible and can reach the same
result in many different ways. Biological systems are designed
through evolutionary mechanisms that have been restricted by
fundamental constraints that all living systems must face.
Therefore, constraints-based modeling strategy embraces these
general realities. Further, the ability to continuously impose
further restrictions on a network model via the tightening of
constraints results in a reduction in the size of the solution
space, thereby enhancing the precision with which physiological
performance or phenotype can be predicted.
[0097] Given the teachings and guidance provided herein, those
skilled in the art will be able to apply various computational
frameworks for metabolic modeling and simulation to design and
implement biosynthesis of 4-HB, BDO, GBL, THF and other BDO family
compounds in host microbial organisms other than E. coli and yeast.
Such metabolic modeling and simulation methods include, for
example, the computational systems exemplified above as
SimPheny.RTM. and OptKnock. For illustration of the invention, some
methods are described herein with reference to the OptKnock
computation framework for modeling and simulation. Those skilled in
the art will know how to apply the identification, design and
implementation of the metabolic alterations using OptKnock to any
of such other metabolic modeling and simulation computational
frameworks and methods well known in the art.
[0098] The ability of a cell or organism to biosynthetically
produce a biochemical product can be illustrated in the context of
the biochemical production limits of a typical metabolic network
calculated using an in silico model. These limits are obtained by
fixing the uptake rate(s) of the limiting substrate(s) to their
experimentally measured value(s) and calculating the maximum and
minimum rates of biochemical production at each attainable level of
growth. The production of a desired biochemical generally is in
direct competition with biomass formation for intracellular
resources. Under these circumstances, enhanced rates of biochemical
production will necessarily result in sub-maximal growth rates. The
knockouts suggested by the above metabolic modeling and simulation
programs such as OptKnock are designed to restrict the allowable
solution boundaries forcing a change in metabolic behavior from the
wild-type strain. Although the actual solution boundaries for a
given strain will expand or contract as the substrate uptake
rate(s) increase or decrease, each experimental point will lie
within its calculated solution boundary. Plots such as these enable
accurate predictions of how close the designed strains are to their
performance limits which also indicates how much room is available
for improvement.
[0099] The OptKnock mathematical framework is exemplified herein
for pinpointing gene deletions leading to product biosynthesis and,
particularly, growth-coupled product biosynthesis. The procedure
builds upon constraint-based metabolic modeling which narrows the
range of possible phenotypes that a cellular system can display
through the successive imposition of governing physico-chemical
constraints, Price et al., Nat. Rev. Microbiol., 2: 886-97 (2004).
As described above, constraint-based models and simulations are
well known in the art and generally invoke the optimization of a
particular cellular objective, subject to network stoichiometry, to
suggest a likely flux distribution.
[0100] Briefly, the maximization of a cellular objective quantified
as an aggregate reaction flux for a steady state metabolic network
comprising a set N={1, . . . , N} of metabolites and a set M={1, .
. . , M} of metabolic reactions is expressed mathematically as
follows:
maximize v cellular objective ##EQU00001## subject to j = 1 M S ij
v j = 0 , .A-inverted. i .di-elect cons. N ##EQU00001.2## v
substrate = v substrate_uptake mmol / gDQ hr ##EQU00001.3##
.A-inverted. i .di-elect cons. { limiting substrate ( s ) }
##EQU00001.4## v atp .gtoreq. v atp_main mmol / gDW hr
##EQU00001.5## v j .gtoreq. 0 , .A-inverted. j .di-elect cons. {
irrev . reactions } ##EQU00001.6##
[0101] where S.sub.ij is the stoichiometric coefficient of
metabolite i in reaction j, v.sub.j is the flux of reaction j,
v.sub.substrate_uptake represents the assumed or measured uptake
rate(s) of the limiting substrate(s), and v.sub.atp_main is the
non-growth associated ATP maintenance requirement. The vector v
includes both internal and external fluxes. In this study, the
cellular objective is often assumed to be a drain of biosynthetic
precursors in the ratios required for biomass formation, Neidhardt,
F. C. et al., Escherichia coli and Salmonella: Cellular and
Molecular Biology, 2nd ed. 1996, Washington, D.C.: ASM Press. 2 v.
(xx, 2822, lxxvi). The fluxes are generally reported per 1 gDWhr
(gram of dry weight times hour) such that biomass formation is
expressed as g biomass produced/gDWhr or l/hr.
[0102] The modeling of gene deletions, and thus reaction
elimination, first employs the incorporation of binary variables
into the constraint-based approach framework, Burgard et al.,
Biotechnol. Bioeng., 74: 364-375 (2001), Burgard et al.,
Biotechnol. Prog., 17: 791-797 (2001). These binary variables,
y j = { 1 , if reaction flux v j is active 0 , if reaction flux v j
is not active , .A-inverted. j .di-elect cons. M ##EQU00002##
assume a value of 1 if reaction j is active and a value of 0 if it
is inactive. The following constraint,
v.sub.j.sup.miny.sub.j.ltoreq.v.sub.j.ltoreq.v.sub.j.sup.maxy.sub.j,.A-i-
nverted.j.di-elect cons.M
ensures that reaction flux v.sub.j is set to zero only if variable
y.sub.j is equal to zero. Alternatively, when y.sub.j is equal to
one, v.sub.j is free to assume any value between a lower
v.sub.j.sup.min and an upper v.sub.j.sup.max bound. Here,
v.sub.j.sup.min and v.sub.j.sup.max are identified by minimizing
and maximizing, respectively, every reaction flux subject to the
network constraints described above, Mahadevan et al., Metab. Eng.,
5: 264-76 (2003).
[0103] Optimal gene/reaction knockouts are identified by solving a
bilevel optimization problem that chooses the set of active
reactions (y.sub.j=1) such that an optimal growth solution for the
resulting network overproduces the chemical of interest.
Mathematically, this bilevel optimization problem is expressed as
the following bilevel mixed-integer optimization problem:
maximize y j v chemical ( OptKnock ) ##EQU00003## ( subject to v j
maximize v biomass subject to j = 1 M S ij v j = 0 , .A-inverted. i
.di-elect cons. N v substrate = v substrate_uptake .A-inverted. i
.di-elect cons. { limiting substrate ( s ) } v atp .gtoreq. v
atp_main ) ##EQU00003.2## v biomass .gtoreq. v biomass target
##EQU00003.3## v j min y j .ltoreq. v j .ltoreq. v j max y j ,
.A-inverted. j .di-elect cons. M ##EQU00003.4## j .di-elect cons. M
forward ( 1 - y j ) = K ##EQU00003.5## y j .di-elect cons. { 0 , 1
} , .A-inverted. j .di-elect cons. M ##EQU00003.6##
where v.sub.chemical is the production of the desired target
product, for example succinate or other biochemical product, and K
is the number of allowable knockouts. Note that setting K equal to
zero returns the maximum biomass solution of the complete network,
while setting K equal to one identifies the single gene/reaction
knockout (y.sub.j=0) such that the resulting network involves the
maximum overproduction given its maximum biomass yield. The final
constraint ensures that the resulting network meets a minimum
biomass yield. Burgard et al., Biotechnol. Bioeng., 84: 647-57
(2003), provide a more detailed description of the model
formulation and solution procedure. Problems containing hundreds of
binary variables can be solved in the order of minutes to hours
using CPLEX 8.0, GAMS: The Solver Manuals. 2003: GAMS Development
Corporation, accessed via the GAMS, Brooke et al., GAMS Development
Corporation (1998), modeling environment on an IBM RS6000-270
workstation. The OptKnock framework has already been able to
identify promising gene deletion strategies for biochemical
overproduction, Burgard et al., Biotechnol. Bioeng., 84: 647-57
(2003), Pharkya et al., Biotechnol. Bioeng., 84: 887-899 (2003),
and establishes a systematic framework that will naturally
encompass future improvements in metabolic and regulatory modeling
frameworks.
[0104] Any solution of the above described bilevel OptKnock problem
will provide one set of metabolic reactions to disrupt. Elimination
of each reaction within the set or metabolic modification can
result in 4-HB or BDO as an obligatory product during the growth
phase of the organism. Because the reactions are known, a solution
to the bilevel OptKnock problem also will provide the associated
gene or genes encoding one or more enzymes that catalyze each
reaction within the set of reactions. Identification of a set of
reactions and their corresponding genes encoding the enzymes
participating in each reaction is generally an automated process,
accomplished through correlation of the reactions with a reaction
database having a relationship between enzymes and encoding
genes.
[0105] Once identified, the set of reactions that are to be
disrupted in order to achieve 4-HB or BDO production are
implemented in the target cell or organism by functional disruption
of at least one gene encoding each metabolic reaction within the
set. One particularly useful means to achieve functional disruption
of the reaction set is by deletion of each encoding gene. However,
in some instances, it can be beneficial to disrupt the reaction by
other genetic aberrations including, for example, mutation,
deletion of regulatory regions such as promoters or cis binding
sites for regulatory factors, or by truncation of the coding
sequence at any of a number of locations. These latter aberrations,
resulting in less than total deletion of the gene set can be
useful, for example, when rapid assessments of the succinate
coupling are desired or when genetic reversion is less likely to
occur.
[0106] To identify additional productive solutions to the above
described bilevel OptKnock problem which lead to further sets of
reactions to disrupt or metabolic modifications that can result in
the biosynthesis, including growth-coupled biosynthesis of 4-HB or
other biochemical product, an optimization method, termed integer
cuts, can be implemented. This method proceeds by iteratively
solving the OptKnock problem exemplified above with the
incorporation of an additional constraint referred to as an integer
cut at each iteration. Integer cut constraints effectively prevent
the solution procedure from choosing the exact same set of
reactions identified in any previous iteration that obligatory
couples product biosynthesis to growth. For example, if a
previously identified growth-coupled metabolic modification
specifies reactions 1, 2, and 3 for disruption, then the following
constraint prevents the same reactions from being simultaneously
considered in subsequent solutions:
y.sub.1+y.sub.2+y.sub.3.gtoreq.1. The integer cut method is well
known in the art and can be found described in, for example,
reference, Burgard et al., Biotechnol. Prog., 17: 791-797 (2001).
As with all methods described herein with reference to their use in
combination with the OptKnock computational framework for metabolic
modeling and simulation, the integer cut method of reducing
redundancy in iterative computational analysis also can be applied
with other computational frameworks well known in the art
including, for example, SimPheny.RTM..
[0107] Constraints of the above form preclude identification of
larger reaction sets that include previously identified sets. For
example, employing the integer cut optimization method above in a
further iteration would preclude identifying a quadruple reaction
set that specified reactions 1, 2, and 3 for disruption since these
reactions had been previously identified. To ensure identification
of all possible reaction sets leading to biosynthetic production of
a product, a modification of the integer cut method can be
employed.
[0108] Briefly, the modified integer cut procedure begins with
iteration `zero` which calculates the maximum production of the
desired biochemical at optimal growth for a wild-type network. This
calculation corresponds to an OptKnock solution with K equaling 0.
Next, single knockouts are considered and the two parameter sets,
objstore.sub.iter and ystore.sub.iter,j, are introduced to store
the objective function (v.sub.chemical) and reaction on-off
information (y.sub.j), respectively, at each iteration, iter. The
following constraints are then successively added to the OptKnock
formulation at each iteration.
v.sub.chemical.gtoreq.objstore.sub.iter+.epsilon.-M.SIGMA..sub.j.di-elec-
t cons.ystore.sub.iter,j.sub.=0y.sub.j
[0109] In the above equation, .epsilon. and M are a small and a
large numbers, respectively. In general, .epsilon. can be set at
about 0.01 and M can be set at about 1000. However, numbers smaller
and/or larger then these numbers also can be used. M ensures that
the constraint can be binding only for previously identified
knockout strategies, while .epsilon. ensures that adding knockouts
to a previously identified strategy must lead to an increase of at
least .epsilon. in biochemical production at optimal growth. The
approach moves onto double deletions whenever a single deletion
strategy fails to improve upon the wild-type strain. Triple
deletions are then considered when no double deletion strategy
improves upon the wild-type strain, and so on. The end result is a
ranked list, represented as desired biochemical production at
optimal growth, of distinct deletion strategies that differ from
each other by at least one knockout. This optimization procedure as
well as the identification of a wide variety of reaction sets that,
when disrupted, lead to the biosynthesis, including growth-coupled
production, of a biochemical product. Given the teachings and
guidance provided herein, those skilled in the art will understand
that the methods and metabolic engineering designs exemplified
herein are equally applicable to identify new biosynthetic pathways
and/or to the obligatory coupling of cell or microorganism growth
to any biochemical product.
[0110] The methods exemplified above and further illustrated in the
Examples below enable the construction of cells and organisms that
biosynthetically produce, including obligatory couple production of
a target biochemical product to growth of the cell or organism
engineered to harbor the identified genetic alterations. In this
regard, metabolic alterations have been identified that result in
the biosynthesis of 4-HB and 1,4-butanediol. Microorganism strains
constructed with the identified metabolic alterations produce
elevated levels of 4-HB or BDO compared to unmodified microbial
organisms. These strains can be beneficially used for the
commercial production of 4-HB, BDO, THF and GBL, for example, in
continuous fermentation process without being subjected to the
negative selective pressures.
[0111] Therefore, the computational methods described herein enable
the identification and implementation of metabolic modifications
that are identified by an in silico method selected from OptKnock
or SimPheny. The set of metabolic modifications can include, for
example, addition of one or more biosynthetic pathway enzymes
and/.or functional disruption of one or more metabolic reactions
including, for example, disruption by gene deletion.
[0112] Application of the OptKock method to identify pathways
suitable for growth-coupled production of 1,4-butanediol (BDO) is
described in Examples V-VII. As discussed above, the OptKnock
methodology was developed on the premise that mutant microbial
networks can be evolved towards their computationally predicted
maximum-growth phenotypes when subjected to long periods of growth
selection. In other words, the approach leverages an organism's
ability to self-optimize under selective pressures. The OptKnock
framework allows for the exhaustive enumeration of gene deletion
combinations that force a coupling between biochemical production
and cell growth based on network stoichiometry. The identification
of optimal gene/reaction knockouts requires the solution of a
bilevel optimization problem that chooses the set of active
reactions such that an optimal growth solution for the resulting
network overproduces the biochemical of interest (Burgard et al.
Biotechnol. Bioeng. 84:647-657 (2003)).
[0113] Growth-coupled biochemical production can be visualized in
the context of the biochemical production limits of a typical
metabolic network. These limits are obtained from an in silico
metabolic model by fixing the uptake rate(s) of the limiting
substrate(s) to their experimentally measured value(s) and
calculating the maximum and minimum rates of biochemical production
at each attainable level of growth. The production envelopes
essentially bracket what is possible by encompassing all potential
biological phenotypes available to a given strain. Although
exceptions exist, typically the production of a desired biochemical
is in direct competition with biomass formation for intracellular
resources (see FIG. 5, gray region). Thus increased biochemical
yields will necessarily result in sub-maximal growth. Furthermore,
the application of growth selective pressures may drive the
performance of a non-growth-coupled production strain towards a low
producing phenotype (point A, FIG. 5), regardless of its initial
starting point. The knockouts suggested by OptKnock are designed to
restrict the allowable solution boundary, forcing a change in
metabolic behavior as depicted in FIG. 5 (cyan region).
Evolutionary engineering approaches can thus be applied to drive
the performance of an OptKnock designed strain to the rightmost
boundary. This will result in a high producing strain that should
be inherently stable to further growth selective pressures (point
B, FIG. 5).
[0114] An in silico stoichiometric model of E. coli metabolism was
employed to identify essential genes for metabolic pathways as
exemplified previously and described in, for example, U.S. patent
publications US 2002/0012939, US 2003/0224363, US 2004/0029149, US
2004/0072723, US 2003/0059792, US 2002/0168654 and US 2004/0009466,
and in U.S. Pat. No. 7,127,379. As disclosed herein, the OptKnock
mathematical framework was applied to pinpoint gene deletions
leading to the growth-coupled production of BDO (see Examples
V-VII). The strategies were initially determined by employing a
reduced model of the E. coli metabolic network. This "small" model
captures the key metabolic functionalities in the network, thus
eliminating the redundancy associated with the genome-scale
metabolic networks. Care was taken to ensure that the model was not
reduced to the point where potentially active pathways possessing
viable targets were neglected. Overall, the reduced model contained
262 reactions and its implementation reduced OptKnock CPU times
approximately ten-fold when compared to the application of OptKnock
to the genome-scale E. coli model (Reed et al., Genome Biol. 4:R54
(2003)).
[0115] Further, the solution of the bilevel OptKnock problem
provides only one set of deletions. To enumerate all meaningful
solutions, that is, all sets of knockouts leading to growth-coupled
production formation, an optimization technique, termed integer
cuts, can be implemented. This entails iteratively solving the
OptKnock problem with the incorporation of an additional constraint
referred to as an integer cut at each iteration, as discussed
above.
[0116] For biochemical pathways to 1,4-butanediol, the conversion
of glucose to BDO in E. coli is expected to derive from a total of
three intracellular reduction steps from succinate semialdehyde
(see FIG. 6). Succinate semialdehyde is natively produced by E.
coli through the TCA cycle intermediate, alpha-ketoglutarate, via
the action of two enzymes, glutamate:succinic semialdehyde
transaminase and glutamate decarboxylase. An alternative pathway,
used by the obligate anaerobe Clostridium kluyveri to degrade
succinate, activates succinate to succinyl-CoA, and then converts
succinyl-CoA to succinic semialdehyde using a CoA-dependant
succinic semialdehyde dehydrogenase (Sohling and Gottschalk, Eur.
J. Biochem. 212:121-127 (1993)). The conversion of succinate
semialdehyde to BDO first requires the activity of
4-hydroxybutanoate (4-HB) dehydrogenase, an enzyme which is not
native to E. coli or yeast but is found in various bacteria such as
C. kluyveri and Ralstonia eutropha (Lutke-Eversloh and Steinbuchel,
FEMS Microbiol. Lett. 181:63-71 (1999); Sohling and Gottschalk, J.
Bacteriol. 178:871-880 (1996); Valentin et al., Eur. J. Biochem.
227:43-60 (1995); Wolff and Kenealy, Protein Expr. Purif. 6:206-212
(1995)). Precedent for the 4-HB to BDO conversion has been
demonstrated in the strict anaerobe, Clostridium acetobutylicum,
which when fed 4-HB, was shown to quantitatively produce BDO
(Jewell et al., Current Microbiology 13:215-219 (1986)). The
required biotransformations from 4-HB to BDO are assumed to be
similar to those of the butyric acid to butanol conversion common
to Clostridia species which proceeds via a CoA-derivative (Girbal
et al., FEMS Microbiology Reviews 17:287-297 (1995)).
[0117] In an additional embodiment, the invention provides a
non-naturally occurring microorganism comprising one or more gene
disruptions, the one or more gene disruptions occurring in genes
encoding an enzyme obligatory to coupling 1,4-butanediol production
to growth of the microorganism when the gene disruption reduces an
activity of the enzyme, whereby the one or more gene disruptions
confers stable growth-coupled production of 1,4-butanediol onto the
non-naturally occurring microorganism. The one or more gene
disruptions can be, for example, those disclosed in Table 6 or 7.
The one or more gene disruptions can comprise a deletion of the one
or more genes, such as those genes disclosed in Table 6 or 7.
[0118] As disclosed herein, the non-naturally occurring
microorganism can be a bacterium, yeast or fungus. For example, the
non-naturally occurring microorganism can be a bacterium such as
Escherichia coli, Klebsiella oxytoca, Anaerobiospirillum
succiniciproducens, Actinobacillus succinogenes, Mannheimia
succiniciproducens, Rhizobium etli, Bacillus subtilis,
Corynebacterium glutamicum, Gluconobacter oxydans, Zymomonas
mobilis, Lactococcus lactis, Lactobacillus plantarum, Streptomyces
coelicolor, Clostridium acetobutylicum, Pseudomonas fluorescens,
and Pseudomonas putida. The non-naturally occurring microorganism
can also be a yeast such as Saccharomyces cerevisiae,
Schizosaccharomyces pombe, Kluyveromyces lactis, Kluyveromyces
marxianus, Aspergillus terreus, Aspergillus niger, and Pichia
pastoris.
[0119] A non-naturally occurring microorganism of the invention can
comprise a set of metabolic modifications obligatory coupling
1,4-butanediol production to growth of the microorganism, the set
of metabolic modifications comprising disruption of one or more
genes selected from the set of genes comprising adhE, ldhA, pflAB;
adhE, ldhA, pflAB, mdh; adhE, ldhA, pflAB, mdh, mqo; adhE, ldhA,
pflAB, mdh, aspA; adhE, mdh, ldhA, pflAB, sfcA; adhE, mdh, ldhA,
pflAB, maeB; adhE, mdh, ldhA, pflAB, sfcA, maeB; adhE, ldhA, pflAB,
mdh, pntAB; adhE, ldhA, pflAB, mdh, gdhA; adhE, ldhA, pflAB, mdh,
pykA, pykF, dhaKLM, deoC, edd, yiaE, ycdW; and adhE, ldhA, pflAB,
mdh, pykA, pykF, dhaKLM, deoC, edd, yiaE, ycdW, prpC, gsk, or an
ortholog thereof, wherein the microorganism exhibits stable
growth-coupled production of 1,4-butanediol.
[0120] In one embodiment, the invention provides a non-naturally
occurring microorganism comprising a set of metabolic modifications
obligatory to coupling 1,4-butanediol production to growth of the
microorganism, the set of metabolic modifications comprising
disruption of one or more genes, or an ortholog thereof, wherein
the set of metabolic modifications comprises disruption of adhE and
ldhA, wherein the microorganism exhibits stable growth-coupled
production of 1,4-butanediol. In an additional embodiment, the set
of metabolic modifications can further comprise disruption of mdh.
In another embodiment, the set of metabolic modifications can
further comprise disruption of one or more genes selected from the
set of genes comprising mqo, aspA, sfcA, maeB, pntAB, and gdhA and
can include, for example, disruption of sfcA and maeB. In still
another embodiment, the set of metabolic modifications can further
comprise disruption of one or more genes selected from the set of
genes comprising pykA, pykF, dhaKLM, deoC, edd, yiaE, ycdW, prpC,
and gsk, including disruption of all of pykA, pykF, dhaKLM, deoC,
edd, yiaE and ycdW, and can further comprise disruption of prpC and
gsk. Any of the above-described set of metabolic modifications can
further comprise disruption of pflAB. In a particular embodiment,
the set of metabolic modifications comprise disruption of one or
more genes selected from the set of genes comprising adhE, ldhA,
pflAB, mdh, and aspA, including up to all of genes adhE, ldhA,
pflAB, mdh, and aspA.
[0121] A non-naturally occurring microorganism of the invention can
further comprise a 1,4-butanediol (BDO) biosynthetic pathway
comprising at least one exogenous nucleic acid encoding
4-hydroxybutanoate dehydrogenase, CoA-independent succinic
semialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependent
succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA
transferase, glutamate:succinic semialdehyde transaminase,
glutamate decarboxylase, CoA-independent aldehyde dehydrogenase,
CoA-dependent aldehyde dehydrogenase or alcohol dehydrogenase,
wherein the exogenous nucleic acid is expressed in sufficient
amounts to produce 1,4-butanediol (BDO), as disclosed herein.
[0122] The invention additionally provides a method of producing a
non-naturally occurring microorganism having stable growth-coupled
production of 1,4-butanediol by identifying in silico a set of
metabolic modifications requiring 1,4-butanediol production during
exponential growth under a defined set of conditions, and
genetically modifying a microorganism to contain the set of
metabolic modifications requiring 1,4-butanediol production. Such
methods can additionally include the addition of exogenous genes
expressing desired enzyme activities to a microorganism. Such a set
of metabolic modifications can be identified by an in silico method
selected from OptKnock or SimPheny (see above and Examples
V-VII).
[0123] The invention additionally provides a microorganism produced
by the methods disclosed herein. Furthermore, the invention
provides a method of producing 1,4-butanediol coupled to the growth
of a microorganism. The method can include the steps of culturing
under exponential growth phase in a sufficient amount of nutrients
and media a non-naturally occurring microorganism comprising a set
of metabolic modifications obligatorily coupling 1,4-butanediol
production to growth of the microorganism, wherein the
microorganism exhibits stable growth-coupled production of
1,4-butanediol, and isolating 1,4-butanediol produced from the
non-naturally occurring microorganism. The set of metabolic
modifications comprising disruption of one or more genes can be
selected from the set of genes comprising adhE, ldhA, pflAB; adhE,
ldhA, pflAB, mdh; adhE, ldhA, pflAB, mdh, mqo; adhE, ldhA, pflAB,
mdh, aspA; adhE, mdh, ldhA, pflAB, sfcA; adhE, mdh, ldhA, pflAB,
maeB; adhE, mdh, ldhA, pflAB, sfcA, maeB; adhE, ldhA, pflAB, mdh,
pntAB; adhE, ldhA, pflAB, mdh, gdhA; adhE, ldhA, pflAB, mdh, pykA,
pykF, dhaKLM, deoC, edd, yiaE, ycdW; and adhE, ldhA, pflAB, mdh,
pykA, pykF, dhaKLM, deoC, edd, yiaE, ycdW, prpC, gsk, or an
ortholog thereof.
[0124] In one embodiment, the invention provides a method of
producing 1,4-butanediol coupled to the growth of a microorganism.
The method can include the steps of culturing under exponential
growth phase in a sufficient amount of nutrients and media a
non-naturally occurring microorganism comprising a set of metabolic
modifications obligatorily coupling 1,4-butanediol production to
growth of the microorganism, the set of metabolic modifications
comprising disruption of one or more genes, or an ortholog thereof,
wherein the set of metabolic modifications comprises disruption of
adhE and ldhA, wherein the microorganism exhibits stable
growth-coupled production of 1,4-butanediol; and isolating
1,4-butanediol produced from the non-naturally occurring
microorganism. In an additional embodiment, the set of metabolic
modifications can further comprise disruption of mdh. In another
embodiment, the set of metabolic modifications can further comprise
disruption of one or more genes selected from the set of genes
comprising mqo, aspA, sfcA, maeB, pntAB, and gdhA and can include,
for example, disruption of sfcA and maeB. In still another
embodiment, the set of metabolic modifications can further comprise
disruption of one or more genes selected from the set of genes
comprising pykA, pykF, dhaKLM, deoC, edd, yiaE, ycdW, prpC, and
gsk, including disruption of all of pykA, pykF, dhaKLM, deoC, edd,
yiaE and ycdW, and can further comprise disruption of prpC and gsk.
Any of the above-described set of metabolic modifications can
further comprise disruption of pflAB.
[0125] In a method of producing BDO, the non-naturally occurring
microorganism can further comprise a 1,4-butanediol (BDO)
biosynthetic pathway comprising at least one exogenous nucleic acid
encoding 4-hydroxybutanoate dehydrogenase, CoA-independent succinic
semialdehyde dehydrogenase, succinyl-CoA synthetase, CoA-dependent
succinic semialdehyde dehydrogenase, 4-hydroxybutyrate:CoA
transferase, glutamate:succinic semialdehyde transaminase,
glutamate decarboxylase, CoA-independent aldehyde dehydrogenase,
CoA-dependent aldehyde dehydrogenase or alcohol dehydrogenase,
wherein the exogenous nucleic acid is expressed in sufficient
amounts to produce 1,4-butanediol (BDO).
[0126] It is understood that modifications which do not
substantially affect the activity of the various embodiments of
this invention are also included within the definition of the
invention provided herein. Accordingly, the following examples are
intended to illustrate but not limit the present invention.
Example I
Biosynthesis of 4-Hydroxybutanoic Acid
[0127] This Example describes the biochemical pathways for 4-HB
production.
[0128] Previous reports of 4-HB synthesis in microbes have focused
on this compound as an intermediate in production of the
biodegradable plastic poly-hydroxyalkanoate (PHA) (U.S. Pat. No.
6,117,658). The use of 4-HB/3-HB copolymers can be advantageous
over the more traditional poly-3-hydroxybutyrate polymer (PHB)
because the resulting plastic is less brittle (Saito and Doi, Intl.
J. Biol. Macromol. 16:99-104 (1994)). The production of monomeric
4-HB described herein is a fundamentally different process for
several reasons: 1). The product is secreted, as opposed to PHA
which is produced intracellularly and remains in the cell; 2) for
organisms that produce hydroxybutanoate polymers, free 4-HB is not
produced, but rather the Coenzyme A derivative is used by the
polyhydroxyalkanoate synthase; 3) in the case of the polymer,
formation of the granular product changes thermodynamics; and 4)
extracellular pH is not an issue for production of the polymer,
whereas it will affect whether 4-HB is present in the free acid or
conjugate base state, and also the equilibrium between 4-HB and
GBL.
[0129] 4-HB can be produced in two enzymatic reduction steps from
succinate, a central metabolite of the TCA cycle, with succinic
semialdehyde as the intermediate (FIG. 2). The first of these
enzymes, succinic semialdehyde dehydrogenase, is native to many
organisms including E. coli, in which both NADH- and
NADPH-dependent enzymes have been found (Donnelly and Cooper, Eur.
J. Biochem. 113:555-561 (1981); Donnelly and Cooper, J. Bacteriol.
145:1425-1427 (1981); Marek and Henson, J. Bacteriol. 170:991-994
(1988)). There is also evidence supporting succinic semialdehyde
dehydrogenase activity in S. cerevisiae (Ramos et al., Eur. J.
Biochem. 149:401-404 (1985)), and a putative gene has been
identified by sequence homology. However, most reports indicate
that this enzyme proceeds in the direction of succinate synthesis,
opposite to that shown in FIG. 2 (Donnelly and Cooper, supra;
Lutke-Eversloh and Steinbuchel, FEMS Microbiol. Lett. 181:63-71
(1999)), participating in the degradation pathway of 4-HB and
gamma-aminobutyrate. An alternative pathway, used by the obligate
anaerobe Clostridium kluyveri to degrade succinate, activates
succinate to succinyl-CoA, then converts succinyl-CoA to succinic
semialdehyde using an alternative succinic semialdehyde
dehydrogenase which is known to function in this direction (Sohling
and Gottschalk, Eur. J. Biochem. 212:121-127 (1993)). However, this
route has the energetic cost of ATP required to convert succinate
to succinyl-CoA.
[0130] The second enzyme of the pathway, 4-hydroxybutanoate
dehydrogenase, is not native to E. coli or yeast but is found in
various bacteria such as C. kluyveri and Ralstonia eutropha
(Lutke-Eversloh and Steinbuchel, supra; Sohling and Gottschalk, J.
Bacteriol. 178:871-880 (1996); Valentin et al., Eur. J. Biochem.
227:43-60 (1995); Wolff and Kenealy, Protein Expr. Purif. 6:206-212
(1995)). These enzymes are known to be NADH-dependent, though
NADPH-dependent forms can exist. An additional pathway to 4-HB from
alpha-ketoglutarate was demonstrated in E. coli resulting in the
accumulation of poly(4-hydroxybutyric acid) (Song et al., Wei Sheng
Wu Xue.Bao. 45:382-386 (2005)). The recombinant strain required the
overexpression of three heterologous genes, PHA synthase (R.
eutropha), 4-hydroxybutyrate dehydrogenase (R. eutropha) and
4-hydroxybutyrate:CoA transferase (C. kluyveri), along with two
native E. coli genes: glutamate:succinic semialdehyde transaminase
and glutamate decarboxylase. Steps 4 and 5 in FIG. 2 can
alternatively be carried out by an alpha-ketoglutarate
decarboxylase such as the one identified in Euglena gracilis
(Shigeoka et al., Biochem. J. 282(Pt2):319-323 (1992); Shigeoka and
Nakano, Arch. Biochem. Biophys. 288:22-28 (1991); Shigeoka and
Nakano, Biochem J. 292(Pt 2):463-467 (1993)). However, this enzyme
has not yet been applied to impact the production of 4-HB or
related polymers in any organism.
[0131] The reported directionality of succinic semialdehyde
dehydrogenase led to the investigation of the thermodynamics of
4-HB metabolism. Specifically, this study investigated whether or
not the reactions involved in the conversion of succinate or
succinyl-CoA to 4-HB are thermodynamically favorable (i.e.,
.DELTA.G.sub.r<0) under the typical physiological conditions
present in E. coli and S. cerevisiae. All oxidation/reduction
reactions were assumed to utilize NADH, although the results for
assuming NADPH utilization would be similar. Standard Gibbs free
energies of formation (.DELTA.G.sub.f.sup.o) were calculated for
each compound in FIG. 2 based on the group contribution method
(Mavrovouniotis, M. L., J. Biol. Chem.266:14440-14445 (1991)). Each
standard Gibbs energy of formation was then transformed in order to
obtain a criterion of spontaneous change at specified pressure,
temperature, pH, and ionic strength (Alberty, R. A., Biochem.
Biophys. Acta 1207:1-11 (1994) (equation 1).
.DELTA. G f ' ( I , pH ) = .DELTA. G f o ( I = 0 ) + N H RT ln ( 10
pH ) - 2.915 I z 2 - N H 1 + B I ( 1 ) ##EQU00004##
[0132] Where .DELTA.G.sub.f.sup.o is the standard Gibbs energy of
formation, NH is the number of hydrogen atoms in the compound, R is
the universal gas constant, T is constant at 298K, z is the charge
of the molecule at the pH of interest, I is the ionic strength in
M, and B is a constant equal to 1.6 L.sup.0.5/mol.sup.0.5.
[0133] Equation 1 reveals that both intracellular pH and ionic
strength play a role in determining thermodynamic feasibility.
Normally, intracellular pH of cells is very well regulated, even
when there are large variations in the culture pH. The
intracellular pH of E. coli and S. cerevisiae have both been
reported in the literature. E. coli maintains an intracellular pH
of 7.4-7.7 during typical growth conditions in neutral buffers, but
can drop to 7.2 in pH 6 medium, and even go as low as 6.9 for
external pH of 5 (Riondet et al., Biotechnology Tech. 11:735-738
(1997)). However, growth of E. coli is severely inhibited at
external pH below 6. Yeast pH exhibits more variation. During
exponential growth phase, S. cerevisiae internal pH has been
measured to be in the range of 6.7-7.0 with external pH controlled
at 5.0 (Dombek and Ingram, Appl. Environ. Microbiol. 53:1286-1291
(1987)). On the other hand, in resting cells the internal pH drops
to below 6 when the external pH is 6 or less (Imai and Ohno, J.
Biotechnol. 38:165-172 (1995)). This analysis assumes an
intracellular pH of 7.4 for E. coli and 6.8 for S. cerevisiae. An
ionic strength of 0.15 also was assumed (Valenti et al.,
supra).
[0134] Transformed Gibbs energies of formation were calculated at
the standard state (pH=7.0, I=0) and at physiological states of E.
coli (pH=7.4, I=0.15) and S. cerevisiae (pH=6.8, I=0.15).
Transformed Gibbs energies of reaction (.DELTA.G.sub.r') were then
calculated by taking the difference in .DELTA.G.sub.f' between the
products and reactants. The transformed Gibbs energies of the
reactions necessary to convert succinate or succinyl-CoA to 4-HB
are provided in Table 2. Note that the standard error, U.sub.f,est,
on .DELTA.G.sub.f calculated by the group contribution theory is 4
kcal/mol. The uncertainty in .DELTA.G.sub.r, U.sub.r,est, can be
calculated as the Euclidean norm of the uncertainty for
.DELTA.G.sub.f of each compound (Equation).
U r , est = i = 1 m n i 2 * U f , est 2 = i = 1 m 16 n i 2 ( 2 )
##EQU00005##
[0135] Where n is the stochiometric coefficient and i is the
compound. For the examined reactions, this uncertainty is on the
order of 8 kcal/mol.
TABLE-US-00002 TABLE 2 Gibbs free energy of reaction (kcal/mole) at
different pH and ionic strength values. The first column is under
standard conditions, while the others are adjusted according to
equation 1. Temperature is constant at 298 K. Error bars for these
values are on the order of 8 kcal/mol, as calculated by equation 2.
.DELTA.G.sub.r.sup.o' .DELTA.G.sub.r' .DELTA.G.sub.r' pH = 7.0 pH =
7.4 pH = 6.8 Reaction IS = 0 IS = 0.15M IS = 0.15M succ + NADH + 2
H+ .fwdarw. sucsa + NAD + h2o 12.0 14.4 12.8 succ + coa + ATP
.fwdarw. succoa + ADP + Pi 0.30 -0.03 -0.03 succoa + NADH + H+
.fwdarw. sucsa + NAD + coa 4.4 7.0 6.2 sucsa + NADH + H+ .fwdarw.
4hb + NAD -5.0 -3.8 -4.6 Abbreviations: suc, succinate; sucsa,
succinic semialdehyde; succoa, succinyl-CoA; Pi, inorganic
phosphate.
[0136] Table 2 reveals that the reaction most likely to encounter a
thermodynamic barrier after considering potential uncertainty in
our calculations is succinic semialdehyde dehydrogenase (step 1 in
FIG. 2). Whether this reaction can be driven closer to
thermodynamic feasibility by varying the assumed concentrations of
the participating metabolites also was studied. For example, the
standard Gibbs energies assume concentrations of 1 M for all
participating compounds (except water). In an anaerobic
environment, NADH will be present at a several-fold higher
concentration than NAD. Assuming [NADH]=5.times.[NAD], we
calculated the effect on .DELTA.G.sub.r' using the equation
.DELTA. G f ' = .DELTA. G f 0 ' + RT ln [ prod ] [ react ] ( 3 )
##EQU00006##
[0137] This change results in a difference of only about 1 kcal/mol
in the delta G values for succinic semialdehyde dehydrogenase.
Equation 3 was also used to calculate other effects on
.DELTA.G.sub.r, such as high succinate concentration to drive the
reactions. A 1000-fold difference in the concentrations of
succinate and succinic semialdehyde will contribute about 5
kcal/mol to delta G. Taken together with an assumed uncertainty of
8 kcal/mol, the possibility that succinic semialdehyde
dehydrogenase will operate in the direction towards succinic
semialdehyde under some set of physiological conditions cannot be
eliminated. Thus we still consider the direct route from succinate
to 4-HB in our subsequent theoretical analysis.
[0138] The microbial production capabilities of 4-hydroxybutyrate
were explored in two microbes, Escherichia coli and Saccharomyces
cerevisiae, using in silico metabolic models of each organism.
Potential pathways to 4-HB proceed via a succinate, succinyl-CoA,
or alpha-ketoglutarate intermediate as shown in FIG. 2.
[0139] The first step in the 4-HB production pathway from succinate
involves the conversion of succinate to succinic semialdehyde via
an NADH- or NADPH-dependant succinic semialdehyde dehydrogenase. In
E. coli, gabD is an NADP-dependant succinic semialdehyde
dehydrogenase and is part of a gene cluster involved in
4-aminobutyrate uptake and degradation (Niegemann et al., Arch.
Microbiol. 160:454-460 (1993); Schneider et al., J. Bacteriol.
184:6976-6986 (2002)). sad is believed to encode the enzyme for
NAD-dependant succinic semialdehyde dehydrogenase activity (Marek
and Henson, supra). S. cerevisiae contains only the NADPH-dependant
succinic semialdehyde dehydrogenase, putatively assigned to UGA2,
which localizes to the cytosol (Huh et al., Nature 425:686-691
(2003)). The maximum yield calculations assuming the succinate
pathway to 4-HB in both E. coli and S. cerevisiae require only the
assumption that a non-native 4-HB dehydrogenase has been added to
their metabolic networks.
[0140] The pathway from succinyl-CoA to 4-hydroxybutyrate was
described in U.S. Pat. No. 6,117,658 as part of a process for
making polyhydroxyalkanoates comprising 4-hydroxybutyrate monomer
units. Clostridium kluyveri is one example organism known to
possess CoA-dependant succinic semialdehyde dehydrogenase activity
(Sohling and Gottschalk, supra; Sohling and Gottschalk, supra). In
this study, it is assumed that this enzyme, from C. kluyveri or
another organism, is expressed in E. coli or S. cerevisiae along
with a non-native or heterologous 4-HB dehydrogenase to complete
the pathway from succinyl-CoA to 4-HB. The pathway from
alpha-ketoglutarate to 4-HB was demonstrated in E. coli resulting
in the accumulation of poly(4-hydroxybutyric acid) to 30% of dry
cell weight (Song et al., supra). As E. coli and S. cerevisiae
natively or endogenously possess both glutamate:succinic
semialdehyde transaminase and glutamate decarboxylase (Coleman et
al., J. Biol. Chem. 276:244-250 (2001)), the pathway from AKG to
4-HB can be completed in both organisms by assuming only that a
non-native 4-HB dehydrogenase is present.
Example II
Production of 4-Hydroxybutanoic Acid in E. coli
[0141] This Example describes the biosynthetic yields for
4-hydroxybutanoic acid resulting from each biochemical pathway.
[0142] In this section, the maximum theoretical yields of 4-HB from
glucose are calculated assuming that each of the three metabolic
pathways depicted in FIG. 2 are functional in E. coli. A
genome-scale metabolic model of E. coli, similar to the one
described in Reed et al., Genome Biol. 4:R54 (2003), was used as
the basis for the analysis. The energetic gain, in terms of ATP
molecules produced, of each maximum yielding pathway is calculated
assuming anaerobic conditions, unless otherwise stated.
4-Hydroxybutyrate is assumed to exit in E. coli via proton symport,
as is the case with most organic acids. It is also possible that
GBL is secreted by simple diffusion, and in this case the
energetics would be more favorable than in the case considered
here. The impact of cofactor specificity (i.e., NADH or
NADPH-dependence) of the participating enzymes on the maximum yield
and energetics of each pathway also was investigated.
[0143] The results from the analysis are shown in Tables 3 A-C.
From an energetic and yield standpoint, the succinate to 4-HB
pathway is the most promising provided that the thermodynamic
concerns raised in Example I can be overcome. Specifically, the
calculations reveal that the maximum theoretical yield of 4-HB from
glucose is 1.33 mol/mol (0.77 g/g; 0.89 Cmol/Cmol) assuming the
succinate to 4-HB pathway is functional. In addition, the anaerobic
production of 4-HB via succinate would result in the net production
of either 1.8, 1.5, or 1.1 mol of ATP per glucose depending upon
the assumed cofactor specificity of the participating enzymes.
These energetic yields are comparable to the 2.0 ATP per glucose
that can be obtained via substrate level phosphorylation by the
production of ethanol or lactate suggesting the potential for
anaerobic homo-4-HB production in E. coli.
[0144] The succinyl-CoA route to 4-HB is the second most promising
pathway when considering maximum yield and energetics. A 1.33
mol/mol yield of 4-HB is achievable in E. coli if at least one of
the pathway steps is assumed NADH-dependant. However, because this
pathway requires the formation of succinyl-CoA, its energetic yield
is considerably lower than that of the succinate pathway. An oxygen
requirement is anticipated at high 4-HB yields if both the
CoA-dependant succinic semialdehyde dehydrogenase and 4-HB
dehydrogenase steps are assumed NADPH-dependant. In this case, the
production of 4-HB at the maximum yield would result in no net ATP
gain and could not support the energetic maintenance demands needed
for E. coli survival. Thus, some energy would have to originate
from oxidative phosphorylation to enable homo-fermentative 4-HB
production. The alpha-ketoglutarate pathway toward 4-HB is the
least favorable of the three potential routes with a maximum
achievable yield of 1.0 mol 4-HB per mol of glucose. In addition to
the lower maximum yield, this pathway requires the utilization of
1.5 moles of oxygen per mol of glucose converted to 4-HB. The
energetics of this pathway are unaffected by the assumed cofactor
specificity of 4-HB dehydrogenase.
TABLE-US-00003 TABLE 3 The overall substrate conversion
stoichiometry to 4-HB assuming the A) succinate, B) succinyl-CoA,
or C) alpha-ketoglutarate production routes are functional in E.
coli. Glucose and oxygen are taken up while all other molecules are
produced. A) Succinate Pathway Cofactor 1 NADH step Specificity 2
NADH steps 1 NADPH step 2 NADPH steps Glucose -1.000 -1.000 -1.000
Oxygen 0.000 0.000 0.000 Protons 1.333 1.333 1.333 4HB 1.333 1.333
1.333 CO2 0.667 0.667 0.667 H2O 0.667 0.667 0.667 ATP 1.800 1.510
1.097 B) Succinyl-CoA Pathway Cofactor 1 NADH step Specificity 2
NADH steps 1 NADPH step 2 NADPH steps 2 NADPH steps Glucose -1.000
-1.000 -1.000 -1.000 Oxygen 0.000 0.000 -0.036 0.000 Protons 1.333
1.333 1.325 1.294 4HB 1.333 1.333 1.325 1.294 CO2 0.667 0.667 0.698
0.082 H2O 0.667 0.667 0.698 0.470 ATP 0.467 0.177 0.000 0.000 C)
Alpha-ketoglutarate Pathway Cofactor Specificity 1 NADH step 1
NADPH step Glucose -1.000 -1.000 Oxygen -1.500 -1.500 Protons 1.000
1.000 4HB 1.000 1.000 CO2 2.000 2.000 H2O 2.000 2.000 ATP 5.500
5.500
[0145] In order to corroborate the computational predictions
proposed in this report, the strains expressing a complete pathway
to 4-HB can be constructed and tested. Corroboration is performed
with both E. coli (Example II) and S. cerevisiae (Example III). In
E. coli, the relevant genes are expressed in a synthetic operon
behind an inducible promoter on a medium- or high-copy plasmid; for
example the P.sub.BAD promoter which is induced by arabinose, on a
plasmid of the pBAD series (Guzman et al., J. Bacteriol.
177:4121-4130 (1995)). In S. cerevisiae, genes are integrated into
the chromosome behind the PDC1 promoter, replacing the native
pyruvate carboxylase gene. It has been reported that this results
in higher expression of foreign genes than from a plasmid (Ishida
et al., Appl. Environ. Microbiol. 71:1964-1970 (2005)), and will
also ensure expression during anaerobic conditions.
[0146] Cells containing the relevant constructs are grown in
minimal media containing glucose, with addition of arabinose in the
case of E. coli containing genes expressed under the P.sub.BAD
promoter. Periodic samples are taken for both gene expression and
enzyme activity analysis. Enzyme activity assays are performed on
crude cell extracts using procedures well known in the art.
Alternatively, assays based on the oxidation of NAD(P)H, which is
produced in all dehydrogenase reaction steps and detectable by
spectrophotometry can be utilized. In addition, antibodies can be
used to detect the level of particular enzymes. In lieu of or in
addition to enzyme activity measurements, RNA can be isolated from
parallel samples and transcript of the gene of interest measured by
reverse transcriptase PCR. Any constructs lacking detectable
transcript expression are reanalyzed to ensure the encoding nucleic
acids are harbored in an expressible form. Where transcripts are
detected, this result indicates either a lack of translation or
production of inactive enzyme. A variety of methods well known in
the art can additionally be employed, such as codon optimization,
engineering a strong ribosome binding site, use of a gene from a
different species, and prevention of N-glycosylation (for
expression of bacterial enzymes in yeast) by conversion of Asn
residues to Asp. Once all required enzyme activities are detected,
the next step is to measure the production of 4-HP in vivo.
Triplicate shake flask cultures are grown either anaerobically or
microaerobically, depending on the conditions required (see above),
and periodic samples taken. Organic acids present in the culture
supernatants are analyzed by HPLC using the Aminex AH-87X column.
The elution time of 4-HB will be determined using a standard
purchased from a chemical supplier.
[0147] The CoA-independent pathway can be implemented and tested
for corroboration. In this case, the genes overexpressed are the
native succinic semialdehyde dehydrogenase from each organism, and
the 4-hydroxybutanoate dehydrogenase from Ralstonia eutropha. Once
both enzyme activities are detected as discussed above, the strains
are tested for 4-HB production. Corroboration also can be obtained
from implementing the CoA-dependent pathway. The CoA-dependent
succinic semialdehyde dehydrogenase and the 4-hydroxybutanoate
dehydrogenase from Clostridium kluyveri are expressed as described
above. In addition, overexpression of the native succinyl-CoA
synthetase also can be performed, to funnel more succinate into the
heterologous pathway. Finally, if 4-HB production is unfavorable,
different culture conditions can be tested, such as a change in
oxygenation status which can manipulate the NAD(P)H/NAD(P)
ratio.
Example III
Production of 4-Hydroxybutanoic Acid in Yeast
[0148] This Example describes the biosynthetic yields for
4-hydroxybutanoic acid resulting from each biochemical pathway in
S. cerevisiae.
[0149] In this section, the maximum theoretical yields of 4-HB from
glucose are calculated assuming that each of the three metabolic
pathways depicted in FIG. 2 are functional in S. cerevisiae. A
genome-scale metabolic model of S. cerevisiae, similar to the one
described in Forster et al. (Genome Res. 13:244-253 (2003)) was
used as the basis for the analysis. The energetic gain of each
maximum yielding pathway is calculated assuming anaerobic
conditions unless otherwise stated. 4-hydroxybutyrate is assumed to
exit S. cerevisiae via proton symport, as is the case with most
organic acids. The impact of cofactor specificity (i.e., NADH or
NADPH-dependence) of the participating enzymes on the maximum yield
and energetics of each pathway was also investigated.
[0150] The results from the analysis are shown in Tables 4 A-C. As
with E. coli, the succinate to 4-HB pathway is the most promising
provided that the thermodynamic concerns raised in Example I can be
overcome. The calculations reveal that the maximum theoretical
yield of 4-HB from glucose is 1.33 mol/mol (0.77 g/g; 0.89
Cmol/Cmol) in S. cerevisiae. In addition, the anaerobic production
of 4-HB via succinate would result in the net production of either
1.4, 1.1, or 0.5 mol of ATP per glucose depending upon the assumed
cofactor specificity of the participating enzymes.
[0151] The succinyl-CoA route to 4-HB is the second most favorable
pathway. A maximum yield of 1.33 mol 4-HB/mol glucose is achievable
in S. cerevisiae regardless of cofactor specificity. However, net
energy generation at the maximum theoretical yield is possible only
if both the CoA-dependant succinic semialdehyde dehydrogenase and
4-HB dehydrogenase steps are assumed to be NADH-dependant. If
either step is NADPH-dependant, no net ATP will be gained from
anaerobic 4-HB production and an alternate energy source (e.g.,
oxidative phosphorylation) would be required to support cell growth
and maintenance. The alpha-ketoglutarate route toward 4-HB is the
least favorable of the three potential pathways in S. cerevisiae
although the maximum yield of 1.1-1.2 mol 4-HB per mol glucose is
slightly higher than was found in E. coli. Nevertheless, this
pathway requires an oxygen uptake of 0.8-0.9 mol oxygen per mol
glucose to become energetically neutral.
TABLE-US-00004 TABLE 4 The overall substrate conversion
stoichiometry to 4-HB in S. cerevisiae., assuming the A) succinate,
B) succinyl-CoA, or C) alpha-ketoglutarate production routes are
functional in S. cerevisiae. Glucose and oxygen are taken up while
all other molecules are produced. Cofactor 1 NADH step Specificity
2 NADH steps 1 NADPH step 2 NADPH steps A) Succinate Pathway
Glucose -1.000 -1.000 -1.000 Oxygen 0.000 0.000 0.000 Protons 1.333
1.333 1.333 4HB 1.333 1.333 1.333 CO2 0.667 0.667 0.667 H2O 0.667
0.667 0.667 ATP 1.444 1.067 0.533 B) Succinyl-CoA Pathway Glucose
-1.000 -1.000 -1.000 Oxygen 0.000 0.000 0.000 Protons 1.333 1.333
1.333 4HB 1.333 1.333 1.333 CO2 0.667 0.667 0.667 H2O 0.667 0.667
0.667 ATP 0.533 0.000 0.000 C) Alpha-ketoglutarate Pathway Cofactor
Specificity 1 NADH step 1 NADPH step Glucose -1.000 -1.000 Oxygen
-0.785 -0.879 Protons 1.159 1.138 4HB 1.159 1.138 CO2 1.364 1.448
H2O 1.364 1.448 ATP 0.000 0.000
Example IV
Biosynthesis of 4-Hydroxybutanoic Acid, .gamma.-Butyrolactone and
1,4-Butanediol
[0152] This Example describes the biosynthetic production of
4-hydroxybutanoic acid, .gamma.-butyrolactone and 1,4-butanediol
using fermentation and other bioprocesses.
[0153] Methods for the integration of the 4-HB fermentation step
into a complete process for the production of purified GBL,
1,4-butanediol (BDO) and tetrahydrofuran (THF) are described below.
Since 4-HB and GBL are in equilibrium, the fermentation broth will
contain both compounds. At low pH this equilibrium is shifted to
favor GBL. Therefore, the fermentation can operate at pH 7.5 or
less. After removal of biomass, the product stream enters into a
separation step in which GBL is removed and the remaining stream
enriched in 4-HB is recycled. Finally, GBL is distilled to remove
any impurities. The process operates in one of three ways: 1)
fed-batch fermentation and batch separation; 2) fed-batch
fermentation and continuous separation; 3) continuous fermentation
and continuous separation. The first two of these modes are shown
schematically in FIG. 4. The integrated fermentation procedures
described below also are used for the BDO producing cells of the
invention for biosynthesis of BDO and subsequent BDO family
products.
[0154] Fermentation Protocol to Produce 4-HB/GBL (Batch):
[0155] The production organism is grown in a 10 L bioreactor
sparged with an N.sub.2/CO.sub.2 mixture, using 5 L broth
containing 5 g/L potassium phosphate, 2.5 g/L ammonium chloride,
0.5 g/L magnesium sulfate, and 30 g/L corn steep liquor, and an
initial glucose concentration of 20 g/L. As the cells grow and
utilize the glucose, additional 70% glucose is fed into the
bioreactor at a rate approximately balancing glucose consumption.
The temperature of the bioreactor is maintained at 30 degrees C.
Growth continues for approximately 24 hours, until 4-HB reaches a
concentration of between 20-200 g/L, with the cell density being
between 5 and 10 g/L. The pH is not controlled, and will typically
decrease to pH 3-6 by the end of the run. Upon completion of the
cultivation period, the fermenter contents are passed through a
cell separation unit (e.g., centrifuge) to remove cells and cell
debris, and the fermentation broth is transferred to a product
separations unit. Isolation of 4-HB and/or GBL would take place by
standard separations procedures employed in the art to separate
organic products from dilute aqueous solutions, such as
liquid-liquid extraction using a water immiscible organic solvent
(e.g., toluene) to provide an organic solution of 4-HB/GBL. The
resulting solution is then subjected to standard distillation
methods to remove and recycle the organic solvent and to provide
GBL (boiling point 204-205.degree. C.) which is isolated as a
purified liquid.
[0156] Fermentation Protocol to Produce 4-HB/GBL (Fully
Continuous):
[0157] The production organism is first grown up in batch mode
using the apparatus and medium composition described above, except
that the initial glucose concentration is 30-50 g/L. When glucose
is exhausted, feed medium of the same composition is supplied
continuously at a rate between 0.5 L/hr and 1 L/hr, and liquid is
withdrawn at the same rate. The 4-HB concentration in the
bioreactor remains constant at 30-40 g/L, and the cell density
remains constant between 3-5 g/L. Temperature is maintained at 30
degrees C., and the pH is maintained at 4.5 using concentrated NaOH
and HCl, as required. The bioreactor is operated continuously for
one month, with samples taken every day to assure consistency of
4-HB concentration. In continuous mode, fermenter contents are
constantly removed as new feed medium is supplied. The exit stream,
containing cells, medium, and products 4-HB and/or GBL, is then
subjected to a continuous product separations procedure, with or
without removing cells and cell debris, and would take place by
standard continuous separations methods employed in the art to
separate organic products from dilute aqueous solutions, such as
continuous liquid-liquid extraction using a water immiscible
organic solvent (e.g., toluene) to provide an organic solution of
4-HB/GBL. The resulting solution is subsequently subjected to
standard continuous distillation methods to remove and recycle the
organic solvent and to provide GBL (boiling point 204-205.degree.
C.) which is isolated as a purified liquid.
[0158] GBL Reduction Protocol:
[0159] Once GBL is isolated and purified as described above, it
will then be subjected to reduction protocols such as those well
known in the art (references cited) to produce 1,4-butanediol or
tetrahydrofuran (THF) or a mixture thereof. Heterogeneous or
homogeneous hydrogenation catalysts combined with GBL under
hydrogen pressure are well known to provide the products
1,4-butanediol or tetrahydrofuran (THF) or a mixture thereof. It is
important to note that the 4-HB/GBL product mixture that is
separated from the fermentation broth, as described above, may be
subjected directly, prior to GBL isolation and purification, to
these same reduction protocols to provide the products
1,4-butanediol or tetrahydrofuran or a mixture thereof. The
resulting products, 1,4-butanediol and THF are then isolated and
purified by procedures well known in the art.
[0160] Fermentation and Hydrogenation Protocol to Produce BDO or
THF Directly (Batch):
[0161] Cells are grown in a 10 L bioreactor sparged with an
N.sub.2/CO.sub.2 mixture, using 5 L broth containing 5 g/L
potassium phosphate, 2.5 g/L ammonium chloride, 0.5 g/L magnesium
sulfate, and 30 g/L corn steep liquor, and an initial glucose
concentration of 20 g/L. As the cells grow and utilize the glucose,
additional 70% glucose is fed into the bioreactor at a rate
approximately balancing glucose consumption. The temperature of the
bioreactor is maintained at 30 degrees C. Growth continues for
approximately 24 hours, until 4-HB reaches a concentration of
between 20-200 g/L, with the cell density being between 5 and 10
g/L. The pH is not controlled, and will typically decrease to pH
3-6 by the end of the run. Upon completion of the cultivation
period, the fermenter contents are passed through a cell separation
unit (e.g., centrifuge) to remove cells and cell debris, and the
fermentation broth is transferred to a reduction unit (e.g.,
hydrogenation vessel), where the mixture 4-HB/GBL is directly
reduced to either 1,4-butanediol or THF or a mixture thereof.
Following completion of the reduction procedure, the reactor
contents are transferred to a product separations unit. Isolation
of 1,4-butanediol and/or THF would take place by standard
separations procedures employed in the art to separate organic
products from dilute aqueous solutions, such as liquid-liquid
extraction using a water immiscible organic solvent (e.g., toluene)
to provide an organic solution of 1,4-butanediol and/or THF. The
resulting solution is then subjected to standard distillation
methods to remove and recycle the organic solvent and to provide
1,4-butanediol and/or THF which are isolated as a purified
liquids.
[0162] Fermentation and Hydrogenation Protocol to Produce BDO or
THF Directly (Fully Continuous):
[0163] The cells are first grown up in batch mode using the
apparatus and medium composition described above, except that the
initial glucose concentration is 30-50 g/L. When glucose is
exhausted, feed medium of the same composition is supplied
continuously at a rate between 0.5 L/hr and 1 L/hr, and liquid is
withdrawn at the same rate. The 4-HB concentration in the
bioreactor remains constant at 30-40 g/L, and the cell density
remains constant between 3-5 g/L. Temperature is maintained at 30
degrees C., and the pH is maintained at 4.5 using concentrated NaOH
and HCl, as required. The bioreactor is operated continuously for
one month, with samples taken every day to assure consistency of
4-HB concentration. In continuous mode, fermenter contents are
constantly removed as new feed medium is supplied. The exit stream,
containing cells, medium, and products 4-HB and/or GBL, is then
passed through a cell separation unit (e.g., centrifuge) to remove
cells and cell debris, and the fermentation broth is transferred to
a continuous reduction unit (e.g., hydrogenation vessel), where the
mixture 4-HB/GBL is directly reduced to either 1,4-butanediol or
THF or a mixture thereof. Following completion of the reduction
procedure, the reactor contents are transferred to a continuous
product separations unit. Isolation of 1,4-butanediol and/or THF
would take place by standard continuous separations procedures
employed in the art to separate organic products from dilute
aqueous solutions, such as liquid-liquid extraction using a water
immiscible organic solvent (e.g., toluene) to provide an organic
solution of 1,4-butanediol and/or THF. The resulting solution is
then subjected to standard continuous distillation methods to
remove and recycle the organic solvent and to provide
1,4-butanediol and/or THF which are isolated as a purified
liquids.
[0164] Fermentation Protocol to Produce BDO Directly (Batch):
[0165] The production organism is grown in a 10 L bioreactor
sparged with an N.sub.2/CO.sub.2 mixture, using 5 L broth
containing 5 g/L potassium phosphate, 2.5 g/L ammonium chloride,
0.5 g/L magnesium sulfate, and 30 g/L corn steep liquor, and an
initial glucose concentration of 20 g/L. As the cells grow and
utilize the glucose, additional 70% glucose is fed into the
bioreactor at a rate approximately balancing glucose consumption.
The temperature of the bioreactor is maintained at 30 degrees C.
Growth continues for approximately 24 hours, until BDO reaches a
concentration of between 20-200 g/L, with the cell density
generally being between 5 and 10 g/L. Upon completion of the
cultivation period, the fermenter contents are passed through a
cell separation unit (e.g., centrifuge) to remove cells and cell
debris, and the fermentation broth is transferred to a product
separations unit. Isolation of BDO would take place by standard
separations procedures employed in the art to separate organic
products from dilute aqueous solutions, such as liquid-liquid
extraction using a water immiscible organic solvent (e.g., toluene)
to provide an organic solution of BDO. The resulting solution is
then subjected to standard distillation methods to remove and
recycle the organic solvent and to provide BDO (boiling point
228-229.degree. C.) which is isolated as a purified liquid.
[0166] Fermentation Protocol to Produce BDO Directly (Fully
Continuous):
[0167] The production organism is first grown up in batch mode
using the apparatus and medium composition described above, except
that the initial glucose concentration is 30-50 g/L.
[0168] When glucose is exhausted, feed medium of the same
composition is supplied continuously at a rate between 0.5 L/hr and
1 L/hr, and liquid is withdrawn at the same rate. The BDO
concentration in the bioreactor remains constant at 30-40 g/L, and
the cell density remains constant between 3-5 g/L. Temperature is
maintained at 30 degrees C., and the pH is maintained at 4.5 using
concentrated NaOH and HCl, as required. The bioreactor is operated
continuously for one month, with samples taken every day to assure
consistency of BDO concentration. In continuous mode, fermenter
contents are constantly removed as new feed medium is supplied. The
exit stream, containing cells, medium, and the product BDO, is then
subjected to a continuous product separations procedure, with or
without removing cells and cell debris, and would take place by
standard continuous separations methods employed in the art to
separate organic products from dilute aqueous solutions, such as
continuous liquid-liquid extraction using a water immiscible
organic solvent (e.g., toluene) to provide an organic solution of
BDO. The resulting solution is subsequently subjected to standard
continuous distillation methods to remove and recycle the organic
solvent and to provide BDO (boiling point 228-229.degree. C.) which
is isolated as a purified liquid (mpt 20.degree. C.).
Example V
In Silico Derived Knockout Strategies for Growth-Coupled Production
of 1,4-Butanediol in Escherichia coli
[0169] This example describes the in silico design of knockout
strategies to generate growth-coupled production of 1,4-butanediol
in E. coli.
[0170] The strategy for generating growth-coupled production of BDO
first involves the demonstration of a functional pathway which is
subsequently optimized through adaptive evolution and targeted gene
insertions, deletions, and overexpressions. Described below in more
detail are strain-engineering strategies identified via OptKnock
for generating growth-coupled BDO production strains of Escherichia
coli. All implementations of OptKnock assume that sufficient
activities of all enzymes outlined in FIG. 6 are available to E.
coli under all conditions. In addition, all reduction steps in this
pathway are assumed to be NADH-dependent although many of the
designs are expected to be applicable regardless of cofactor
specificity.
[0171] The BDO yield potential of the biochemical pathways to
1,4-butanediol in E. coli is detailed below. Three conditions were
used, anaerobic, anaerobic+nitrate addition, and aerobic. The
maximum theoretical yields for each scenario assuming that the
production of BDO must be energetically neutral are shown in Table
5. Table 5 shows the maximum theoretical yields of 1,4-butanediol
(BDO) for five sets of environmental conditions: 1) aerobic
respiration, 2) anaerobic fermentation with acetate co-production,
3) anaerobic fermentation with ethanol co-production, 4) nitrate
respiration leading to nitrite formation, and 5) nitrate
respiration leading to ammonia formation. Negative values indicate
metabolites taken up; positive values indicate metabolites
secreted. Molar units are assumed along with pathway energetic
neutrality. The maximum theoretical yield without assuming
energetic neutrality is 1.091 mol/mol (0.545 g/g) glucose for all
cases. The highest yield is obtained under aerobic conditions where
enough ATP to render the pathway energetically neutral can be
generated with minimal loss of carbon through respiration. In the
anaerobic case, the yield drops as ATP is made via substrate level
phosphorylation and either acetate or ethanol is made as a
byproduct. Controlled nitrate addition can provide nearly the same
yield as oxygen, the exact amount depending upon whether further
reduction of nitrite to ammonia occurs.
TABLE-US-00005 TABLE 5 Maximum theoretical yields of 1,4-butanediol
(BDO) for five sets of environmental conditions. Anerobic Acetate
Ethanol Copro- Copro- Nitrate to Nitrate to Aerobic duction duction
Nitrite Ammonia Glucose -1.000 -1.000 -1.000 -1.000 1.000 Oxygen
-0.068 Nitrate -0.147 -0.092 H.sup.+ 0.144 -0.184 H.sub.2O 0.607
0.519 0.498 0.612 0.621 CO.sub.2 1.686 1.558 1.668 1.690 1.770
Nitrite 0.147 Ammonia 0.092 Acetate 0.144 Ethanol 0.173 BDO 1.079
1.039 0.997 1.078 1.058
[0172] If phosphoenolpyruvate (PEP) carboxykinase is assumed to
operate in the direction of phosphoenolpyruvate to oxaloacetate,
BDO production becomes an energy-generating endeavor and the
maximum theoretical yield for all scenarios becomes 0.545 g/g with
CO.sub.2 as the only byproduct. PEP carboxykinase is known to
produce oxaloacetate from PEP in rumen bacteria such as Mannheimia
succiniciproducens (Hong et al., Nat. Biotechnol. 22:1275-1281
(2004)). However, the role of PEP carboxykinase in producing
oxaloacetate in E. coli is believed to be minor as compared to PEP
carboxylase possibly due to the higher Km for bicarbonate of PEP
carboxykinase (Kim et al., Appl. Environ. Microbiol. 70:1238-1241
(2004)). Nevertheless, activity of the native E. coli PEP
carboxykinase from PEP towards oxaloacetate has been recently
demonstrated in ppc mutants of K-12 (Kwon et al., J. Microbiol.
Biotechnol. 16:1448-1452 (2006)).
[0173] In more detail, the designs identified for increasing BDO
production in E. coli are described below. A non-growth associated
energetic maintenance requirement of 7.6 mmol/gDW/hr was assumed
along with a maximum specific glucose uptake rate of 20
mmol/gDW/hr. BDO was assumed to be exported via diffusion. Knockout
strategies were identified assuming that 1) PEP carboxykinase is
irreversible and functions only to convert oxaloacetate to PEP and
2) PEP carboxykinase is reversible. For both cases, the OptKnock
code described in Burgard et al. (Biotechnol. Bioeng. 84:647-657
(2003)) was modified to allow for limited, unlimited or no nitrate
respiration. Adding the possibility of nitrate respiration serves
to increase the maximum theoretical yield of BDO when PEP
carboxykinase is assumed irreversible and also, in some cases,
enables the selection of knockout strategies that otherwise would
not be selected under anaerobic conditions due to unfavorable
energetics. Specifically, six nitrate uptake reactions were added
to the E. coli network with lower bounds of 0, -2, -5, -10, -20, or
-1000 mmol/gDW/hr (the negative values signify metabolite uptake in
the stoichiometric model). The dual constraints of OptKnock were
adjusted accordingly and an additional constraint was added that
allowed only one nitrate uptake reaction to be active at a time.
OptKnock selects the optimum amount of nitrate respiration for each
identified knockout strategy. Finally, all simulations assume that
the reaction catalyzed by adhE in E. coli (that is,
acetyl-CoA+NADH.fwdarw.ethanol+NAD) has been removed. Because
acetyl-CoA is a necessary intermediate for BDO production, nearly
all OptKnock designs would include this deletion anyway to prevent
ethanol production from competing with BDO production. Including
this deletion a priori was done to lower the CPU time of the
computational procedure.
[0174] The knockout strategies derived by OptKnock are given in
Tables 6 and 7 assuming PEP carboxykinase to be irreversible and
reversible, respectively. In this description, the knockout
strategies are listed by reaction abbreviations in all capital
letters for simplicity. The corresponding genes that would have to
be knocked out to prevent a particular reaction from occurring in
E. coli are provided in Table 8 along with the reaction
stoichiometry. The metabolite names corresponding to Table 8 are
listed in Table 9.
TABLE-US-00006 TABLE 6 Knockout strategies derived by OptKnock,
assuming PEP carboxykinase to be irreversible. Metabolic
Transformations Targeted for Removal .dagger., .dagger-dbl., # BDO
BIO AC ALA CO2 FOR GLC GLY H+ 1 ADHEr 5.67 0.57 25.42 0.00 -5.74
28.42 -20.00 0.00 57.91 2 ADHEr, 9.95 0.50 22.31 0.00 14.90 0.00
-20.00 0.00 25.85 PFLi 3 ADHEr, 6.92 0.52 25.03 0.00 -0.07 20.82
-20.00 0.00 49.54 NADH6 4 ADHEr, 6.55 0.56 23.75 0.00 -3.54 26.67
-20.00 0.00 54.38 THD2 5 ADHEr, 6.21 0.30 28.99 0.00 -5.98 30.57
-20.00 0.00 61.72 PGI 6 ADHEr, 6.13 0.31 29.10 0.00 -6.17 30.70
-20.00 0.00 61.98 PFK 7 ADHEr, 6.13 0.31 29.10 0.00 -6.17 30.70
-20.00 0.00 61.98 FBA 8 ADHEr, 6.15 0.61 23.38 0.00 -3.10 26.55
-20.00 0.00 54.24 ATPS4r 9 ADHEr, 6.13 0.31 29.10 0.00 -6.17 30.70
-20.00 0.00 61.98 TPI 10 ADHEr, 5.85 0.47 26.89 0.00 -5.92 29.33
-20.00 0.00 59.54 SUCD4 11 ADHEr, 5.78 0.57 25.20 0.00 -5.45 28.19
-20.00 0.00 57.44 RPE 12 ADHEr, 5.78 0.51 26.33 0.00 -5.85 28.99
-20.00 0.00 58.92 GLCpts 13 ADHEr, 5.77 0.52 26.20 0.00 -5.84 28.90
-20.00 0.00 58.78 GLUDy 14 ADHEr, 5.73 0.57 25.30 0.00 -5.59 28.30
-20.00 0.00 57.67 TAL 15 ADHEr, 5.73 0.54 25.90 0.00 -5.80 28.72
-20.00 0.00 58.44 MDH 16 ADHEr, 5.73 0.54 25.90 0.00 -5.80 28.72
-20.00 0.00 58.44 FUM 17 ADHEr, 5.68 0.57 25.48 0.00 -5.75 28.46
-20.00 0.00 57.99 CBMK2 18 ADHEr, 11.82 0.22 18.07 0.00 8.10 19.23
-20.00 0.00 38.88 HEX1, PGI 19 ADHEr, 11.82 0.22 18.05 0.00 8.10
19.22 -20.00 0.00 38.86 EDA, PGI 20 ADHEr, 10.87 0.20 25.96 0.00
16.29 0.00 -20.00 0.00 27.38 PFLi, PGI 21 ADHEr, 10.83 0.20 26.02
0.00 16.23 0.00 -20.00 0.00 27.46 FBA, PFLi 22 ADHEr, 10.83 0.20
26.02 0.00 16.23 0.00 -20.00 0.00 27.46 PFLi, TPI 23 ADHEr, 10.83
0.20 26.02 0.00 16.23 0.00 -20.00 0.00 27.46 PFK, PFLi 24 ADHEr,
10.49 0.49 21.02 0.00 15.71 0.00 -20.00 0.00 24.49 PFLi, THD2 25
ADHEr, 10.15 0.43 23.16 0.00 15.20 0.00 -20.00 0.00 26.22 GLCpts,
PFLi 26 ADHEr, 10.10 0.45 22.94 0.00 15.12 0.00 -20.00 0.00 26.12
GLUDy, PFLi 27 ADHEr, 10.02 0.50 22.14 0.00 15.00 0.00 -20.00 0.00
25.67 PFLi, RPE 28 ADHEr, 9.99 0.50 22.22 0.00 14.95 0.00 -20.00
0.00 25.76 PFLi, TAL 29 ADHEr, 9.96 0.49 22.36 0.00 14.92 0.00
-20.00 0.00 25.87 CBMK2, PFLi 30 ADHEr, 8.97 0.26 23.60 0.00 0.95
24.98 -20.00 0.00 50.46 ATPS4r, PGI 31 ADHEr, 8.62 0.48 23.04 0.00
6.28 13.23 -20.00 0.00 39.68 ATPS4r, NADH6 32 ADHEr, 8.26 0.26
27.70 0.00 3.99 16.78 -20.00 0.00 46.33 ATPS4r, TPI 33 ADHEr, 8.26
0.26 27.70 0.00 3.99 16.78 -20.00 0.00 46.33 ATPS4r, PFK 34 ADHEr,
8.26 0.26 27.70 0.00 3.99 16.78 -20.00 0.00 46.33 ATPS4r, FBA 35
ADHEr, 7.88 0.50 23.49 0.00 2.67 18.21 -20.00 0.00 45.24 NADH6,
THD2 36 ADHEr, 7.86 0.49 22.44 0.00 0.04 23.43 -20.00 0.00 49.33
ATPS4r, FUM 37 ADHEr, 7.86 0.49 22.44 0.00 0.04 23.43 -20.00 0.00
49.33 ATPS4r, MDH 38 ADHEr, 7.58 0.23 28.71 0.00 0.18 22.36 -20.00
0.00 52.73 NADH6, PGI 39 ADHEr, 7.55 0.49 22.66 0.00 -1.33 25.25
-20.00 0.00 51.43 FUM, THD2 40 ADHEr, 7.55 0.49 22.66 0.00 -1.33
25.25 -20.00 0.00 51.43 MDH, THD2 41 ADHEr, 7.50 0.24 28.80 0.00
-0.03 22.54 -20.00 0.00 53.03 NADH6, PFK 42 ADHEr, 7.50 0.24 28.80
0.00 -0.03 22.54 -20.00 0.00 53.03 FBA, NADH6 43 ADHEr, 7.50 0.24
28.80 0.00 -0.03 22.54 -20.00 0.00 53.03 NADH6, TPI 44 ADHEr, 7.06
0.45 25.94 0.00 -0.06 21.24 -20.00 0.00 50.38 GLCpts, NADH6 45
ADHEr, 7.05 0.52 24.82 0.00 0.30 20.47 -20.00 0.00 48.96 NADH6, RPE
46 ADHEr, 7.02 0.47 25.73 0.00 -0.06 21.14 -20.00 0.00 50.18 GLUDy,
NADH6 47 ADHEr, 7.01 0.47 25.63 0.00 -0.06 21.10 -20.00 0.00 50.09
FUM, NADH6 48 ADHEr, 7.01 0.47 25.63 0.00 -0.06 21.10 -20.00 0.00
50.09 MDH, NADH6 49 ADHEr, 6.98 0.52 24.92 0.00 0.12 20.64 -20.00
0.00 49.23 NADH6, TAL 50 ADHEr, 6.56 0.49 24.86 0.00 -3.91 27.44
-20.00 0.00 55.80 GLCpts, THD2 51 ADHEr, 6.56 0.50 24.67 0.00 -3.84
27.31 -20.00 0.00 55.57 GLUDy, THD2 52 ADHEr, 6.33 0.22 30.11 0.00
-6.16 31.28 -20.00 0.00 62.99 PGI, SUCD4 53 ADHEr, 6.28 0.26 29.59
0.00 -6.08 30.96 -20.00 0.00 62.41 GLCpts, PGI 54 ADHEr, 6.28 0.22
30.24 0.00 -6.31 31.41 -20.00 0.00 63.24 HEX1, PFK 55 ADHEr, 6.28
0.22 30.24 0.00 -6.31 31.41 -20.00 0.00 63.24 FBA, HEX1 56 ADHEr,
6.28 0.22 30.24 0.00 -6.31 31.41 -20.00 0.00 63.24 HEX1, TPI 57
ADHEr, 6.27 0.23 30.17 0.00 -6.30 31.37 -20.00 0.00 63.17 PFK,
SUCD4 58 ADHEr, 6.27 0.23 30.17 0.00 -6.30 31.37 -20.00 0.00 63.17
FBA, SUCD4 59 ADHEr, 6.27 0.23 30.17 0.00 -6.30 31.37 -20.00 0.00
63.17 SUCD4, TPI 60 ADHEr, 6.26 0.27 29.44 0.00 -6.06 30.86 -20.00
0.00 62.23 GLUDy, PGI 61 ADHEr, 6.21 0.26 29.69 0.00 -6.24 31.07
-20.00 0.00 62.64 GLCpts, TPI 62 ADHEr, 6.21 0.26 29.69 0.00 -6.24
31.07 -20.00 0.00 62.64 GLCpts, PFK 63 ADHEr, 6.21 0.26 29.69 0.00
-6.24 31.07 -20.00 0.00 62.64 FBA, GLCpts 64 ADHEr, 6.20 0.30 29.01
0.00 -6.02 30.60 -20.00 0.00 61.77 PFK, RPE 65 ADHEr, 6.20 0.30
29.01 0.00 -6.02 30.60 -20.00 0.00 61.77 FBA, RPE 66 ADHEr, 6.20
0.30 29.01 0.00 -6.02 30.60 -20.00 0.00 61.77 RPE, TPI 67 ADHEr,
6.19 0.27 29.54 0.00 -6.23 30.98 -20.00 0.00 62.47 GLUDy, TPI 68
ADHEr, 6.19 0.27 29.54 0.00 -6.23 30.98 -20.00 0.00 62.47 FBA,
GLUDy 69 ADHEr, 6.19 0.27 29.54 0.00 -6.23 30.98 -20.00 0.00 62.47
GLUDy, PFK 70 ADHEr, 6.17 0.31 29.05 0.00 -6.09 30.65 -20.00 0.00
61.87 TAL, TPI 71 ADHEr, 6.17 0.31 29.05 0.00 -6.09 30.65 -20.00
0.00 61.87 PFK, TAL 72 ADHEr, 6.17 0.31 29.05 0.00 -6.09 30.65
-20.00 0.00 61.87 FBA, TAL 73 ADHEr, 6.17 0.55 24.36 0.00 -3.40
27.23 -20.00 0.00 55.50 ATPS4r, GLUDy 74 ADHEr, 6.00 0.38 28.04
0.00 -6.05 30.04 -20.00 0.00 60.81 PYK, SUCD4 75 ADHEr, 5.97 0.40
27.84 0.00 -6.03 29.92 -20.00 0.00 60.59 GLCpts, SUCD4 76 ADHEr,
5.95 0.46 26.74 0.00 -5.68 29.16 -20.00 0.00 59.19 RPE, SUCD4 77
ADHEr, 5.94 0.42 27.58 0.00 -6.00 29.76 -20.00 0.00 60.30 FUM,
GLUDy 78 ADHEr, 5.94 0.42 27.58 0.00 -6.00 29.76 -20.00 0.00 60.30
GLUDy, MDH 79 ADHEr, 5.94 0.42 27.54 0.00 -5.99 29.74 -20.00 0.00
60.27 GLUDy, SUCD4 80 ADHEr, 5.90 0.46 26.81 0.00 -5.79 29.24
-20.00 0.00 59.36 SUCD4, TAL 81 ADHEr, 5.89 0.51 26.14 0.00 -5.59
28.78 -20.00 0.00 58.51 GLCpts, RPE 82 ADHEr, 5.87 0.46 27.02 0.00
-5.93 29.42 -20.00 0.00 59.69 GLCpts, GLUDy 83 ADHEr, 5.85 0.47
26.83 0.00 -5.91 29.29 -20.00 0.00 59.47 GLCpts, MDH 84 ADHEr, 5.85
0.47 26.83 0.00 -5.91 29.29 -20.00 0.00 59.47 FUM, GLCpts 85 ADHEr,
5.87 0.52 26.00 0.00 -5.57 28.69 -20.00 0.00 58.35 GLUDy, RPE 86
ADHEr, 5.84 0.51 26.23 0.00 -5.72 28.88 -20.00 0.00 58.71 GLCpts,
TAL 87 ADHEr, 5.84 0.54 25.70 0.00 -5.53 28.50 -20.00 0.00 58.02
FUM, RPE 88 ADHEr, 5.84 0.54 25.70 0.00 -5.53 28.50 -20.00 0.00
58.02 MDH, RPE 89 ADHEr, 5.82 0.47 26.84 0.00 -5.98 29.28 -20.00
0.00 59.61 MDH, PYK 90 ADHEr, 5.82 0.47 26.84 0.00 -5.98 29.28
-20.00 0.00 59.61 FUM, PYK 91 ADHEr, 5.79 0.54 25.79 0.00 -5.66
28.61 -20.00 0.00 58.22 MDH, TAL 92 ADHEr, 5.79 0.54 25.79 0.00
-5.66 28.61 -20.00 0.00 58.22
FUM, TAL 93 ADHEr, 5.82 0.52 26.09 0.00 -5.70 28.79 -20.00 0.00
58.55 GLUDy, TAL 94 ADHEr, 5.68 0.57 25.51 0.00 -5.76 28.48 -20.00
0.00 58.02 CBMK2, GLU5K 95 ADHEr, 5.68 0.57 25.51 0.00 -5.76 28.48
-20.00 0.00 58.02 CBMK2, G5SD 96 ADHEr, 5.68 0.57 25.51 0.00 -5.76
28.48 -20.00 0.00 58.02 ASNS2, CBMK2 97 ADHEr, 5.68 0.57 25.50 0.00
-5.75 28.47 -20.00 0.00 58.00 CBMK2, SO4t2 98 ADHEr, 5.68 0.57
25.48 0.00 -5.75 28.46 -20.00 0.00 57.99 CBMK2, HEX1 99 ADHEr,
14.76 0.16 16.11 0.00 22.13 0.00 -20.00 0.00 17.24 EDA, PFLi, PGI
100 ADHEr, 14.39 0.13 16.96 0.00 21.96 1.24 -20.00 0.00 19.11 EDA,
NADH6, PGI 101 ADHEr, 12.91 0.12 12.29 0.00 -0.02 38.75 -20.00 0.00
51.90 FRD2, GLUDy, LDH_D 102 ADHEr, 12.89 0.13 12.25 0.00 -0.02
38.70 -20.00 0.00 51.85 FRD2, LDH_D, THD2 103 ADHEr, 12.62 0.11
12.68 0.00 0.33 39.20 -20.00 0.00 52.67 ACKr, ACS, PPC 104 ADHEr,
12.60 0.16 11.81 0.00 0.47 38.84 -20.00 0.00 51.79 GLUDy, LDH_D,
PPC 105 ADHEr, 12.60 0.16 11.72 0.00 0.49 38.81 -20.00 0.00 51.71
LDH_D, PPC, THD2 106 ADHEr, 11.95 0.15 19.06 0.00 8.00 19.85 -20.00
0.00 39.98 ATPS4r, EDA, PGI 107 ADHEr, 11.95 0.15 19.03 0.00 8.00
19.83 -20.00 0.00 39.94 EDA, GLCpts, PGI 108 ADHEr, 11.86 0.20
18.37 0.00 8.07 19.42 -20.00 0.00 39.21 EDA, GLUDy, PGI 109 ADHEr,
11.85 0.20 18.38 0.00 8.05 19.43 -20.00 0.00 39.24 GLUDy, HEX1, PGI
110 ADHEr, 11.72 0.28 7.78 0.00 9.70 25.16 -20.00 0.00 37.14
ATPS4r, FRD2, LDH_D 111 ADHEr, 11.48 0.56 11.79 0.00 27.19 0.00
-20.00 0.00 15.78 ACKr, NADH6, PYK 112 ADHEr, 11.02 0.64 11.37 0.00
26.49 0.00 -20.00 0.00 15.94 ACKr, LDH_D, NADH6 113 ADHEr, 10.95
0.17 26.34 0.00 16.41 0.00 -20.00 0.00 27.55 GLCpts, PFLi, PGI 114
ADHEr, 10.93 0.18 26.24 0.00 16.38 0.00 -20.00 0.00 27.51 GLUDy,
PFLi, PGI 115 ADHEr, 10.91 0.17 26.39 0.00 16.36 0.00 -20.00 0.00
27.62 FBA, GLCpts, PFLi 116 ADHEr, 10.91 0.17 26.39 0.00 16.36 0.00
-20.00 0.00 27.62 GLCpts, PFK, PFLi 117 ADHEr, 10.91 0.17 26.39
0.00 16.36 0.00 -20.00 0.00 27.62 GLCpts, PFLi, TPI 118 ADHEr,
10.89 0.18 26.30 0.00 16.33 0.00 -20.00 0.00 27.58 FBA, GLUDy, PFLi
119 ADHEr, 10.89 0.18 26.30 0.00 16.33 0.00 -20.00 0.00 27.58
GLUDy, PFK, PFLi 120 ADHEr, 10.89 0.18 26.30 0.00 16.33 0.00 -20.00
0.00 27.58 GLUDy, PFLi, TPI 121 ADHEr, 10.86 0.20 25.97 0.00 16.28
0.00 -20.00 0.00 27.40 FBA, PFLi, RPE 122 ADHEr, 10.86 0.20 25.97
0.00 16.28 0.00 -20.00 0.00 27.40 PFLi, RPE, TPI 123 ADHEr, 10.86
0.20 25.97 0.00 16.28 0.00 -20.00 0.00 27.40 PFK, PFLi, RPE 124
ADHEr, 10.85 0.20 26.00 0.00 16.25 0.00 -20.00 0.00 27.43 PFK,
PFLi, TAL 125 ADHEr, 10.85 0.20 26.00 0.00 16.25 0.00 -20.00 0.00
27.43 PFLi, TAL, TPI 126 ADHEr, 10.85 0.20 26.00 0.00 16.25 0.00
-20.00 0.00 27.43 FBA, PFLi, TAL 127 ADHEr, 10.64 0.17 23.71 0.00
8.96 13.97 -20.00 0.00 38.90 ATPS4r, NADH6, PGI 128 ADHEr, 10.62
0.42 22.05 0.00 15.90 0.00 -20.00 0.00 25.05 GLCpts, PFLi, THD2 129
ADHEr, 10.59 0.44 21.77 0.00 15.85 0.00 -20.00 0.00 24.89 GLUDy,
PFLi, THD2 130 ADHEr, 10.53 0.30 24.78 0.00 15.78 0.00 -20.00 0.00
26.92 LDH_D, PFLi, SUCD4 131 ADHEr, 10.31 0.38 23.85 0.00 15.45
0.00 -20.00 0.00 26.52 LDH_D, NADH6, PFLi 132 ADHEr, 10.30 0.35
21.88 0.00 12.39 8.06 -20.00 0.00 32.44 ATPS4r, LDH_D, SUCD4 133
ADHEr, 10.29 0.38 23.75 0.00 15.41 0.00 -20.00 0.00 26.47 FUM,
PFLi, PYK 134 ADHEr, 10.29 0.38 23.75 0.00 15.41 0.00 -20.00 0.00
26.47 MDH, PFLi, PYK 135 ADHEr, 10.28 0.39 23.71 0.00 15.40 0.00
-20.00 0.00 26.46 GLCpts, GLUDy, PFLi 136 ADHEr, 10.21 0.43 23.02
0.00 15.29 0.00 -20.00 0.00 26.07 GLCpts, PFLi, RPE 137 ADHEr,
10.18 0.43 23.09 0.00 15.25 0.00 -20.00 0.00 26.14 GLCpts, PFLi,
TAL 138 ADHEr, 10.16 0.45 22.79 0.00 15.22 0.00 -20.00 0.00 25.96
GLUDy, PFLi, RPE 139 ADHEr, 10.16 0.43 23.21 0.00 15.22 0.00 -20.00
0.00 26.24 CBMK2, GLCpts, PFLi 140 ADHEr, 10.15 0.43 23.14 0.00
15.19 0.00 -20.00 0.00 26.21 LDH_D, MDH, PFLi 141 ADHEr, 10.15 0.43
23.14 0.00 15.19 0.00 -20.00 0.00 26.21 FUM, LDH_D, PFLi 142 ADHEr,
10.13 0.45 22.86 0.00 15.17 0.00 -20.00 0.00 26.04 GLUDy, PFLi, TAL
143 ADHEr, 10.11 0.44 22.98 0.00 15.14 0.00 -20.00 0.00 26.14
CBMK2, GLUDy, PFLi 144 ADHEr, 10.04 0.49 22.20 0.00 15.02 0.00
-20.00 0.00 25.69 CBMK2, PFLi, RPE 145 ADHEr, 10.00 0.49 22.28 0.00
14.97 0.00 -20.00 0.00 25.78 CBMK2, PFLi, TAL 146 ADHEr, 9.96 0.49
22.35 0.00 14.91 0.00 -20.00 0.00 25.87 ASNS2, G5SD, PFLi 147
ADHEr, 9.96 0.49 22.35 0.00 14.91 0.00 -20.00 0.00 25.87 ASNS2,
GLU5K, PFLi 148 ADHEr, 9.96 0.37 21.98 0.00 9.04 11.74 -20.00 0.00
36.36 ATPS4r, GLCpts, MDH 149 ADHEr, 9.96 0.37 21.98 0.00 9.04
11.74 -20.00 0.00 36.36 ATPS4r, FUM, GLCpts 150 ADHEr, 9.87 0.17
27.44 0.00 11.15 7.29 -20.00 0.00 35.96 ATPS4r, FBA, NADH6 151
ADHEr, 9.87 0.17 27.44 0.00 11.15 7.29 -20.00 0.00 35.96 ATPS4r,
NADH6, TPI 152 ADHEr, 9.87 0.17 27.44 0.00 11.15 7.29 -20.00 0.00
35.96 ATPS4r, NADH6, PFK 153 ADHEr, 9.82 0.44 23.35 0.00 13.64 2.12
-20.00 0.00 28.60 ATPS4r, FUM, PGL 154 ADHEr, 9.82 0.44 23.35 0.00
13.64 2.12 -20.00 0.00 28.60 ATPS4r, MDH, PGDH 155 ADHEr, 9.82 0.44
23.35 0.00 13.64 2.12 -20.00 0.00 28.60 ATPS4r, FUM, G6PDHy 156
ADHEr, 9.82 0.44 23.35 0.00 13.64 2.12 -20.00 0.00 28.60 ATPS4r,
FUM, PGDH 157 ADHEr, 9.82 0.44 23.35 0.00 13.64 2.12 -20.00 0.00
28.60 ATPS4r, MDH, PGL 158 ADHEr, 9.82 0.44 23.35 0.00 13.64 2.12
-20.00 0.00 28.60 ATPS4r, G6PDHy, MDH 159 ADHEr, 9.81 0.44 12.15
0.00 1.32 36.78 -20.00 0.00 52.07 ATPS4r, LDH_D, PPC 160 ADHEr,
9.77 0.44 23.32 0.00 13.29 2.67 -20.00 0.00 29.13 ATPS4r, FUM, TAL
161 ADHEr, 9.77 0.44 23.32 0.00 13.29 2.67 -20.00 0.00 29.13
ATPS4r, MDH, TAL 162 ADHEr, 9.76 0.42 22.90 0.00 13.62 4.00 -20.00
0.00 29.90 ATPS4r, GLCpts, NADH6 163 ADHEr, 9.72 0.44 23.30 0.00
12.97 3.18 -20.00 0.00 29.62 ATPS4r,
MDH, RPE 164 ADHEr, 9.72 0.44 23.30 0.00 12.97 3.18 -20.00 0.00
29.62 ATPS4r, FUM, RPE 165 ADHEr, 9.39 0.46 23.42 0.00 11.71 4.69
-20.00 0.00 31.40 ATPS4r, NADH6, PGDH 166 ADHEr, 9.39 0.46 23.42
0.00 11.71 4.69 -20.00 0.00 31.40 ATPS4r, NADH6, PGL 167 ADHEr,
9.39 0.46 23.42 0.00 11.71 4.69 -20.00 0.00 31.40 ATPS4r, G6PDHy,
NADH6 168 ADHEr, 9.34 0.46 23.40 0.00 11.35 5.25 -20.00 0.00 31.94
ATPS4r, NADH6, TAL 169 ADHEr, 9.29 0.46 23.37 0.00 11.02 5.76
-20.00 0.00 32.44 ATPS4r, NADH6, RPE 170 ADHEr, 9.08 0.43 19.50
0.00 0.35 26.49 -20.00 0.00 49.05 G6PDHy, ME2, THD2 171 ADHEr, 9.08
0.43 19.50 0.00 0.35 26.49 -20.00 0.00 49.05 ME2, PGL, THD2 172
ADHEr, 8.99 0.47 0.00 0.00 6.58 33.74 -20.00 0.00 45.14 G6PDHy,
PPC, THD2 173 ADHEr, 8.99 0.47 0.00 0.00 6.58 33.74 -20.00 0.00
45.14 PGL, PPC, THD2 174 ADHEr, 8.65 0.43 23.82 0.00 6.11 13.67
-20.00 0.00 40.57 ATPS4r, GLUDy, NADH6 175 ADHEr, 8.46 0.64 8.81
0.00 9.05 20.63 -20.00 0.00 37.61 ACKr, FRD2, LDH_D 176 ADHEr, 8.29
0.26 27.65 0.00 3.98 16.88 -20.00 0.00 46.36 ATPS4r, FBA, RPE 177
ADHEr, 8.29 0.26 27.65 0.00 3.98 16.88 -20.00 0.00 46.36 ATPS4r,
RPE, TPI 178 ADHEr, 8.29 0.26 27.65 0.00 3.98 16.88 -20.00 0.00
46.36 ATPS4r, PFK, RPE 179 ADHEr, 8.28 0.23 28.10 0.00 3.79 17.22
-20.00 0.00 46.98 ATPS4r, GLUDy, PFK 180 ADHEr, 8.28 0.23 28.10
0.00 3.79 17.22 -20.00 0.00 46.98 ATPS4r, GLUDy, TPI 181 ADHEr,
8.28 0.23 28.10 0.00 3.79 17.22 -20.00 0.00 46.98 ATPS4r, FBA,
GLUDy 182 ADHEr, 8.28 0.26 27.67 0.00 3.98 16.83 -20.00 0.00 46.35
ATPS4r, PFK, TAL 183 ADHEr, 8.28 0.26 27.67 0.00 3.98 16.83 -20.00
0.00 46.35 ATPS4r, TAL, TPI 184 ADHEr, 8.28 0.26 27.67 0.00 3.98
16.83 -20.00 0.00 46.35 ATPS4r, FBA, TAL 185 ADHEr, 8.16 0.28 23.57
0.00 -4.24 32.94 -20.00 0.00 58.48 ASPT, MDH, PYK 186 ADHEr, 8.00
0.71 13.36 0.00 7.12 24.68 -20.00 0.00 43.08 MDH, PGL, THD2 187
ADHEr, 8.00 0.71 13.36 0.00 7.12 24.68 -20.00 0.00 43.08 G6PDHy,
MDH, THD2 188 ADHEr, 7.89 0.43 24.60 0.00 2.33 18.97 -20.00 0.00
46.64 GLCpts, NADH6, THD2 189 ADHEr, 7.89 0.45 24.31 0.00 2.42
18.77 -20.00 0.00 46.27 GLUDy, NADH6, THD2 190 ADHEr, 7.71 0.45
21.65 0.00 -4.12 31.31 -20.00 0.00 56.15 ASPT, LDH_D, MDH 191
ADHEr, 7.65 0.19 29.27 0.00 0.15 22.63 -20.00 0.00 53.28 GLCpts,
NADH6, PGI 192 ADHEr, 7.64 0.40 24.07 0.00 -1.65 26.18 -20.00 0.00
53.11 LDH_D, SUCD4, THD2 193 ADHEr, 7.62 0.21 29.06 0.00 0.16 22.53
-20.00 0.00 53.08 GLUDy, NADH6, PGI 194 ADHEr, 7.62 0.32 7.79 0.00
-2.23 9.47 -20.00 0.00 37.34 ACKr, FUM, LDH_D 195 ADHEr, 7.59 0.20
29.34 0.00 -0.03 22.79 -20.00 0.00 53.53 FBA, GLCpts, NADH6 196
ADHEr, 7.59 0.20 29.34 0.00 -0.03 22.79 -20.00 0.00 53.53 GLCpts,
NADH6, TPI 197 ADHEr, 7.62 0.32 7.79 0.00 -2.23 9.47 -20.00 0.00
37.34 ACKr, LDH_D, MDH 198 ADHEr, 7.59 0.20 29.34 0.00 -0.03 22.79
-20.00 0.00 53.53 GLCpts, NADH6, PFK 199 ADHEr, 7.58 0.43 23.76
0.00 -1.67 26.01 -20.00 0.00 52.82 GLCpts, MDH, THD2 200 ADHEr,
7.58 0.43 23.76 0.00 -1.67 26.01 -20.00 0.00 52.82 FUM, GLCpts,
THD2 201 ADHEr, 7.57 0.23 28.73 0.00 0.14 22.39 -20.00 0.00 52.79
NADH6, PFK, RPE 202 ADHEr, 7.57 0.23 28.73 0.00 0.14 22.39 -20.00
0.00 52.79 FBA, NADH6, RPE 203 ADHEr, 7.57 0.23 28.73 0.00 0.14
22.39 -20.00 0.00 52.79 NADH6, RPE, TPI 204 ADHEr, 7.56 0.21 29.14
0.00 -0.03 22.70 -20.00 0.00 53.35 GLUDy, NADH6, TPI 205 ADHEr,
7.56 0.21 29.14 0.00 -0.03 22.70 -20.00 0.00 53.35 GLUDy, NADH6,
PFK 206 ADHEr, 7.56 0.21 29.14 0.00 -0.03 22.70 -20.00 0.00 53.35
FBA, GLUDy, NADH6 207 ADHEr, 7.54 0.24 28.76 0.00 0.06 22.46 -20.00
0.00 52.90 NADH6, PFK, TAL 208 ADHEr, 7.54 0.24 28.76 0.00 0.06
22.46 -20.00 0.00 52.90 FBA, NADH6, TAL 209 ADHEr, 7.54 0.24 28.76
0.00 0.06 22.46 -20.00 0.00 52.90 NADH6, TAL, TPI 210 ADHEr, 7.24
0.58 7.55 0.00 52.00 0.00 -20.00 0.00 11.65 ACKr, AKGD, ATPS4r 211
ADHEr, 7.17 0.45 25.76 0.00 0.26 20.93 -20.00 0.00 49.87 GLCpts,
NADH6, RPE 212 ADHEr, 7.15 0.40 26.55 0.00 -0.06 21.52 -20.00 0.00
50.95 FUM, GLCpts, NADH6 213 ADHEr, 7.15 0.40 26.55 0.00 -0.06
21.52 -20.00 0.00 50.95 GLCpts, MDH, NADH6 214 ADHEr, 7.15 0.41
26.54 0.00 -0.06 21.51 -20.00 0.00 50.94 GLCpts, GLUDy, NADH6 215
ADHEr, 7.15 0.41 26.54 0.00 -0.06 21.51 -20.00 0.00 50.94 FUM,
NADH6, PYK 216 ADHEr, 7.15 0.41 26.54 0.00 -0.06 21.51 -20.00 0.00
50.94 MDH, NADH6, PYK 217 ADHEr, 7.14 0.58 7.46 0.00 16.55 35.80
-20.00 0.00 47.39 ACKr, ATPS4r, SUCOAS 218 ADHEr, 7.14 0.46 25.54
0.00 0.27 20.82 -20.00 0.00 49.66 GLUDy, NADH6, RPE 219 ADHEr, 7.13
0.47 25.45 0.00 0.27 20.78 -20.00 0.00 49.57 MDH, NADH6, RPE 220
ADHEr, 7.13 0.47 25.45 0.00 0.27 20.78 -20.00 0.00 49.57 FUM,
NADH6, RPE 221 ADHEr, 7.12 0.45 25.84 0.00 0.11 21.08 -20.00 0.00
50.11 GLCpts, NADH6, TAL 222 ADHEr, 7.09 0.46 25.63 0.00 0.11 20.98
-20.00 0.00 49.91 GLUDy, NADH6, TAL 223 ADHEr, 7.07 0.47 25.53 0.00
0.11 20.93 -20.00 0.00 49.82 FUM, NADH6, TAL 224 ADHEr, 7.07 0.47
25.53 0.00 0.11 20.93 -20.00 0.00 49.82 MDH, NADH6, TAL 225 ADHEr,
6.93 0.51 25.11 0.00 -0.07 20.86 -20.00 0.00 49.62 CBMK2, GLU5K,
NADH6 226 ADHEr, 6.93 0.51 25.11 0.00 -0.07 20.86 -20.00 0.00 49.62
CBMK2, G5SD, NADH6 227 ADHEr, 6.93 0.51 25.10 0.00 -0.07 20.86
-20.00 0.00 49.60 CBMK2, NADH6, SO4t2 228 ADHEr, 6.93 0.51 25.11
0.00 -0.07 20.86 -20.00 0.00 49.61 ASNS2, CBMK2, NADH6 229 ADHEr,
6.69 0.36 19.85 0.00 7.01 7.99 -20.00 0.00 38.27 ATPS4r, PYK,
SUCD4 230 ADHEr, 6.40 0.18 30.74 0.00 -6.26 31.69 -20.00 0.00 63.71
GLCpts, PGI, SUCD4 231 ADHEr, 6.38 0.19 30.56 0.00 -6.23 31.57
-20.00 0.00 63.51 FUM, GLUDy, PGI 232 ADHEr, 6.38 0.19 30.56 0.00
-6.23 31.57 -20.00 0.00 63.51 GLUDy, MDH, PGI 233 ADHEr, 6.37 0.20
30.45 0.00 -6.21 31.50 -20.00 0.00 63.38 GLUDy, PGI, SUCD4 234
ADHEr, 6.35 0.18 30.80 0.00 -6.37 31.76 -20.00 0.00 63.86 GLCpts,
SUCD4, TPI 235 ADHEr, 6.35 0.18 30.80 0.00 -6.37 31.76 -20.00 0.00
63.86 GLCpts, PFK, SUCD4 236 ADHEr, 6.35 0.18 30.80 0.00 -6.37
31.76 -20.00 0.00 63.86 FBA, GLCpts, SUCD4 237 ADHEr, 6.32 0.22
30.18 0.00 -6.20 31.34 -20.00 0.00 63.09 HEX1, RPE, TPI 238 ADHEr,
6.32 0.22 30.18 0.00 -6.20 31.34 -20.00 0.00 63.09 FBA, HEX1, RPE
239 ADHEr, 6.32 0.22 30.18 0.00 -6.20 31.34 -20.00 0.00 63.09 HEX1,
PFK, RPE 240 ADHEr, 6.32 0.20 30.62 0.00 -6.35 31.65 -20.00 0.00
63.67 FUM, GLUDy, TPI 241 ADHEr, 6.32 0.20 30.62 0.00 -6.35 31.65
-20.00 0.00 63.67 FBA, GLUDy, MDH 242 ADHEr, 6.32 0.20 30.62 0.00
-6.35 31.65 -20.00 0.00 63.67 FUM, GLUDy, PFK 243 ADHEr, 6.32 0.20
30.62 0.00 -6.35 31.65 -20.00 0.00 63.67 GLUDy, MDH, PFK 244 ADHEr,
6.32 0.20 30.62 0.00 -6.35 31.65 -20.00 0.00 63.67 GLUDy, MDH, TPI
245 ADHEr, 6.32 0.20 30.62 0.00 -6.35 31.65 -20.00 0.00 63.67 FBA,
FUM, GLUDy 246 ADHEr, 6.32 0.23 29.99 0.00 -6.14 31.21 -20.00 0.00
62.85 GLCpts, GLUDy, PGI 247 ADHEr, 6.32 0.23 30.12 0.00 -6.19
31.30 -20.00 0.00 63.02 RPE, SUCD4, TPI 248 ADHEr, 6.32 0.23 30.12
0.00 -6.19 31.30 -20.00 0.00 63.02 FBA, RPE, SUCD4 249 ADHEr, 6.32
0.23 30.12 0.00 -6.19 31.30 -20.00 0.00 63.02 PFK, RPE, SUCD4 250
ADHEr, 6.31 0.20 30.55 0.00 -6.34 31.60 -20.00 0.00 63.59 GLUDy,
HEX1, PFK 251 ADHEr, 6.31 0.20 30.55 0.00 -6.34 31.60 -20.00 0.00
63.59 GLUDy, HEX1, TPI 252 ADHEr, 6.31 0.20 30.55 0.00 -6.34 31.60
-20.00 0.00 63.59 FBA, GLUDy, HEX1 253 ADHEr, 6.31 0.20 30.51 0.00
-6.34 31.58 -20.00 0.00 63.55 FBA, GLUDy, SUCD4 254 ADHEr, 6.31
0.20 30.51 0.00 -6.34 31.58 -20.00 0.00 63.55 GLUDy, SUCD4, TPI 255
ADHEr, 6.31 0.20 30.51 0.00 -6.34 31.58 -20.00 0.00 63.55 GLUDy,
PFK, SUCD4 256 ADHEr, 6.30 0.22 30.21 0.00 -6.25 31.37 -20.00 0.00
63.16 FBA, HEX1, TAL 257 ADHEr, 6.30 0.22 30.21 0.00 -6.25 31.37
-20.00 0.00 63.16 HEX1, PFK, TAL 258 ADHEr, 6.30 0.22 30.21 0.00
-6.25 31.37 -20.00 0.00 63.16 HEX1, TAL, TPI 259 ADHEr, 6.29 0.23
30.14 0.00 -6.24 31.33 -20.00 0.00 63.09 PFK, SUCD4, TAL 260 ADHEr,
6.29 0.23 30.14 0.00 -6.24 31.33 -20.00 0.00 63.09 SUCD4, TAL, TPI
261 ADHEr, 6.29 0.23 30.14 0.00 -6.24 31.33 -20.00 0.00 63.09 FBA,
SUCD4, TAL 262 ADHEr, 6.28 0.22 6.40 0.00 -6.31 7.56 -20.00 0.00
39.41 ACKr, LDH_D, SUCD4 263 ADHEr, 6.26 0.26 29.61 0.00 -6.11
30.98 -20.00 0.00 62.45 GLCpts, RPE, TPI 264 ADHEr, 6.26 0.26 29.61
0.00 -6.11 30.98 -20.00 0.00 62.45 GLCpts, PFK, RPE 265 ADHEr, 6.26
0.26 29.61 0.00 -6.11 30.98 -20.00 0.00 62.45 FBA, GLCpts, RPE 266
ADHEr, 6.26 0.23 0.00 0.00 -6.29 1.22 -20.00 0.00 32.99 ACt6,
LDH_D, MDH 267 ADHEr, 6.26 0.24 30.08 0.00 -6.29 31.31 -20.00 0.00
63.07 GLCpts, GLUDy, TPI 268 ADHEr, 6.26 0.23 0.00 0.00 -6.29 1.22
-20.00 0.00 32.99 ACt6, FUM, LDH_D 269 ADHEr, 6.26 0.24 30.08 0.00
-6.29 31.31 -20.00 0.00 63.07 FBA, GLCpts, GLUDy 270 ADHEr, 6.26
0.24 30.08 0.00 -6.29 31.31 -20.00 0.00 63.07 GLCpts, GLUDy, PFK
271 ADHEr, 6.25 0.27 29.46 0.00 -6.09 30.88 -20.00 0.00 62.28
GLUDy, RPE, TPI 272 ADHEr, 6.25 0.27 29.46 0.00 -6.09 30.88 -20.00
0.00 62.28 FBA, GLUDy, RPE 273 ADHEr, 6.25 0.27 29.46 0.00 -6.09
30.88 -20.00 0.00 62.28 GLUDy, PFK, RPE 274 ADHEr, 6.24 0.26 29.65
0.00 -6.17 31.02 -20.00 0.00 62.54 GLCpts, TAL, TPI 275 ADHEr, 6.24
0.26 29.65 0.00 -6.17 31.02 -20.00 0.00 62.54 FBA, GLCpts, TAL 276
ADHEr, 6.24 0.26 29.65 0.00 -6.17 31.02 -20.00 0.00 62.54 GLCpts,
PFK, TAL 277 ADHEr, 6.22 0.27 29.50 0.00 -6.15 30.93 -20.00 0.00
62.37 GLUDy, PFK, TAL 278 ADHEr, 6.22 0.27 29.50 0.00 -6.15 30.93
-20.00 0.00 62.37 FBA, GLUDy, TAL 279 ADHEr, 6.22 0.27 29.50 0.00
-6.15 30.93 -20.00 0.00 62.37 GLUDy, TAL, TPI 280 ADHEr, 6.09 0.33
28.74 0.00 -6.13 30.48 -20.00 0.00 61.59 GLUDy, MDH, PYK 281 ADHEr,
6.09 0.33 28.74 0.00 -6.13 30.48 -20.00 0.00 61.59 FUM, GLUDy, PYK
282 ADHEr, 6.08 0.38 27.93 0.00 -5.86 29.92 -20.00 0.00 60.54 PYK,
RPE, SUCD4 283 ADHEr, 6.08 0.34 28.65 0.00 -6.12 30.43 -20.00 0.00
61.49 GLUDy, PYK, SUCD4 284 ADHEr, 6.06 0.35 28.51 0.00 -6.10 30.34
-20.00 0.00 61.33 MDH, PYK, SUCD4 285 ADHEr, 6.06 0.35 28.51 0.00
-6.10 30.34 -20.00 0.00 61.33 FUM, PYK, SUCD4 286 ADHEr, 6.06 0.40
27.70 0.00 -5.83 29.77 -20.00 0.00 60.29 GLCpts, RPE, SUCD4 287
ADHEr, 6.05 0.35 28.44 0.00 -6.10 30.30 -20.00 0.00 61.26 GLCpts,
GLUDy, MDH 288 ADHEr, 6.05 0.35 28.44 0.00 -6.10 30.30 -20.00 0.00
61.26 FUM, GLCpts, GLUDy 289 ADHEr, 6.04 0.36 28.39 0.00 -6.09
30.27 -20.00 0.00 61.21 GLCpts, GLUDy, SUCD4 290 ADHEr, 6.04 0.38
27.98 0.00 -5.95 29.98 -20.00 0.00 60.67 PYK, SUCD4, TAL 291 ADHEr,
6.03 0.41 27.44 0.00 -5.79 29.60 -20.00 0.00 59.98 GLUDy, MDH, RPE
292 ADHEr, 6.03 0.41 27.44 0.00 -5.79 29.60 -20.00 0.00 59.98 FUM,
GLUDy, RPE 293 ADHEr, 6.02 0.42 27.40 0.00 -5.78 29.58 -20.00 0.00
59.94 GLUDy, RPE, SUCD4 294 ADHEr, 6.02 0.40 27.77 0.00 -5.92 29.84
-20.00 0.00 60.43 GLCpts, SUCD4, TAL 295 ADHEr, 5.99 0.42 27.50
0.00 -5.89 29.68 -20.00 0.00 60.14 GLUDy, MDH, TAL 296 ADHEr, 5.99
0.42 27.50 0.00 -5.89 29.68 -20.00 0.00 60.14 FUM, GLUDy, TAL 297
ADHEr, 5.98 0.42 27.47 0.00 -5.88 29.66 -20.00 0.00 60.10 GLUDy,
SUCD4,
TAL 298 ADHEr, 5.96 0.46 26.84 0.00 -5.70 29.23 -20.00 0.00 59.31
GLCpts, GLUDy, RPE 299 ADHEr, 5.94 0.47 26.66 0.00 -5.67 29.11
-20.00 0.00 59.10 FUM, GLCpts, RPE 300 ADHEr, 5.94 0.47 26.66 0.00
-5.67 29.11 -20.00 0.00 59.10 GLCpts, MDH, RPE 301 ADHEr, 5.92 0.43
27.41 0.00 -5.98 29.66 -20.00 0.00 60.12 FUM, LDH_D, SUCD4 302
ADHEr, 5.92 0.43 27.41 0.00 -5.98 29.66 -20.00 0.00 60.12 LDH_D,
MDH, SUCD4 303 ADHEr, 5.92 0.46 26.93 0.00 -5.81 29.32 -20.00 0.00
59.49 GLCpts, GLUDy, TAL 304 ADHEr, 5.90 0.47 26.74 0.00 -5.78
29.20 -20.00 0.00 59.28 FUM, GLCpts, TAL 305 ADHEr, 5.90 0.47 26.74
0.00 -5.78 29.20 -20.00 0.00 59.28 GLCpts, MDH, TAL 306 ADHEr, 5.86
0.46 26.97 0.00 -5.93 29.38 -20.00 0.00 59.63 CBMK2, GLU5K, SUCD4
307 ADHEr, 5.86 0.46 26.97 0.00 -5.93 29.38 -20.00 0.00 59.63
CBMK2, G5SD, SUCD4 308 ADHEr, 5.79 0.56 25.29 0.00 -5.47 28.25
-20.00 0.00 57.55 CBMK2, GLU5K, RPE 309 ADHEr, 5.79 0.56 25.29 0.00
-5.47 28.25 -20.00 0.00 57.55 CBMK2, G5SD, RPE 310 ADHEr, 5.79 0.50
26.42 0.00 -5.86 29.04 -20.00 0.00 59.02 CBMK2, GLCpts, GLU5K 311
ADHEr, 5.79 0.56 25.29 0.00 -5.47 28.25 -20.00 0.00 57.55 ASNS2,
CBMK2, RPE 312 ADHEr, 5.79 0.50 26.42 0.00 -5.86 29.04 -20.00 0.00
59.02 CBMK2, G5SD, GLCpts 313 ADHEr, 5.79 0.50 26.42 0.00 -5.86
29.04 -20.00 0.00 59.02 ASNS2, CBMK2, GLCpts 314 ADHEr, 5.74 0.57
25.40 0.00 -5.60 28.36 -20.00 0.00 57.77 CBMK2, GLU5K, TAL 315
ADHEr, 5.74 0.57 25.40 0.00 -5.60 28.36 -20.00 0.00 57.77 CBMK2,
G5SD, TAL 316 ADHEr, 5.74 0.57 25.40 0.00 -5.60 28.36 -20.00 0.00
57.77 ASNS2, CBMK2, TAL 317 ADHEr, 5.74 0.53 25.99 0.00 -5.81 28.77
-20.00 0.00 58.54 CBMK2, GLU5K, MDH 318 ADHEr, 5.74 0.53 25.99 0.00
-5.81 28.77 -20.00 0.00 58.54 CBMK2, FUM, G5SD 319 ADHEr, 5.74 0.53
25.99 0.00 -5.81 28.77 -20.00 0.00 58.54 CBMK2, G5SD, MDH 320
ADHEr, 5.74 0.53 25.99 0.00 -5.81 28.77 -20.00 0.00 58.54 CBMK2,
FUM, GLU5K 321 ADHEr, 5.74 0.53 25.99 0.00 -5.81 28.77 -20.00 0.00
58.54 ASNS2, CBMK2, MDH 322 ADHEr, 5.74 0.53 25.99 0.00 -5.81 28.77
-20.00 0.00 58.54 ASNS2, CBMK2, FUM 323 ADHEr, 5.68 0.57 25.48 0.00
-5.75 28.46 -20.00 0.00 57.99 ASNS2, GLU5K, SO4t2 324 ADHEr, 14.96
0.23 14.07 0.00 22.42 0.00 -20.00 0.00 15.71 ASPT, LDH_D, MDH, PFLi
325 ADHEr, 14.81 0.14 16.32 0.00 22.21 0.00 -20.00 0.00 17.33 EDA,
GLUDy, PFLi, PGI 326 ADHEr, 14.63 0.18 15.95 0.00 21.94 0.00 -20.00
0.00 17.25 ATPS4r, G6PDHy, GLCpts, MDH 327 ADHEr, 14.63 0.18 15.95
0.00 21.94 0.00 -20.00 0.00 17.25 ATPS4r, GLCpts, MDH, PGL 328
ADHEr, 14.42 0.11 17.14 0.00 21.96 1.32 -20.00 0.00 19.28 EDA,
GLUDy, NADH6, PGI 329 ADHEr, 13.73 0.10 9.55 0.00 1.40 40.38 -20.00
0.00 50.65 G6PDHy, LDH_D, PPC, THD2 330 ADHEr, 13.73 0.10 9.55 0.00
1.40 40.38 -20.00 0.00 50.65 LDH_D, PGL, PPC, THD2 331 ADHEr, 13.37
0.17 4.27 6.42 13.23 18.61 -20.00 0.00 30.48 ATPS4r, FRD2, LDH_D,
NADH6 332 ADHEr, 13.09 0.16 10.61 0.00 1.33 38.58 -20.00 0.00 50.31
EDA, LDH_D, PPC, THD2 333 ADHEr, 12.96 0.12 12.19 0.00 0.07 38.74
-20.00 0.00 51.78 FRD2, GLUDy, LDH_D, RPE 334 ADHEr, 12.95 0.12
12.13 0.00 0.07 38.68 -20.00 0.00 51.70 FRD2, LDH_D, RPE, THD2 335
ADHEr, 12.94 0.11 12.36 0.00 -0.02 38.83 -20.00 0.00 52.00 FRD2,
GLUDy, LDH_D, THD2 336 ADHEr, 12.94 0.12 12.24 0.00 0.03 38.74
-20.00 0.00 51.84 FRD2, GLUDy, LDH_D, TAL 337 ADHEr, 12.92 0.13
12.19 0.00 0.03 38.69 -20.00 0.00 51.77 FRD2, LDH_D, TAL, THD2 338
ADHEr, 12.83 0.28 9.13 0.00 9.66 29.15 -20.00 0.00 40.26 ATPS4r,
LDH_D, NADH6, PPC 339 ADHEr, 12.77 0.13 12.57 0.00 -0.13 38.55
-20.00 0.00 52.04 ASPT, FUM, GLUDy, LDH_D 340 ADHEr, 12.75 0.13
12.54 0.00 -0.14 38.50 -20.00 0.00 51.99 ASPT, FUM, LDH_D, THD2 341
ADHEr, 12.69 0.38 17.31 0.00 19.01 0.00 -20.00 0.00 19.99 ME2,
PFLi, PGL, THD2 342 ADHEr, 12.69 0.38 17.31 0.00 19.01 0.00 -20.00
0.00 19.99 G6PDHy, ME2, PFLi, THD2 343 ADHEr, 12.67 0.10 12.72 0.00
0.38 39.23 -20.00 0.00 52.69 ACKr, ACS, PPC, RPE 344 ADHEr, 12.67
0.16 11.69 0.00 0.58 38.82 -20.00 0.00 51.63 GLUDy, LDH_D, PPC, RPE
345 ADHEr, 12.67 0.16 11.57 0.00 0.60 38.78 -20.00 0.00 51.52
LDH_D, PPC, RPE, THD2 346 ADHEr, 12.64 0.16 11.75 0.00 0.53 38.83
-20.00 0.00 51.71 GLUDy, LDH_D, PPC, TAL 347 ADHEr, 12.63 0.16
11.65 0.00 0.55 38.79 -20.00 0.00 51.61 LDH_D, PPC, TAL, THD2 348
ADHEr, 12.62 0.10 12.80 0.00 0.31 39.24 -20.00 0.00 52.79 GLCpts,
GLUDy, LDH_D, PPC 349 ADHEr, 12.62 0.11 12.74 0.00 0.32 39.22
-20.00 0.00 52.73 GLCpts, LDH_D, PPC, THD2 350 ADHEr, 12.38 0.13
12.45 0.00 9.92 10.79 -20.00 0.00 30.65 ACKr, GLUDy, LDH_D, PGI 351
ADHEr, 12.20 0.20 10.35 0.00 18.28 0.00 -20.00 0.00 19.76 ASPT,
ATPS4r, GLCpts, MDH 352 ADHEr, 12.11 0.25 21.60 0.00 18.15 0.00
-20.00 0.00 23.35 LDH_D, PFLi, SUCD4, THD2 353 ADHEr, 12.05 0.24
12.18 0.00 12.02 25.48 -20.00 0.00 39.36 ACKr, AKGD, ATPS4r, PYK
354 ADHEr, 12.00 0.24 12.13 0.00 12.20 25.51 -20.00 0.00 39.36
ACKr, ATPS4r, PYK, SUCOAS
355 ADHEr, 11.98 0.13 19.29 0.00 7.97 19.98 -20.00 0.00 40.22
ATPS4r, EDA, GLUDy, PGI 356 ADHEr, 11.98 0.14 19.25 0.00 7.98 19.96
-20.00 0.00 40.18 EDA, GLCpts, GLUDy, PGI 357 ADHEr, 11.91 0.20
12.01 3.31 5.46 24.78 -20.00 0.00 41.50 ACKr, LDH_D, MDH, SUCD4 358
ADHEr, 11.90 0.49 12.16 0.00 27.81 0.00 -20.00 0.00 15.63 ACKr,
GLUDy, NADH6, PYK 359 ADHEr, 11.76 0.39 19.51 0.00 17.62 0.00
-20.00 0.00 22.30 FUM, LDH_D, PFLi, THD2 360 ADHEr, 11.76 0.39
19.51 0.00 17.62 0.00 -20.00 0.00 22.30 LDH_D, MDH, PFLi, THD2 361
ADHEr, 11.67 0.14 0.00 0.00 1.15 24.89 -19.46 0.00 38.65 ACt6,
ATPS4r, LDH_D, PPC 362 ADHEr, 11.66 0.21 11.78 0.00 8.63 21.96
-20.00 0.00 37.33 ACKr, LDH_D, PYRt2, SUCD4 363 ADHEr, 11.59 0.23
20.54 0.00 13.84 9.06 -20.00 0.00 31.25 ATPS4r, FUM, LDH_D, SUCD4
364 ADHEr, 11.59 0.23 20.54 0.00 13.84 9.06 -20.00 0.00 31.25
ATPS4r, LDH_D, MDH, SUCD4 365 ADHEr, 11.52 0.55 11.82 0.00 27.24
0.00 -20.00 0.00 15.76 ACKr, CBMK2, NADH6, PYK 366 ADHEr, 11.50
0.56 11.81 0.00 27.22 0.00 -20.00 0.00 15.77 ACKr, NADH6, PYK, RPE
367 ADHEr, 11.50 0.56 11.80 0.00 27.21 0.00 -20.00 0.00 15.77 ACKr,
ASNS2, NADH6, PYK 368 ADHEr, 11.49 0.56 11.80 0.00 27.20 0.00
-20.00 0.00 15.77 ACKr, NADH6, PYK, TAL 369 ADHEr, 11.46 0.30 8.96
0.00 8.56 25.90 -20.00 0.00 38.85 ASPT, ATPS4r, FUM, LDH_D 370
ADHEr, 11.43 0.34 17.13 0.00 8.27 17.71 -20.00 0.00 37.24 ASPT,
ATPS4r, LDH_D, MDH 371 ADHEr, 11.38 0.38 20.90 0.00 17.05 0.00
-20.00 0.00 23.58 MDH, PFLi, PYK, THD2 372 ADHEr, 11.38 0.38 20.90
0.00 17.05 0.00 -20.00 0.00 23.58 FUM, PFLi, PYK, THD2 373 ADHEr,
11.25 0.53 11.54 0.00 23.26 0.00 -20.00 0.00 17.50 ACKr, FUM,
LDH_D, NADH6 374 ADHEr, 11.24 0.27 20.72 0.00 13.49 8.70 -20.00
0.00 31.38 ATPS4r, G6PDHy, LDH_D, SUCD4 375 ADHEr, 11.24 0.27 20.72
0.00 13.49 8.70 -20.00 0.00 31.38 ATPS4r, LDH_D, PGL, SUCD4 376
ADHEr, 11.24 0.27 20.72 0.00 13.49 8.70 -20.00 0.00 31.38 ATPS4r,
LDH_D, PGDH, SUCD4 377 ADHEr, 11.24 0.14 2.95 0.00 3.54 28.60
-20.00 0.02 42.95 GLYCL, PGL, PPC, THD2 378 ADHEr, 11.24 0.14 2.95
0.00 3.54 28.60 -20.00 0.02 42.95 G6PDHy, GLYCL, PPC, THD2 379
ADHEr, 11.22 0.14 2.94 0.00 3.54 28.55 -20.00 0.00 42.92 FTHFD,
G6PDHy, PPC, THD2 380 ADHEr, 11.22 0.14 2.94 0.00 3.54 28.55 -20.00
0.00 42.92 FTHFD, PGL, PPC, THD2 381 ADHEr, 11.22 0.14 2.94 0.00
3.54 28.55 -20.00 0.00 42.92 MTHFC, PGL, PPC, THD2 382 ADHEr, 11.22
0.14 2.94 0.00 3.54 28.55 -20.00 0.00 42.92 G6PDHy, MTHFC, PPC,
THD2 383 ADHEr, 11.20 0.32 22.07 0.00 17.78 0.00 -20.00 0.00 24.34
ATPS4r, LDH_D, PFLi, SUCD4 384 ADHEr, 11.18 0.28 20.84 0.00 13.41
8.69 -20.00 0.00 31.49 ATPS4r, LDH_D, SUCD4, TAL 385 ADHEr, 11.13
0.28 23.11 0.00 17.68 0.00 -20.00 0.00 25.09 ATPS4r, LDH_D, RPE,
SUCD4 386 ADHEr, 11.13 0.27 19.49 0.00 16.68 0.00 -20.00 0.00 24.27
ATPS4r, FUM, GLCpts, NADH6 387 ADHEr, 11.13 0.27 19.49 0.00 16.68
0.00 -20.00 0.00 24.27 ATPS4r, GLCpts, MDH, NADH6 388 ADHEr, 11.12
0.34 22.41 0.00 16.65 0.00 -20.00 0.00 24.82 ATPS4r, LDH_D, NADH6,
PFLi 389 ADHEr, 11.06 0.63 11.41 0.00 26.56 0.00 -20.00 0.00 15.92
ACKr, MALS, NADH12, NADH6 390 ADHEr, 11.06 0.63 11.41 0.00 26.56
0.00 -20.00 0.00 15.92 ACKr, ICL, NADH12, NADH6 391 ADHEr, 11.06
0.64 11.40 0.00 26.54 0.00 -20.00 0.00 15.93 ACKr, CBMK2, LDH_D,
NADH6 392 ADHEr, 11.04 0.35 22.48 0.00 16.54 0.00 -20.00 0.00 24.94
ATPS4r, GLCpts, MDH, PFLi 393 ADHEr, 11.04 0.35 22.48 0.00 16.54
0.00 -20.00 0.00 24.94 ATPS4r, FUM, GLCpts, PFLi 394 ADHEr, 11.03
0.64 11.38 0.00 26.51 0.00 -20.00 0.00 15.93 ACKr, ASNS2, LDH_D,
NADH6 395 ADHEr, 11.00 0.38 11.21 0.00 5.04 32.88 -20.00 0.00 46.77
ACKr, ATPS4r, LDH_D, THD2 396 ADHEr, 11.00 0.15 26.58 0.00 16.49
0.00 -20.00 0.00 27.66 GLCpts, GLUDy, PFLi, PGI 397 ADHEr, 10.97
0.15 26.63 0.00 16.45 0.00 -20.00 0.00 27.73 GLCpts, GLUDy, PFK,
PFLi 398 ADHEr, 10.97 0.15 26.63 0.00 16.45 0.00 -20.00 0.00 27.73
FBA, GLCpts, GLUDy, PFLi 399 ADHEr, 10.97 0.15 26.63 0.00 16.45
0.00 -20.00 0.00 27.73 GLCpts, GLUDy, PFLi, TPI 400 ADHEr, 10.97
0.11 27.23 0.00 15.95 0.99 -20.00 0.00 29.02 ATPS4r, GLCpts, NADH6,
PGI 401 ADHEr, 10.95 0.12 27.26 0.00 15.95 0.94 -20.00 0.00 29.02
ATPS4r, GLCpts, NADH6, TPI 402 ADHEr, 10.95 0.12 27.26 0.00 15.95
0.94 -20.00 0.00 29.02 ATPS4r, GLCpts, NADH6, PFK 403 ADHEr, 10.95
0.12 27.26 0.00 15.95 0.94 -20.00 0.00 29.02 ATPS4r, FBA, GLCpts,
NADH6 404 ADHEr, 10.94 0.17 26.35 0.00 16.40 0.00 -20.00 0.00 27.57
GLCpts, PFLi, RPE, TPI 405 ADHEr, 10.94 0.17 26.35 0.00 16.40 0.00
-20.00 0.00 27.57 GLCpts, PFK, PFLi, RPE 406 ADHEr, 10.94 0.17
26.35 0.00 16.40 0.00 -20.00 0.00 27.57 FBA, GLCpts, PFLi, RPE 407
ADHEr, 10.93 0.28 24.13 0.00 16.38 0.00 -20.00 0.00 26.12
ATPS4r,
NADH6, PFLi, PYK 408 ADHEr, 10.93 0.17 26.37 0.00 16.38 0.00 -20.00
0.00 27.59 GLCpts, PFK, PFLi, TAL 409 ADHEr, 10.93 0.17 26.37 0.00
16.38 0.00 -20.00 0.00 27.59 FBA, GLCpts, PFLi, TAL 410 ADHEr,
10.93 0.17 26.37 0.00 16.38 0.00 -20.00 0.00 27.59 GLCpts, PFLi,
TAL, TPI 411 ADHEr, 10.92 0.18 26.25 0.00 16.37 0.00 -20.00 0.00
27.52 FBA, GLUDy, PFLi, RPE 412 ADHEr, 10.92 0.18 26.25 0.00 16.37
0.00 -20.00 0.00 27.52 GLUDy, PFK, PFLi, RPE 413 ADHEr, 10.92 0.18
26.25 0.00 16.37 0.00 -20.00 0.00 27.52 GLUDy, PFLi, RPE, TPI 414
ADHEr, 10.91 0.18 26.27 0.00 16.35 0.00 -20.00 0.00 27.55 GLUDy,
PFLi, TAL, TPI 415 ADHEr, 10.91 0.18 26.27 0.00 16.35 0.00 -20.00
0.00 27.55 GLUDy, PFK, PFLi, TAL 416 ADHEr, 10.91 0.18 26.27 0.00
16.35 0.00 -20.00 0.00 27.55 FBA, GLUDy, PFLi, TAL 417 ADHEr, 10.79
0.32 21.77 0.00 14.53 5.29 -20.00 0.00 29.37 ATPS4r, LDH_D, NADH6,
SUCD4 418 ADHEr, 10.76 0.23 25.74 0.00 16.13 0.00 -20.00 0.00 27.34
FUM, LDH_D, PFLi, SUCD4 419 ADHEr, 10.76 0.23 25.74 0.00 16.13 0.00
-20.00 0.00 27.34 LDH_D, MDH, PFLi, SUCD4 H2O LAC NH4 NO3 PI PYR
SO4 SUC VAL 1 -0.90 0.00 -4.96 0.00 -0.61 0.00 -0.10 0.00 0.00 2
14.07 0.00 -4.31 0.00 -0.53 0.00 -0.09 0.00 0.00 3 2.51 0.00 -4.48
0.00 -0.55 0.00 -0.09 0.00 0.00 4 0.13 0.00 -4.83 0.00 -0.60 0.00
-0.10 0.00 0.00 5 -6.65 0.00 -2.62 0.00 -0.32 0.00 -0.05 0.00 0.00
6 -6.68 0.00 -2.66 0.00 -0.33 0.00 -0.05 0.00 0.00 7 -6.68 0.00
-2.66 0.00 -0.33 0.00 -0.05 0.00 0.00 8 1.88 0.00 -5.25 -2.00 -0.65
0.00 -0.11 0.00 0.00 9 -6.68 0.00 -2.66 0.00 -0.33 0.00 -0.05 0.00
0.00 10 -3.22 0.00 -4.04 0.00 -0.50 0.00 -0.08 0.00 0.00 11 -0.76
0.00 -4.95 0.00 -0.61 0.00 -0.10 0.00 0.00 12 -2.34 0.00 -4.39 0.00
-0.54 0.00 -0.09 0.00 0.00 13 -2.13 0.00 -4.47 0.00 -0.55 0.00
-0.09 0.00 0.00 14 -0.83 0.00 -4.95 0.00 -0.61 0.00 -0.10 0.00 0.00
15 -1.66 0.00 -4.66 0.00 -0.58 0.00 -0.09 0.00 0.00 16 -1.66 0.00
-4.66 0.00 -0.58 0.00 -0.09 0.00 0.00 17 -1.01 0.00 -4.92 0.00
-0.61 0.00 -0.10 0.00 0.00 18 0.36 0.00 -1.93 0.00 -0.24 0.00 -0.04
0.00 0.00 19 0.38 0.00 -1.94 0.00 -0.24 0.00 -0.04 0.00 0.00 20
9.09 0.00 -1.73 0.00 -0.21 0.00 -0.03 0.00 0.00 21 9.11 0.00 -1.75
0.00 -0.22 0.00 -0.04 0.00 0.00 22 9.11 0.00 -1.75 0.00 -0.22 0.00
-0.04 0.00 0.00 23 9.11 0.00 -1.75 0.00 -0.22 0.00 -0.04 0.00 0.00
24 14.16 0.00 -4.23 0.00 -0.52 0.00 -0.09 0.00 0.00 25 12.93 0.00
-3.72 0.00 -0.46 0.00 -0.08 0.00 0.00 26 13.23 0.00 -3.87 0.00
-0.48 0.00 -0.08 0.00 0.00 27 14.08 0.00 -4.30 0.00 -0.53 0.00
-0.09 0.00 0.00 28 14.07 0.00 -4.30 0.00 -0.53 0.00 -0.09 0.00 0.00
29 13.99 0.00 -4.27 0.00 -0.53 0.00 -0.09 0.00 0.00 30 -3.19 0.00
-2.28 0.00 -0.28 0.00 -0.05 0.00 0.00 31 6.46 0.00 -4.15 0.00 -0.51
0.00 -0.08 0.00 0.00 32 0.48 0.00 -2.25 0.00 -0.28 0.00 -0.05 0.00
0.00 33 0.48 0.00 -2.25 0.00 -0.28 0.00 -0.05 0.00 0.00 34 0.48
0.00 -2.25 0.00 -0.28 0.00 -0.05 0.00 0.00 35 3.90 0.00 -4.30 0.00
-0.53 0.00 -0.09 0.00 0.00 36 1.11 0.00 -4.22 0.00 -0.52 0.00 -0.09
0.00 0.00 37 1.11 0.00 -4.22 0.00 -0.52 0.00 -0.09 0.00 0.00 38
-3.12 0.00 -2.02 0.00 -0.25 0.00 -0.04 0.00 0.00 39 0.18 0.00 -4.28
0.00 -0.53 0.00 -0.09 0.00 0.00 40 0.18 0.00 -4.28 0.00 -0.53 0.00
-0.09 0.00 0.00 41 -3.17 0.00 -2.06 0.00 -0.25 0.00 -0.04 0.00 0.00
42 -3.17 0.00 -2.06 0.00 -0.25 0.00 -0.04 0.00 0.00 43 -3.17 0.00
-2.06 0.00 -0.25 0.00 -0.04 0.00 0.00 44 1.14 0.00 -3.90 0.00 -0.48
0.00 -0.08 0.00 0.00 45 2.70 0.00 -4.46 0.00 -0.55 0.00 -0.09 0.00
0.00 46 1.46 0.00 -4.03 0.00 -0.50 0.00 -0.08 0.00 0.00 47 1.61
0.00 -4.10 0.00 -0.51 0.00 -0.08 0.00 0.00 48 1.61 0.00 -4.10 0.00
-0.51 0.00 -0.08 0.00 0.00 49 2.61 0.00 -4.47 0.00 -0.55 0.00 -0.09
0.00 0.00 50 -1.43 0.00 -4.27 0.00 -0.53 0.00 -0.09 0.00 0.00 51
-1.17 0.00 -4.36 0.00 -0.54 0.00 -0.09 0.00 0.00 52 -8.37 0.00
-1.95 0.00 -0.24 0.00 -0.04 0.00 0.00 53 -7.58 0.00 -2.25 0.00
-0.28 0.00 -0.05 0.00 0.00 54 -8.47 0.00 -1.94 0.00 -0.24 0.00
-0.04 0.00 0.00 55 -8.47 0.00 -1.94 0.00 -0.24 0.00 -0.04 0.00 0.00
56 -8.47 0.00 -1.94 0.00 -0.24 0.00 -0.04 0.00 0.00 57 -8.37 0.00
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0.00 298 -3.31 0.00 -3.94 0.00 -0.49 0.00 -0.08 0.00 0.00 299 -3.02
0.00 -4.06 0.00 -0.50 0.00 -0.08 0.00 0.00 300 -3.02 0.00 -4.06
0.00 -0.50 0.00 -0.08 0.00 0.00 301 -4.04 0.00 -3.71 0.00 -0.46
0.00 -0.08 0.00 0.00 302 -4.04 0.00 -3.71 0.00 -0.46 0.00 -0.08
0.00 0.00 303 -3.36 0.00 -3.95 0.00 -0.49 0.00 -0.08 0.00 0.00 304
-3.07 0.00 -4.07 0.00 -0.50 0.00 -0.08 0.00 0.00 305 -3.07 0.00
-4.07 0.00 -0.50 0.00 -0.08 0.00 0.00 306 -3.34 0.00 -3.99 0.00
-0.49 0.00 -0.08 0.00 0.00 307 -3.34 0.00 -3.99 0.00 -0.49 0.00
-0.08 0.00 0.00 308 -0.91 0.00 -4.89 0.00 -0.60 0.00 -0.10 0.00
0.00 309 -0.91 0.00 -4.89 0.00 -0.60 0.00 -0.10 0.00 0.00 310 -2.47
0.00 -4.34 0.00 -0.54 0.00 -0.09 0.00 0.00 311 -0.91 0.00 -4.89
0.00 -0.60 0.00 -0.10 0.00 0.00 312 -2.47 0.00 -4.34 0.00 -0.54
0.00 -0.09 0.00 0.00 313 -2.47 0.00 -4.34 0.00 -0.54 0.00 -0.09
0.00 0.00 314 -0.98 0.00 -4.89 0.00 -0.61 0.00 -0.10 0.00 0.00 315
-0.98 0.00 -4.89 0.00 -0.61 0.00 -0.10 0.00 0.00 316 -0.98 0.00
-4.90 0.00 -0.61 0.00 -0.10 0.00 0.00 317 -1.80 0.00 -4.61 0.00
-0.57 0.00 -0.09 0.00 0.00 318 -1.80 0.00 -4.61 0.00 -0.57 0.00
-0.09 0.00 0.00 319 -1.80 0.00 -4.61 0.00 -0.57 0.00 -0.09 0.00
0.00 320 -1.80 0.00 -4.61 0.00 -0.57 0.00 -0.09 0.00 0.00 321 -1.80
0.00 -4.61 0.00 -0.57 0.00 -0.09 0.00 0.00 322 -1.80 0.00 -4.61
0.00 -0.57 0.00 -0.09 0.00 0.00 323 -1.01 0.00 -4.92 0.00 -0.61
0.00 -0.10 0.00 0.00 324 11.71 0.00 -2.00 0.00 -0.25 0.00 -0.04
0.00 0.00 325 10.00 0.00 -1.23 0.00 -0.15 0.00 -0.02 0.00 0.00 326
10.67 0.00 -1.59 0.00 -0.20 0.00 -0.03 0.00 0.00 327 10.67 0.00
-1.59 0.00 -0.20 0.00 -0.03 0.00 0.00 328 9.64 0.00 -0.99 -2.00
-0.12 0.00 -0.02 0.00 0.00 329 -10.49 0.00 -0.87 -2.00 -0.11 0.00
-0.02 0.00 0.00 330 -10.49 0.00 -0.87 -2.00 -0.11 0.00 -0.02 0.00
0.00 331 9.33 0.00 -7.86 -5.00 -0.18 0.00 -0.03 0.00 0.00 332 -8.88
0.00 -1.36 -2.00 -0.17 0.00 -0.03 0.00 0.00 333 -10.71 0.00 -1.03
0.00 -0.13 0.00 -0.02 0.00 0.00 334 -10.58 0.00 -1.08 0.00 -0.13
0.00 -0.02 0.00 0.00 335 -10.88 0.00 -0.98 0.00 -0.12 0.00 -0.02
0.00 0.00 336 -10.71 0.00 -1.04 0.00 -0.13 0.00 -0.02 0.00 0.00 337
-10.59 0.00 -1.09 0.00 -0.13 0.00 -0.02 0.00 0.00 338 1.95 0.00
-2.42 -10.00 -0.30 0.00 -0.05 0.00 0.00 339 -10.53 0.00 -1.12 0.00
-0.14 0.00 -0.02 0.00 0.00 340 -10.42 0.00 -1.16 0.00 -0.14 0.00
-0.02 0.00 0.00 341 13.21 0.00 -3.25 0.00 -0.40 0.00 -0.07 0.00
0.00 342 13.21 0.00 -3.25 0.00 -0.40 0.00 -0.07 0.00 0.00 343
-10.40 0.00 -0.89 -2.00 -0.11 0.00 -0.02 0.00 0.00 344 -9.20 0.00
-1.37 -2.00 -0.17 0.00 -0.03 0.00 0.00 345 -9.06 0.00 -1.42 -2.00
-0.18 0.00 -0.03 0.00 0.00 346 -9.20 0.00 -1.37 -2.00 -0.17 0.00
-0.03 0.00 0.00 347 -9.08 0.00 -1.42 -2.00 -0.18 0.00 -0.03 0.00
0.00 348 -10.40 0.00 -0.91 -2.00 -0.11 0.00 -0.02 0.00 0.00 349
-10.32 0.00 -0.93 -2.00 -0.12 0.00 -0.02 0.00 0.00 350 6.43 0.00
-1.13 0.00 -0.14 6.48 -0.02 0.00 0.00 351 9.70 8.01 -1.71 0.00
-0.21 0.00 -0.03 0.00 0.00 352 10.54 0.00 -2.12 0.00 -0.26 0.00
-0.04 0.00 0.00 353 4.35 0.00 -2.07 -13.40 -0.26 0.00 -0.04 0.00
0.00 354 4.62 0.00 -2.09 -13.93 -0.26 0.00 -0.04 0.00 0.00 355
-1.56 0.00 -1.15 0.00 -0.14 0.00 -0.02 0.00 0.00 356 -1.51 0.00
-1.18 0.00 -0.15 0.00 -0.02 0.00 0.00 357 0.47 0.00 -5.01 0.00
-0.21 0.00 -0.03 0.00 0.00 358 24.87 0.00 -4.23 -20.00 -0.52 0.00
-0.09 0.00 0.00 359 13.05 0.00 -3.40 0.00 -0.42 0.00 -0.07 0.00
0.00 360 13.05 0.00 -3.40 0.00 -0.42 0.00 -0.07 0.00 0.00 361 4.77
0.00 -1.18 -5.00 -0.15 12.79 -0.02 0.00 0.00 362 2.88 0.00 -3.91
0.00 -0.22 0.00 -0.04 0.00 2.12 363 6.51 0.00 -2.01 -2.00 -0.25
0.00 -0.04 0.00 0.00 364 6.51 0.00 -2.01 -2.00 -0.25 0.00 -0.04
0.00 0.00 365 25.89 0.00 -4.80 -20.00 -0.59 0.00 -0.10 0.00 0.00
366 25.93 0.00 -4.82 -20.00 -0.60 0.00 -0.10 0.00 0.00 367 25.95
0.00 -4.83 -20.00 -0.60 0.00 -0.10 0.00 0.00 368 25.96 0.00 -4.84
-20.00 -0.60 0.00 -0.10 0.00 0.00 369 4.50 0.00 -4.47 -5.00 -0.33
0.00 -0.05 0.00 1.84 370 3.02 0.00 -2.92 0.00 -0.36 0.00 -0.06 0.00
0.00 371 12.56 0.00 -3.25 0.00 -0.40 0.00 -0.07 0.00 0.00 372 12.56
0.00 -3.25 0.00 -0.40 0.00 -0.07 0.00 0.00 373 23.94 0.00 -4.61
-15.00 -0.57 2.16 -0.09 0.00 0.00 374 7.29 0.00 -2.38 -2.00 -0.29
0.00 -0.05 0.00 0.00 375 7.29 0.00 -2.38 -2.00 -0.29 0.00 -0.05
0.00 0.00 376 7.29 0.00 -2.38 -2.00 -0.29 0.00 -0.05 0.00 0.00 377
-5.10 10.37 -1.24 -2.00 -0.15 0.00 -0.02 0.00 0.00 378 -5.10 10.37
-1.24 -2.00 -0.15 0.00 -0.02 0.00 0.00 379 -5.08 10.43 -1.22 -2.00
-0.15 0.00 -0.02 0.00 0.00 380 -5.08 10.43 -1.22 -2.00 -0.15 0.00
-0.02 0.00 0.00 381 -5.08 10.43 -1.22 -2.00 -0.15 0.00 -0.02 0.00
0.00 382 -5.08 10.43 -1.22 -2.00 -0.15 0.00 -0.02 0.00 0.00 383
12.44 0.00 -2.76 -2.00 -0.34 0.00 -0.06 0.00 0.00 384 7.30 0.00
-2.40 -2.00 -0.30 0.00 -0.05 0.00 0.00 385 11.65 0.00 -2.41 -2.00
-0.30 0.00 -0.05 0.00 0.00 386 10.51 2.86 -2.34 0.00 -0.29 0.00
-0.05 0.00 0.00 387 10.51 2.86 -2.34 0.00 -0.29 0.00 -0.05 0.00
0.00 388 11.74 0.00 -2.93 0.00 -0.36 0.00 -0.06 0.00 0.00 389 27.12
0.00 -5.49 -20.00 -0.68 0.00 -0.11 0.00 0.00 390 27.12 0.00 -5.49
-20.00 -0.68 0.00 -0.11 0.00 0.00 391 27.15 0.00 -5.51 -20.00 -0.68
0.00 -0.11 0.00 0.00 392 11.83 0.00 -2.99 0.00 -0.37 0.00 -0.06
0.00 0.00 393 11.83 0.00 -2.99 0.00 -0.37 0.00 -0.06 0.00 0.00 394
27.21 0.00 -5.54 -20.00 -0.69 0.00 -0.11 0.00 0.00 395 0.94 0.00
-3.26 -10.00 -0.40 0.00 -0.07 0.00 0.00 396 8.29 0.00 -1.32 0.00
-0.16 0.00 -0.03 0.00 0.00 397 8.31 0.00 -1.34 0.00 -0.17 0.00
-0.03 0.00 0.00 398 8.31 0.00 -1.34 0.00 -0.17 0.00 -0.03 0.00 0.00
399 8.31 0.00 -1.34 0.00 -0.17 0.00 -0.03 0.00 0.00 400 7.07 0.00
-0.99 0.00 -0.12 0.00 -0.02 0.00 0.00 401 7.11 0.00 -1.00 0.00
-0.12 0.00 -0.02 0.00 0.00 402 7.11 0.00 -1.00 0.00 -0.12 0.00
-0.02 0.00 0.00 403 7.11 0.00 -1.00 0.00 -0.12 0.00 -0.02 0.00 0.00
404 8.61 0.00 -1.49 0.00 -0.18 0.00 -0.03 0.00 0.00 405 8.61 0.00
-1.49 0.00 -0.18 0.00 -0.03 0.00 0.00 406 8.61 0.00 -1.49 0.00
-0.18 0.00 -0.03 0.00 0.00 407 10.57 0.00 -2.42 0.00 -0.30 0.00
-0.05 0.00 0.00 408 8.62 0.00 -1.49 0.00 -0.18 0.00 -0.03 0.00 0.00
409 8.62 0.00 -1.49 0.00 -0.18 0.00 -0.03 0.00 0.00 410 8.62 0.00
-1.49 0.00 -0.18 0.00 -0.03 0.00 0.00 411 8.73 0.00 -1.55 0.00
-0.19 0.00 -0.03 0.00 0.00 412 8.73 0.00 -1.55 0.00 -0.19 0.00
-0.03 0.00 0.00 413 8.73 0.00 -1.55 0.00 -0.19 0.00 -0.03 0.00 0.00
414 8.74 0.00 -1.56 0.00 -0.19 0.00 -0.03 0.00 0.00 415 8.74 0.00
-1.56 0.00 -0.19 0.00 -0.03 0.00 0.00 416 8.74 0.00 -1.56 0.00
-0.19 0.00 -0.03 0.00 0.00 417 9.67 0.00 -2.81 -2.00 -0.35 0.00
-0.06 0.00 0.00 418 9.50 0.00 -1.95 0.00 -0.24 0.00 -0.04 0.00 0.00
419 9.50 0.00 -1.95 0.00 -0.24 0.00 -0.04 0.00 0.00
TABLE-US-00007 TABLE 7 Knockout strategies derived by OptKnock
assuming PEP carboxykinase to be reversible. Metabolic
Transformations Targeted For Removal .dagger., .dagger-dbl., # BDO
BIO AC ALA CO2 FOR FUM GLC GLY H+ 1 ADHEr, 6.44 0.72 22.08 0.00
1.90 17.19 0.00 -20.00 0.00 44.88 NADH6 2 ADHEr, 6.29 0.03 33.29
0.00 -6.29 33.43 0.00 -20.00 0.00 66.92 ENO 3 ADHEr, 6.29 0.03
33.29 0.00 -6.29 33.43 0.00 -20.00 0.00 66.92 PGM 4 ADHEr, 5.67
0.57 25.42 0.00 -5.74 28.42 0.00 -20.00 0.00 57.91 PPCK 5 ADHEr,
5.54 0.65 24.41 0.00 -5.63 27.79 0.00 -20.00 0.00 56.80 SUCD4 6
ADHEr, 4.41 0.82 22.86 0.00 -4.52 27.15 0.00 -20.00 0.00 55.83
ATPS4r 7 ADHEr, 1.81 0.52 0.00 0.00 -11.12 2.74 0.00 -20.00 0.00
56.18 PGI 8 ADHEr, 1.32 0.74 14.95 0.00 -13.58 18.85 0.00 -20.00
0.00 63.42 FUM 9 ADHEr, 0.82 0.77 13.74 0.00 -14.47 17.74 0.00
-20.00 0.00 64.01 HEX1 10 ADHEr, 0.72 0.67 14.49 0.00 -15.13 18.02
0.00 -20.00 0.00 65.94 MDH 11 ADHEr, 0.33 0.49 15.60 0.00 -16.86
18.16 0.00 -20.00 0.00 70.17 TPI 12 ADHEr, 0.33 0.49 15.60 0.00
-16.86 18.16 0.00 -20.00 0.00 70.17 FBA 13 ADHEr, 0.33 0.49 15.60
0.00 -16.86 18.16 0.00 -20.00 0.00 70.17 PFK 14 ADHEr, 11.52 0.39
15.75 0.00 8.35 17.80 0.00 -20.00 0.00 36.34 HEX1, PGI 15 ADHEr,
9.95 0.50 22.31 0.00 14.90 0.00 0.00 -20.00 0.00 25.85 PFLi, PPCK
16 ADHEr, 9.81 0.55 21.71 0.00 14.68 0.00 0.00 -20.00 0.00 25.59
PFLi, SUCD4 17 ADHEr, 9.81 0.86 10.27 0.00 24.66 0.00 0.00 -20.00
0.00 16.36 ACKr, NADH6 18 ADHEr, 9.71 0.58 21.27 0.00 14.52 0.00
0.00 -20.00 0.00 25.40 NADH6, PFLi 19 ADHEr, 7.82 0.08 30.86 0.00
9.99 13.48 0.00 -20.00 0.00 44.95 NADH6, PGM 20 ADHEr, 7.82 0.08
30.86 0.00 9.99 13.48 0.00 -20.00 0.00 44.95 ENO, NADH6 21 ADHEr,
7.51 0.53 20.76 0.00 -4.06 30.56 0.00 -20.00 0.00 55.08 ASPT, MDH
22 ADHEr, 7.36 0.35 27.02 0.00 0.27 21.51 0.00 -20.00 0.00 51.05
NADH6, PGI 23 ADHEr, 7.25 0.36 27.17 0.00 -0.05 21.80 0.00 -20.00
0.00 51.52 NADH6, TPI 24 ADHEr, 7.25 0.36 27.17 0.00 -0.05 21.80
0.00 -20.00 0.00 51.52 FBA, NADH6 25 ADHEr, 7.25 0.36 27.17 0.00
-0.05 21.80 0.00 -20.00 0.00 51.52 NADH6, PFK 26 ADHEr, 6.92 0.52
25.03 0.00 -0.07 20.82 0.00 -20.00 0.00 49.54 NADH6, PPCK 27 ADHEr,
6.76 0.59 24.04 0.00 -0.08 20.37 0.00 -20.00 0.00 48.62 MDH, NADH6
28 ADHEr, 6.60 0.67 23.01 0.00 -0.09 19.90 0.00 -20.00 0.00 47.66
FUM, NADH6 29 ADHEr, 6.55 0.56 23.75 0.00 -3.54 26.67 0.00 -20.00
0.00 54.38 PPCK, THD2 30 ADHEr, 6.53 0.77 21.10 0.00 5.45 13.37
0.00 -20.00 0.00 40.40 NADH6, RPE 31 ADHEr, 6.48 0.77 21.41 0.00
5.18 13.97 0.00 -20.00 0.00 40.88 NADH6, TAL 32 ADHEr, 6.21 0.30
28.99 0.00 -5.98 30.57 0.00 -20.00 0.00 61.72 PGI, PPCK 33 ADHEr,
6.18 0.33 28.63 0.00 -5.93 30.35 0.00 -20.00 0.00 61.31 PGI, SUCD4
34 ADHEr, 6.15 0.61 23.38 0.00 -3.10 26.55 0.00 -20.00 0.00 54.24
ATPS4r, PPCK 35 ADHEr, 6.13 0.31 29.10 0.00 -6.17 30.70 0.00 -20.00
0.00 61.98 PFK, PPCK 36 ADHEr, 6.13 0.31 29.10 0.00 -6.17 30.70
0.00 -20.00 0.00 61.98 FBA, PPCK 37 ADHEr, 6.13 0.31 29.10 0.00
-6.17 30.70 0.00 -20.00 0.00 61.98 PPCK, TPI 38 ADHEr, 6.11 0.32
28.91 0.00 -6.15 30.59 0.00 -20.00 0.00 61.78 FBA, HEX1 39 ADHEr,
6.11 0.32 28.91 0.00 -6.15 30.59 0.00 -20.00 0.00 61.78 HEX1, PFK
40 ADHEr, 6.11 0.32 28.91 0.00 -6.15 30.59 0.00 -20.00 0.00 61.78
HEX1, TPI 41 ADHEr, 6.10 0.62 14.90 0.00 -3.07 18.16 0.00 -20.00
0.00 49.89 MDH, THD2 42 ADHEr, 6.09 0.33 28.75 0.00 -6.13 30.48
0.00 -20.00 0.00 61.59 SUCD4, TPI 43 ADHEr, 6.09 0.33 28.75 0.00
-6.13 30.48 0.00 -20.00 0.00 61.59 FBA, SUCD4 44 ADHEr, 6.09 0.33
28.75 0.00 -6.13 30.48 0.00 -20.00 0.00 61.59 PFK, SUCD4 45 ADHEr,
5.96 0.70 15.28 0.00 4.93 0.00 0.00 -20.00 0.00 36.10 FUM, PFLi 46
ADHEr, 5.85 0.47 26.89 0.00 -5.92 29.33 0.00 -20.00 0.00 59.54
PPCK, SUCD4 47 ADHEr, 5.78 0.57 25.20 0.00 -5.45 28.19 0.00 -20.00
0.00 57.44 PPCK, RPE 48 ADHEr, 5.78 0.51 26.33 0.00 -5.85 28.99
0.00 -20.00 0.00 58.92 GLCpts, PPCK 49 ADHEr, 5.78 0.51 26.30 0.00
-5.85 28.97 0.00 -20.00 0.00 58.89 GLUDy, MDH 50 ADHEr, 5.77 0.52
26.20 0.00 -5.84 28.90 0.00 -20.00 0.00 58.78 GLUDy, PPCK 51 ADHEr,
5.74 0.53 25.99 0.00 -5.81 28.78 0.00 -20.00 0.00 58.55 MDH, SUCD4
52 ADHEr, 5.73 0.57 25.30 0.00 -5.59 28.30 0.00 -20.00 0.00 57.67
PPCK, TAL 53 ADHEr, 5.73 0.54 25.90 0.00 -5.80 28.72 0.00 -20.00
0.00 58.44 FUM, PPCK 54 ADHEr, 5.73 0.54 25.90 0.00 -5.80 28.72
0.00 -20.00 0.00 58.44 MDH, PPCK 55 ADHEr, 5.68 0.64 24.22 0.00
-5.31 27.57 0.00 -20.00 0.00 56.34 RPE, SUCD4 56 ADHEr, 5.68 0.57
25.49 0.00 -5.75 28.46 0.00 -20.00 0.00 57.99 ME2, SUCD4 57 ADHEr,
5.66 0.58 25.35 0.00 -5.74 28.38 0.00 -20.00 0.00 57.84 FUM, GLUDy
58 ADHEr, 5.66 0.58 25.34 0.00 -5.74 28.37 0.00 -20.00 0.00 57.83
GLUDy, SUCD4 59 ADHEr, 5.65 0.58 25.28 0.00 -5.73 28.34 0.00 -20.00
0.00 57.77 GLCpts, SUCD4 60 ADHEr, 5.61 0.64 24.31 0.00 -5.46 27.67
0.00 -20.00 0.00 56.56 SUCD4, TAL 61 ADHEr, 5.57 0.63 24.65 0.00
-5.66 27.94 0.00 -20.00 0.00 57.07 FUM, SUCD4 62 ADHEr, 5.56 0.64
24.55 0.00 -5.64 27.88 0.00 -20.00 0.00 56.95 HEX1, SUCD4 63 ADHEr,
5.55 0.64 24.49 0.00 -5.64 27.84 0.00 -20.00 0.00 56.89 CBMK2,
SUCD4 64 ADHEr, 5.44 0.70 23.61 0.00 -5.53 27.29 0.00 -20.00 0.00
55.91 FUM, HEX1 65 ADHEr, 5.24 0.72 14.09 0.00 3.10 0.00 0.00
-20.00 0.00 38.10 HEX1, PFLi 66 ADHEr, 5.06 0.52 26.72 0.00 -4.66
29.44 0.00 -20.00 0.00 59.85 ATPS4r, PGI 67 ADHEr, 4.91 0.53 26.89
0.00 -4.99 29.65 0.00 -20.00 0.00 60.29 ATPS4r, FBA 68 ADHEr, 4.91
0.53 26.89 0.00 -4.99 29.65 0.00 -20.00 0.00 60.29 ATPS4r, PFK 69
ADHEr, 4.91 0.53 26.89 0.00 -4.99 29.65 0.00 -20.00 0.00 60.29
ATPS4r, TPI 70 ADHEr, 4.88 0.44 16.04 0.00 1.18 0.00 0.00 -20.00
0.00 43.62 PFLi, TPI 71 ADHEr, 4.88 0.44 16.04 0.00 1.18 0.00 0.00
-20.00 0.00 43.62 PFK, PFLi 72 ADHEr, 4.88 0.44 16.04 0.00 1.18
0.00 0.00 -20.00 0.00 43.62 FBA, PFLi 73 ADHEr, 4.87 0.74 22.12
0.00 -6.49 25.99 0.00 -20.00 0.00 56.39 HEX1, THD2 74 ADHEr, 4.59
0.81 22.69 0.00 -4.12 26.91 0.00 -20.00 0.00 55.33 ATPS4r, RPE 75
ADHEr, 4.56 0.73 24.10 0.00 -4.66 27.92 0.00 -20.00 0.00 57.20
ATPS4r, GLUDy 76 ADHEr, 4.50 0.81 22.77 0.00 -4.31 27.02 0.00
-20.00 0.00 55.57 ATPS4r, TAL 77 ADHEr, 4.42 0.81 22.97 0.00 -4.53
27.22 0.00 -20.00 0.00 55.95 ATPS4r, CBMK2 78 ADHEr, 4.21 0.51 0.00
0.00 -5.69 2.64 0.00 -20.00 0.00 48.84 EDA, PGI 79 ADHEr, 2.48 0.52
0.00 0.00 -8.45 0.00 0.00 -20.00 0.00 52.22 PFLi, PGI 80 ADHEr,
1.36 0.47 17.83 0.00 -14.86 20.31 0.00 -20.00 0.00 68.38 MDH, PFK
81 ADHEr, 1.36 0.47 17.83 0.00 -14.86 20.31 0.00 -20.00 0.00 68.38
MDH, TPI 82 ADHEr, 1.36 0.47 17.83 0.00 -14.86 20.31 0.00 -20.00
0.00 68.38 FBA, MDH 83 ADHEr, 1.13 0.76 13.77 0.00 -13.77 17.76
0.00 -20.00 0.00 63.09 HEX1, RPE 84 ADHEr, 1.00 0.67 14.51 0.00
-14.51 18.02 0.00 -20.00 0.00 65.11 MDH, RPE 85 ADHEr, 0.98 0.76
13.76 0.00 -14.10 17.75 0.00 -20.00 0.00 63.53 HEX1, TAL 86 ADHEr,
0.86 0.67 14.50 0.00 -14.81 18.02 0.00 -20.00 0.00 65.51 MDH, TAL
87 ADHEr, 0.47 0.48 15.53 0.00 -16.54 18.07 0.00 -20.00 0.00 69.75
RPE, TPI 88 ADHEr, 0.47 0.48 15.53 0.00 -16.54 18.07 0.00 -20.00
0.00 69.75 PFK, RPE 89 ADHEr, 0.47 0.48 15.53 0.00 -16.54 18.07
0.00 -20.00 0.00 69.75 FBA, RPE 90 ADHEr, 0.40 0.49 15.57 0.00
-16.69 18.11 0.00 -20.00 0.00 69.95 PFK, TAL 91 ADHEr, 0.40 0.49
15.57 0.00 -16.69 18.11 0.00 -20.00 0.00 69.95 FBA, TAL
92 ADHEr, 14.20 0.34 13.81 0.00 21.29 0.00 0.00 -20.00 0.00 16.25
HEX1, PFLi, PGI 93 ADHEr, 14.07 0.28 14.98 0.00 19.90 2.36 0.00
-20.00 0.00 19.31 HEX1, NADH6, PGI 94 ADHEr, 14.00 0.31 14.58 0.00
19.89 2.18 0.00 -20.00 0.00 18.94 EDA, NADH6, PGI 95 ADHEr, 13.92
0.34 14.10 0.00 21.85 0.00 0.00 -20.00 0.00 16.55 ACKr, NADH6, PGI
96 ADHEr, 13.31 0.25 5.13 0.00 13.27 19.78 0.00 -20.00 0.00 28.86
FRD2, LDH_D, MDH 97 ADHEr, 13.14 0.27 15.98 0.00 17.23 6.93 0.00
-20.00 0.00 24.84 ATPS4r, PGI, SUCD4 98 ADHEr, 12.33 0.49 15.46
0.00 19.46 0.00 0.00 -20.00 0.00 18.94 ATPS4r, FDH2, NADH6 99
ADHEr, 12.03 0.46 12.29 0.00 28.02 0.00 0.00 -20.00 0.00 15.58
ACKr, NADH6, TPI 100 ADHEr, 12.03 0.46 12.29 0.00 28.02 0.00 0.00
-20.00 0.00 15.58 ACKr, FBA, NADH6 101 ADHEr, 12.03 0.46 12.29 0.00
28.02 0.00 0.00 -20.00 0.00 15.58 ACKr, NADH6, PFK 102 ADHEr, 11.95
0.26 4.09 0.00 7.62 17.42 4.30 -20.00 0.00 33.78 FRD2, LDH_D, ME2
103 ADHEr, 11.82 0.22 18.07 0.00 8.10 19.23 0.00 -20.00 0.00 38.88
HEX1, PGI, PPCK 104 ADHEr, 11.82 0.22 18.05 0.00 8.10 19.22 0.00
-20.00 0.00 38.86 EDA, PGI, PPCK 105 ADHEr, 11.77 0.25 17.66 0.00
8.15 18.98 0.00 -20.00 0.00 38.43 HEX1, PGI, SUCD4 106 ADHEr, 11.76
0.26 17.62 0.00 8.15 18.95 0.00 -20.00 0.00 38.39 EDA, PGI, SUCD4
107 ADHEr, 11.62 0.34 16.52 0.00 8.27 18.28 0.00 -20.00 0.00 37.18
ATPS4r, EDA, PGI 108 ADHEr, 11.59 0.35 16.29 0.00 8.29 18.13 0.00
-20.00 0.00 36.93 GLUDy, HEX1, PGI 109 ADHEr, 11.18 0.48 13.34 0.00
4.81 23.87 0.00 -20.00 0.00 40.62 MDH, PGL, THD2 110 ADHEr, 11.18
0.48 13.34 0.00 4.81 23.87 0.00 -20.00 0.00 40.62 G6PDHy, MDH, THD2
111 ADHEr, 10.87 0.20 25.96 0.00 16.29 0.00 0.00 -20.00 0.00 27.38
PFLi, PGI, PPCK 112 ADHEr, 10.83 0.20 26.02 0.00 16.23 0.00 0.00
-20.00 0.00 27.46 PFLi, PPCK, TPI 113 ADHEr, 10.83 0.20 26.02 0.00
16.23 0.00 0.00 -20.00 0.00 27.46 FBA, PFLi, PPCK 114 ADHEr, 10.83
0.20 26.02 0.00 16.23 0.00 0.00 -20.00 0.00 27.46 PFK, PFLi, PPCK
115 ADHEr, 10.80 0.74 11.20 0.00 23.65 0.00 0.00 -20.00 0.00 16.47
ACKr, MDH, NADH6 116 ADHEr, 10.79 0.23 25.57 0.00 16.16 0.00 0.00
-20.00 0.00 27.21 NADH6, PFLi, PGI 117 ADHEr, 10.79 0.23 25.57 0.00
16.16 0.00 0.00 -20.00 0.00 27.21 PFLi, PGI, SUCD4 118 ADHEr, 10.74
0.23 25.64 0.00 16.09 0.00 0.00 -20.00 0.00 27.30 FBA, PFLi, SUCD4
119 ADHEr, 10.74 0.23 25.64 0.00 16.09 0.00 0.00 -20.00 0.00 27.30
PFK, PFLi, SUCD4 120 ADHEr, 10.74 0.23 25.64 0.00 16.09 0.00 0.00
-20.00 0.00 27.30 NADH6, PFK, PFLi 121 ADHEr, 10.74 0.23 25.64 0.00
16.09 0.00 0.00 -20.00 0.00 27.30 FBA, NADH6, PFLi 122 ADHEr, 10.74
0.23 25.64 0.00 16.09 0.00 0.00 -20.00 0.00 27.30 PFLi, SUCD4, TPI
123 ADHEr, 10.74 0.23 25.64 0.00 16.09 0.00 0.00 -20.00 0.00 27.30
NADH6, PFLi, TPI 124 ADHEr, 10.73 0.24 25.61 0.00 16.08 0.00 0.00
-20.00 0.00 27.28 HEX1, PFK, PFLi 125 ADHEr, 10.73 0.24 25.61 0.00
16.08 0.00 0.00 -20.00 0.00 27.28 FBA, HEX1, PFLi 126 ADHEr, 10.73
0.24 25.61 0.00 16.08 0.00 0.00 -20.00 0.00 27.28 HEX1, PFLi, TPI
127 ADHEr, 10.49 0.49 21.02 0.00 15.71 0.00 0.00 -20.00 0.00 24.49
PFLi, PPCK, THD2 128 ADHEr, 10.40 0.75 10.81 0.00 25.55 0.00 0.00
-20.00 0.00 16.16 ACKr, GLUDy, NADH6 129 ADHEr, 10.28 0.77 10.70
0.00 25.38 0.00 0.00 -20.00 0.00 16.20 ACKr, GLCpts, NADH6 130
ADHEr, 10.24 0.58 10.55 0.00 11.67 22.30 0.00 -20.00 0.00 36.95
ACKr, AKGD, ATPS4r 131 ADHEr, 10.17 0.53 19.34 0.00 15.23 0.00 0.00
-20.00 0.00 24.21 ATPS4r, NADH6, PFLi 132 ADHEr, 10.15 0.43 23.16
0.00 15.20 0.00 0.00 -20.00 0.00 26.22 GLCpts, PFLi, PPCK 133
ADHEr, 10.12 0.58 10.44 0.00 10.60 23.88 0.00 -20.00 0.00 38.45
ACKr, ATPS4r, SUCOAS 134 ADHEr, 10.11 0.77 10.53 0.00 22.07 0.00
0.00 -20.00 0.00 18.19 ACKr, ME2, NADH6 135 ADHEr, 10.10 0.45 22.94
0.00 15.12 0.00 0.00 -20.00 0.00 26.12 GLUDy, PFLi, PPCK 136 ADHEr,
10.05 0.47 22.71 0.00 15.04 0.00 0.00 -20.00 0.00 26.02 ME2, PFLi,
SUCD4 137 ADHEr, 10.04 0.47 22.67 0.00 15.02 0.00 0.00 -20.00 0.00
26.01 MDH, NADH6, PFLi 138 ADHEr, 10.02 0.50 22.14 0.00 15.00 0.00
0.00 -20.00 0.00 25.67 PFLi, PPCK, RPE 139 ADHEr, 9.99 0.50 22.22
0.00 14.95 0.00 0.00 -20.00 0.00 25.76 PFLi, PPCK, TAL 140 ADHEr,
9.98 0.49 22.45 0.00 14.95 0.00 0.00 -20.00 0.00 25.91 GLUDy, PFLi,
SUCD4 141 ADHEr, 9.96 0.49 22.36 0.00 14.92 0.00 0.00 -20.00 0.00
25.87 CBMK2, PFLi, PPCK 142 ADHEr, 9.92 0.57 20.45 0.00 15.84 0.00
0.00 -20.00 0.00 24.48 ATPS4r, LDH_D, SUCD4 143 ADHEr, 9.90 0.54
21.57 0.00 14.81 0.00 0.00 -20.00 0.00 25.41 PFLi, RPE, SUCD4 144
ADHEr, 9.86 0.85 10.32 0.00 24.74 0.00 0.00 -20.00 0.00 16.34 ACKr,
CBMK2, NADH6 145 ADHEr, 9.86 0.54 21.64 0.00 14.75 0.00 0.00 -20.00
0.00 25.50 PFLi, SUCD4, TAL 146 ADHEr, 9.86 0.85 10.32 0.00 24.73
0.00 0.00 -20.00 0.00 16.34 ACKr, NADH6, RPE 147 ADHEr, 9.84 0.85
10.30 0.00 24.70 0.00 0.00 -20.00 0.00 16.35 ACKr, FUM, NADH6 148
ADHEr, 9.83 0.85 10.30 0.00 24.70 0.00 0.00 -20.00 0.00 16.35 ACKr,
NADH6, TAL 149 ADHEr, 9.83 0.85 10.29 0.00 24.69 0.00 0.00 -20.00
0.00 16.35 ACKr, ASNS2, NADH6 150 ADHEr, 9.83 0.54 21.78 0.00 14.70
0.00 0.00 -20.00 0.00 25.62 CBMK2, PFLi, SUCD4 151 ADHEr, 9.83 0.85
10.29 0.00 24.68 0.00 0.00 -20.00 0.00 16.36 ACKr, NADH12, NADH6
152 ADHEr, 9.82 0.85 10.28 0.00 24.67 0.00 0.00 -20.00 0.00 16.36
ACKr, NADH6, SO4t2 153 ADHEr, 9.81 0.55 21.71 0.00 14.68 0.00 0.00
-20.00 0.00 25.59 NADH12, NADH6, PFLi 154 ADHEr, 9.80 0.55 21.69
0.00 14.67 0.00 0.00 -20.00 0.00 25.58 FUM, NADH6, PFLi 155 ADHEr,
9.80 0.22 9.92 0.00 12.33 11.06 0.00 -20.00 0.00 30.56 ACKr, PGI,
SUCD4 156 ADHEr, 9.75 0.58 21.19 0.00 14.60 0.00 0.00 -20.00 0.00
25.30 NADH6, PFLi, TAL 157 ADHEr, 9.72 0.58 21.33 0.00 14.55 0.00
0.00 -20.00 0.00 25.43 CBMK2, NADH6, PFLi 158 ADHEr, 9.55 0.63
20.62 0.00 14.29 0.00 0.00 -20.00 0.00 25.11 FUM, HEX1, PFLi 159
ADHEr, 9.34 0.55 19.65 0.00 7.38 13.18 0.00 -20.00 0.00 36.73 MDH,
NADH6, THD2 160 ADHEr, 9.28 0.55 19.75 0.00 7.20 13.35 0.00 -20.00
0.00 37.01 ATPS4r,
MDH, NADH6 161 ADHEr, 9.03 0.63 21.22 0.00 11.62 3.77 0.00 -20.00
0.00 29.45 ATPS4r, FUM, NADH6 162 ADHEr, 8.97 0.26 23.60 0.00 0.95
24.98 0.00 -20.00 0.00 50.46 ATPS4r, PGI, PPCK 163 ADHEr, 8.65 0.48
19.82 0.00 -0.06 26.01 0.00 -20.00 0.00 49.23 ASPT, MDH, NADH6 164
ADHEr, 8.62 0.48 23.04 0.00 6.28 13.23 0.00 -20.00 0.00 39.68
ATPS4r, NADH6, PPCK 165 ADHEr, 8.60 0.50 18.60 0.00 -1.82 29.38
0.00 -20.00 0.00 51.55 ASPT, MDH, THD2 166 ADHEr, 8.49 0.65 21.41
0.00 14.16 2.07 0.00 -20.00 0.00 28.11 ATPS4r, GLCpts, SUCD4 167
ADHEr, 8.36 0.23 23.87 0.00 -4.10 33.24 0.00 -20.00 0.00 58.77
ASPT, MDH, PGI 168 ADHEr, 8.27 0.24 24.01 0.00 -4.27 33.31 0.00
-20.00 0.00 59.01 ASPT, FBA, MDH 169 ADHEr, 8.27 0.24 24.01 0.00
-4.27 33.31 0.00 -20.00 0.00 59.01 ASPT, MDH, PFK 170 ADHEr, 8.27
0.24 24.01 0.00 -4.27 33.31 0.00 -20.00 0.00 59.01 ASPT, MDH, TPI
171 ADHEr, 8.26 0.26 27.70 0.00 3.99 16.78 0.00 -20.00 0.00 46.33
ATPS4r, PFK, PPCK 172 ADHEr, 8.26 0.26 27.70 0.00 3.99 16.78 0.00
-20.00 0.00 46.33 ATPS4r, FBA, PPCK 173 ADHEr, 8.26 0.26 27.70 0.00
3.99 16.78 0.00 -20.00 0.00 46.33 ATPS4r, PPCK, TPI 174 ADHEr, 8.12
0.45 8.36 0.00 1.85 10.72 0.00 -20.00 0.00 42.04 ACKr, EDA, PGI 175
ADHEr, 7.99 0.67 21.56 0.00 6.75 10.40 0.00 -20.00 0.00 36.71
ATPS4r, HEX1, NADH6 176 ADHEr, 7.88 0.50 23.49 0.00 2.67 18.21 0.00
-20.00 0.00 45.24 NADH6, PPCK, THD2 177 ADHEr, 7.87 0.46 22.87 0.00
-0.07 23.70 0.00 -20.00 0.00 49.84 ATPS4r, GLUDy, MDH 178 ADHEr,
7.86 0.49 22.44 0.00 0.04 23.43 0.00 -20.00 0.00 49.33 ATPS4r, MDH,
PPCK 179 ADHEr, 7.86 0.49 22.44 0.00 0.04 23.43 0.00 -20.00 0.00
49.33 ATPS4r, FUM, PPCK 180 ADHEr, 7.84 0.08 30.83 0.00 10.05 13.42
0.00 -20.00 0.00 44.85 ENO, NADH6, RPE 181 ADHEr, 7.84 0.08 30.83
0.00 10.05 13.42 0.00 -20.00 0.00 44.85 NADH6, PGM, RPE 182 ADHEr,
7.83 0.08 30.84 0.00 10.02 13.45 0.00 -20.00 0.00 44.90 NADH6, PGM,
TAL 183 ADHEr, 7.83 0.08 30.84 0.00 10.02 13.45 0.00 -20.00 0.00
44.90 ENO, NADH6, TAL 184 ADHEr, 7.70 0.46 21.57 0.00 -4.11 31.25
0.00 -20.00 0.00 56.07 ASPT, GLCpts, MDH 185 ADHEr, 7.65 0.52 20.47
0.00 -3.75 30.40 0.00 -20.00 0.00 54.60 ASPT, MDH, RPE 186 ADHEr,
7.65 0.47 21.39 0.00 -4.10 31.10 0.00 -20.00 0.00 55.85 ASPT,
GLUDy, MDH 187 ADHEr, ME2, 7.62 0.87 17.24 0.00 18.87 0.00 0.00
-20.00 0.00 23.43 NADH6, THD2 188 ADHEr, ME2, 7.61 0.52 22.02 0.00
-0.99 24.76 0.00 -20.00 0.00 50.50 SUCD4, THD2 189 ADHEr, 7.58 0.53
20.61 0.00 -3.90 30.48 0.00 -20.00 0.00 54.83 ASPT, MDH, TAL 190
ADHEr, 7.58 0.23 28.71 0.00 0.18 22.36 0.00 -20.00 0.00 52.73
NADH6, PGI, PPCK 191 ADHEr, 7.55 0.49 22.66 0.00 -1.33 25.25 0.00
-20.00 0.00 51.43 FUM, PPCK, THD2 192 ADHEr, 7.55 0.49 22.66 0.00
-1.33 25.25 0.00 -20.00 0.00 51.43 MDH, PPCK, THD2 193 ADHEr, 7.56
0.47 23.10 0.00 -1.46 25.55 0.00 -20.00 0.00 51.99 GLUDy, MDH, THD2
194 ADHEr, 7.52 0.52 20.82 0.00 -4.06 30.61 0.00 -20.00 0.00 55.15
ASPT, CBMK2, MDH 195 ADHEr, 7.51 0.42 25.99 0.00 6.86 13.76 0.00
-20.00 0.00 42.72 ATPS4r, FBA, SUCD4 196 ADHEr, 7.51 0.42 25.99
0.00 6.86 13.76 0.00 -20.00 0.00 42.72 ATPS4r, PFK, SUCD4 197
ADHEr, 7.51 0.42 25.99 0.00 6.86 13.76 0.00 -20.00 0.00 42.72
ATPS4r, SUCD4, TPI 198 ADHEr, FBA, 7.50 0.24 28.80 0.00 -0.03 22.54
0.00 -20.00 0.00 53.03 NADH6, PPCK 199 ADHEr, 7.50 0.24 28.80 0.00
-0.03 22.54 0.00 -20.00 0.00 53.03 NADH6, PFK, PPCK 200 ADHEr, 7.50
0.24 28.80 0.00 -0.03 22.54 0.00 -20.00 0.00 53.03 NADH6, PPCK, TPI
201 ADHEr, 7.47 0.69 14.32 0.00 8.09 0.00 0.00 -20.00 0.00 31.52
HEX1, PFLi, THD2 202 ADHEr, 7.46 0.26 28.53 0.00 -0.04 22.42 0.00
-20.00 0.00 52.78 HEX1, NADH6, PFK 203 ADHEr, FBA, 7.46 0.26 28.53
0.00 -0.04 22.42 0.00 -20.00 0.00 52.78 HEX1, NADH6 204 ADHEr, 7.46
0.26 28.53 0.00 -0.04 22.42 0.00 -20.00 0.00 52.78 HE.chi.1, NADH6,
TPI 205 ADHEr, 7.44 0.58 13.04 0.00 6.96 0.00 0.00 -20.00 0.00
33.76 ATPS4r, G6PDHy, MDH 206 ADHEr, 7.44 0.58 13.04 0.00 6.96 0.00
0.00 -20.00 0.00 33.76 ATPS4r, MDH, PGL 207 ADHEr, 7.43 0.32 27.55
0.00 0.24 21.78 0.00 -20.00 0.00 51.58 GLUDy, NADH6, PGI 208 ADHEr,
7.42 0.97 7.95 0.00 15.46 13.33 0.00 -20.00 0.00 29.24 ACKr, FRD2,
LDH_D 209 ADHEr, 7.42 0.97 7.95 2.12 14.40 13.33 0.00 -20.00 0.00
30.30 ACKr, LDH_D, SUCD4 210 ADHEr, 7.38 0.54 24.25 0.00 2.47 17.14
0.00 -20.00 0.00 45.22 ATPS4r, FUM, GLUDy 211 ADHEr, 7.34 0.36
27.05 0.00 0.21 21.57 0.00 -20.00 0.00 51.14 NADH6, PFK, RPE 212
ADHEr, FBA, 7.34 0.36 27.05 0.00 0.21 21.57 0.00 -20.00 0.00 51.14
NADH6, RPE 213 ADHEr, 7.34 0.36 27.05 0.00 0.21 21.57 0.00 -20.00
0.00 51.14 NADH6, RPE, TPI 214 ADHEr, FBA, 7.33 0.32 27.69 0.00
-0.04 22.04 0.00 -20.00 0.00 52.00 GLUDy, NADH6 215 ADHEr, 7.33
0.32 27.69 0.00 -0.04 22.04 0.00 -20.00 0.00 52.00 GLUDy, NADH6,
PFK 216 ADHEr, 7.33 0.32 27.69 0.00 -0.04 22.04 0.00 -20.00 0.00
52.00 GLUDy, NADH6, TPI 217 ADHEr, 7.31 0.66 21.12 0.00 0.90 20.04
0.00 -20.00 0.00 45.88 ATPS4r, FUM, HEX1 218 ADHEr, 7.30 0.36 27.10
0.00 0.09 21.68 0.00 -20.00 0.00 51.32 NADH6, TAL, TPI 219 ADHEr,
7.30 0.36 27.10 0.00 0.09 21.68 0.00 -20.00 0.00 51.32 NADH6, PFK,
TAL 220 ADHEr, FBA, 7.30 0.36 27.10 0.00 0.09 21.68 0.00 -20.00
0.00 51.32 NADH6, TAL 221 ADHEr, 7.27 0.62 19.64 0.00 -1.10 22.88
0.00 -20.00 0.00 49.00 ATPS4r, MDH, THD2 222 ADHEr, 7.06 0.45 25.98
0.00 -0.06 21.26 0.00 -20.00 0.00 50.42 GLUDy, MDH, NADH6 223
ADHEr, 7.06 0.45 25.94 0.00 -0.06 21.24 0.00 -20.00 0.00 50.38
GLCpts, NADH6, PPCK 224 ADHEr, 7.05 0.52 24.82 0.00 0.30 20.47 0.00
-20.00 0.00 48.96 NADH6, PPCK, RPE 225 ADHEr, 7.02 0.47 25.73 0.00
-0.06 21.14 0.00 -20.00 0.00 50.18 GLUDy, NADH6, PPCK 226 ADHEr,
7.01 0.47 25.63 0.00 -0.06 21.10 0.00 -20.00 0.00 50.09 FUM, NADH6,
PPCK 227 ADHEr, 7.01 0.47 25.63 0.00 -0.06 21.10 0.00 -20.00 0.00
50.09 MDH, NADH6, PPCK 228 ADHEr, 7.00 0.32 0.00 0.00 17.78 8.35
0.00 -20.00 0.00 21.13 ATPS4r, FRD2, LDH_D 229 ADHEr, 6.98 0.52
24.92 0.00 0.12 20.64 0.00 -20.00 0.00 49.23 NADH6, PPCK, TAL 230
ADHEr, 6.92 0.52 25.04 0.00 -0.07 20.83 0.00 -20.00 0.00 49.55 FUM,
GLUDy, NADH6 231 ADHEr, 6.91 0.52 24.98 0.00 -0.07 20.80 0.00
-20.00 0.00 49.49 GLCpts, MDH, NADH6 232 ADHEr, 6.91 0.59 23.79
0.00 0.34 19.96 0.00 -20.00 0.00 47.94 MDH, NADH6, RPE 233 ADHEr,
6.84 0.59 23.91 0.00 0.14 20.16 0.00 -20.00 0.00 48.26 MDH, NADH6,
TAL 234 ADHEr, 6.83 0.92 18.08 0.00 17.16 1.07 0.00 -20.00 0.00
25.70 HEX1, NADH6, THD2 235 ADHEr, 6.77 0.66 22.78 0.00 0.38 19.47
0.00 -20.00 0.00 46.96 FUM,
NADH6, RPE 236 ADHEr, 6.77 0.59 24.11 0.00 -0.08 20.40 0.00 -20.00
0.00 48.68 CBMK2, MDH, NADH6 237 ADHEr, 6.76 0.59 24.04 0.00 -0.08
20.37 0.00 -20.00 0.00 48.62 FUM, ME2, NADH6 238 ADHEr, 6.69 0.66
22.89 0.00 0.16 19.68 0.00 -20.00 0.00 47.29 FUM, NADH6, TAL 239
ADHEr, 6.64 0.60 16.99 0.00 6.42 0.00 0.00 -20.00 0.00 35.28
ATPS4r, MDH, PGDH 240 ADHEr, 6.64 0.65 23.23 0.00 -0.09 20.01 0.00
-20.00 0.00 47.87 FUM, HEX1, NADH6 241 ADHEr, 6.61 0.66 23.09 0.00
-0.09 19.94 0.00 -20.00 0.00 47.74 CBMK2, FUM, NADH6 242 ADHEr,
6.56 0.49 24.86 0.00 -3.91 27.44 0.00 -20.00 0.00 55.80 GLCpts,
PPCK, THD2 243 ADHEr, 6.56 0.50 24.67 0.00 -3.84 27.31 0.00 -20.00
0.00 55.57 GLUDy, PPCK, THD2 244 ADHEr, 6.49 0.77 21.51 0.00 5.18
14.02 0.00 -20.00 0.00 40.97 CBMK2, NADH6, TAL 245 ADHEr, 6.48 0.61
16.56 0.00 5.95 0.00 0.00 -20.00 0.00 35.78 ATPS4r, MDH, TAL 246
ADHEr, 6.45 0.65 22.52 0.00 1.91 16.48 0.00 -20.00 0.00 45.54
ATPS4r, GLUDy, NADH6 247 ADHEr, PGI, 6.33 0.22 30.11 0.00 -6.16
31.28 0.00 -20.00 0.00 62.99 PPCK, SUCD4 248 ADHEr, 6.33 0.61 16.17
0.00 5.52 0.00 0.00 -20.00 0.00 36.25 ATPS4r, MDH, RPE 249 ADHEr,
FBP, 6.33 0.03 33.21 0.00 -6.19 33.35 0.00 -20.00 0.00 66.75 PGM,
THD2 250 ADHEr, ENO, 6.33 0.03 33.21 0.00 -6.19 33.35 0.00 -20.00
0.00 66.75 FBP, THD2 251 ADHEr, FBA, 6.33 0.03 33.21 0.00 -6.19
33.35 0.00 -20.00 0.00 66.75 PGM, THD2 252 ADHEr, ENO, 6.33 0.03
33.21 0.00 -6.19 33.35 0.00 -20.00 0.00 66.75 FBA, THD2 253 ADHEr,
ENO, 6.33 0.03 33.21 0.00 -6.19 33.35 0.00 -20.00 0.00 66.75 THD2,
TPI 254 ADHEr, 6.33 0.03 33.21 0.00 -6.19 33.35 0.00 -20.00 0.00
66.75 PGM, THD2, TPI 255 ADHEr, 6.28 0.26 29.59 0.00 -6.08 30.96
0.00 -20.00 0.00 62.41 GLCpts, PGI, PPCK 256 ADHEr, FBA, 6.28 0.22
30.24 0.00 -6.31 31.41 0.00 -20.00 0.00 63.24 HEX1, PPCK 257 ADHEr,
6.28 0.22 30.24 0.00 -6.31 31.41 0.00 -20.00 0.00 63.24 HEX1, PFK,
PPCK 258 ADHEr, 6.28 0.22 30.24 0.00 -6.31 31.41 0.00 -20.00 0.00
63.24 HEX1, PPCK, TPI 259 ADHEr, 6.27 0.23 30.17 0.00 -6.30 31.37
0.00 -20.00 0.00 63.17 PPCK, SUCD4, TPI 260 ADHEr, PFK, 6.27 0.23
30.17 0.00 -6.30 31.37 0.00 -20.00 0.00 63.17 PPCK, SUCD4 261
ADHEr, FBA, 6.27 0.23 30.17 0.00 -6.30 31.37 0.00 -20.00 0.00 63.17
PPCK, SUCD4 262 ADHEr, 6.26 0.27 29.44 0.00 -6.06 30.86 0.00 -20.00
0.00 62.23 GLUDy, PGI, PPCK 263 ADHEr, 6.24 0.28 29.26 0.00 -6.03
30.74 0.00 -20.00 0.00 62.02 GLCpts, PGI, SUCD4 264 ADHEr, 6.24
0.29 29.24 0.00 -6.02 30.73 0.00 -20.00 0.00 62.00 FUM, GLUDy, PGI
265 ADHEr, 6.24 0.29 29.24 0.00 -6.02 30.73 0.00 -20.00 0.00 62.00
GLUDy, MDH, PGI 266 ADHEr, FBA, 6.24 0.25 29.95 0.00 -6.27 31.23
0.00 -20.00 0.00 62.93 HEX1, SUCD4 267 ADHEr, 6.24 0.25 29.95 0.00
-6.27 31.23 0.00 -20.00 0.00 62.93 HEX1, SUCD4, TPI 268 ADHEr, 6.24
0.25 29.95 0.00 -6.27 31.23 0.00 -20.00 0.00 62.93 HEX1, PFK, SUCD4
269 ADHEr, 6.23 0.29 29.14 0.00 -6.01 30.67 0.00 -20.00 0.00 61.88
GLUDy, PGI, SUCD4 270 ADHEr, 6.21 0.69 22.25 0.00 -3.64 25.83 0.00
-20.00 0.00 52.95 FUM, HEX1, THD2 271 ADHEr, FBA, 6.21 0.26 29.69
0.00 -6.24 31.07 0.00 -20.00 0.00 62.64 GLCpts, PPCK 272 ADHEr,
6.21 0.26 29.69 0.00 -6.24 31.07 0.00 -20.00 0.00 62.64 GLCpts,
PFK, PPCK 273 ADHEr, 6.21 0.26 29.69 0.00 -6.24 31.07 0.00 -20.00
0.00 62.64 GLCpts, PPCK, TPI 274 ADHEr, PFK, 6.20 0.30 29.01 0.00
-6.02 30.60 0.00 -20.00 0.00 61.77 PPCK, RPE 275 ADHEr, 6.20 0.30
29.01 0.00 -6.02 30.60 0.00 -20.00 0.00 61.77 PPCK, RPE, TPI 276
ADHEr, FBA, 6.20 0.30 29.01 0.00 -6.02 30.60 0.00 -20.00 0.00 61.77
PPCK, RPE 277 ADHEr, 6.19 0.27 29.54 0.00 -6.23 30.98 0.00 -20.00
0.00 62.47 GLUDy, PFK, PPCK 278 ADHEr, 6.19 0.27 29.54 0.00 -6.23
30.98 0.00 -20.00 0.00 62.47 GLUDy, PPCK, TPI 279 ADHEr, FBA, 6.19
0.27 29.54 0.00 -6.23 30.98 0.00 -20.00 0.00 62.47 GLUDy, PPCK 280
ADHEr, 6.19 0.71 21.49 0.00 -2.37 25.20 0.00 -20.00 0.00 51.73
ATPS4r, ME2, THD2 281 ADHEr, 6.18 0.32 28.82 0.00 -5.99 30.48 0.00
-20.00 0.00 61.55 HEX1, PFK, RPE 282 ADHEr, 6.18 0.32 28.82 0.00
-5.99 30.48 0.00 -20.00 0.00 61.55 HEX1, RPE, TPI 283 ADHEr, FBA,
6.18 0.32 28.82 0.00 -5.99 30.48 0.00 -20.00 0.00 61.55 HEX1, RPE
284 ADHEr, FBA, 6.17 0.31 29.05 0.00 -6.09 30.65 0.00 -20.00 0.00
61.87 PPCK, TAL 285 ADHEr, 6.17 0.31 29.05 0.00 -6.09 30.65 0.00
-20.00 0.00 61.87 PPCK, TAL, TPI 286 ADHEr, PFK, 6.17 0.31 29.05
0.00 -6.09 30.65 0.00 -20.00 0.00 61.87 PPCK, TAL 287 ADHEr, 6.17
0.55 24.36 0.00 -3.40 27.23 0.00 -20.00 0.00 55.50 ATPS4r, GLUDy,
PPCK 288 ADHEr, FBA, 6.17 0.29 29.36 0.00 -6.20 30.87 0.00 -20.00
0.00 62.28 GLUDy, HEX1 289 ADHEr, 6.17 0.29 29.36 0.00 -6.20 30.87
0.00 -20.00 0.00 62.28 GLUDy, HEX1, TPI 290 ADHEr, 6.17 0.29 29.36
0.00 -6.20 30.87 0.00 -20.00 0.00 62.28 GLUDy, HEX1, PFK 291 ADHEr,
6.16 0.29 29.36 0.00 -6.20 30.86 0.00 -20.00 0.00 62.27 GLCpts,
SUCD4, TPI 292 ADHEr, FBA, 6.16 0.29 29.36 0.00 -6.20 30.86 0.00
-20.00 0.00 62.27 GLCpts, SUCD4 293 ADHEr, 6.16 0.29 29.36 0.00
-6.20 30.86 0.00 -20.00 0.00 62.27 GLCpts, PFK, SUCD4 294 ADHEr,
6.16 0.29 29.34 0.00 -6.20 30.85 0.00 -20.00 0.00 62.25 GLUDy, MDH,
PFK 295 ADHEr, FBA, 6.16 0.29 29.34 0.00 -6.20 30.85 0.00 -20.00
0.00 62.25 GLUDy, MDH 296 ADHEr, FBA, 6.16 0.29 29.34 0.00 -6.20
30.85 0.00 -20.00 0.00 62.25 FUM, GLUDy 297 ADHEr, 6.16 0.29 29.34
0.00 -6.20 30.85 0.00 -20.00 0.00 62.25 FUM, GLUDy, PFK 298 ADHEr,
6.16 0.29 29.34 0.00 -6.20 30.85 0.00 -20.00 0.00 62.25 GLUDy, MDH,
TPI 299 ADHEr, 6.16 0.29 29.34 0.00 -6.20 30.85 0.00 -20.00 0.00
62.25 FUM, GLUDy, TPI 300 ADHEr, RPE, 6.16 0.33 28.65 0.00 -5.97
30.37 0.00 -20.00 0.00 61.36 SUCD4, TPI 301 ADHEr, PFK, 6.16 0.33
28.65 0.00 -5.97 30.37 0.00 -20.00 0.00 61.36 RPE, SUCD4 302 ADHEr,
FBA, 6.16 0.33 28.65 0.00 -5.97 30.37 0.00 -20.00 0.00 61.36 RPE,
SUCD4 303 ADHEr, 6.15 0.30 29.25 0.00 -6.19 30.80 0.00 -20.00 0.00
62.15 GLUDy, PFK, SUCD4 304 ADHEr, FBA, 6.15 0.30 29.25 0.00 -6.19
30.80 0.00 -20.00 0.00 62.15 GLUDy, SUCD4 305 ADHEr, 6.15 0.30
29.25 0.00 -6.19 30.80 0.00 -20.00 0.00 62.15 GLUDy, SUCD4, TPI 306
ADHEr, 6.14 0.32 28.86 0.00 -6.07 30.53 0.00 -20.00 0.00 61.66
HEX1, PFK, TAL 307 ADHEr, FBA, 6.14 0.32 28.86 0.00 -6.07 30.53
0.00 -20.00 0.00 61.66 HEX1, TAL 308 ADHEr, 6.14 0.32 28.86 0.00
-6.07 30.53 0.00 -20.00 0.00 61.66 HEX1, TAL, TPI 309 ADHEr, PFK,
6.13 0.33 28.70 0.00 -6.05 30.43 0.00 -20.00 0.00 61.47 SUCD4, TAL
310 ADHEr, FBA, 6.13 0.33 28.70 0.00 -6.05 30.43 0.00 -20.00 0.00
61.47 SUCD4, TAL 311 ADHEr, 6.13 0.33 28.70 0.00 -6.05 30.43 0.00
-20.00 0.00 61.47 SUCD4, TAL, TPI 312 ADHEr, 6.10 0.62 14.90 0.00
-3.07 18.16 0.00 -20.00 0.00 49.89 FUM, ME2, THD2 313 ADHEr, 6.04
0.65 18.68 0.00 0.48 22.08 0.00 -20.00 0.00 48.37 ATPS4r, ME2,
SUCD4 314 ADHEr, 6.00 0.38 28.04 0.00 -6.05 30.04 0.00 -20.00 0.00
60.81 PPCK, PYK, SUCD4 315 ADHEr, 5.97 0.40 27.84 0.00 -6.03 29.92
0.00 -20.00 0.00 60.59 GLCpts, PPCK, SUCD4 316 ADHEr, 5.95 0.46
26.74 0.00 -5.68 29.16 0.00 -20.00 0.00 59.19 PPCK, RPE, SUCD4 317
ADHEr, 5.94 0.42 27.58 0.00 -6.00 29.76 0.00 -20.00 0.00 60.30 FUM,
GLUDy, PPCK 318 ADHEr, 5.94 0.42 27.58 0.00 -6.00 29.76 0.00 -20.00
0.00 60.30 GLUDy, MDH, PPCK 319 ADHEr, 5.94 0.42 27.54 0.00 -5.99
29.74 0.00 -20.00 0.00 60.27 GLUDy, PPCK, SUCD4 320 ADHEr, 5.90
0.46 26.81 0.00 -5.79 29.24 0.00 -20.00 0.00 59.36 PPCK, SUCD4, TAL
321 ADHEr, 5.89 0.45 27.19 0.00 -5.95 29.52 0.00 -20.00 0.00 59.87
GLCpts, GLUDy, MDH 322 ADHEr, 5.89 0.51 26.14 0.00 -5.59 28.78 0.00
-20.00 0.00 58.51 GLCpts,
PPCK, RPE 323 ADHEr, 5.88 0.51 26.12 0.00 -5.59 28.77 0.00 -20.00
0.00 58.49 GLUDy, MDH, RPE 324 ADHEr, 5.87 0.52 26.00 0.00 -5.57
28.69 0.00 -20.00 0.00 58.35 GLUDy, PPCK, RPE 325 ADHEr, 5.87 0.46
27.02 0.00 -5.93 29.42 0.00 -20.00 0.00 59.69 GLCpts, GLUDy, PPCK
326 ADHEr, 5.86 0.46 26.97 0.00 -5.93 29.38 0.00 -20.00 0.00 59.63
GLCpts, MDH, SUCD4 327 ADHEr, 5.85 0.53 25.81 0.00 -5.54 28.57 0.00
-20.00 0.00 58.14 MDH, RPE, SUCD4 328 ADHEr, 5.85 0.47 26.83 0.00
-5.91 29.29 0.00 -20.00 0.00 59.47 GLCpts, MDH, PPCK 329 ADHEr,
5.85 0.47 26.83 0.00 -5.91 29.29 0.00 -20.00 0.00 59.47 FUM,
GLCpts, PPCK 330 ADHEr, 5.84 0.54 25.70 0.00 -5.53 28.50 0.00
-20.00 0.00 58.02 MDH, PPCK, RPE 331 ADHEr, 5.84 0.54 25.70 0.00
-5.53 28.50 0.00 -20.00 0.00 58.02 FUM, PPCK, RPE 332 ADHEr, 5.84
0.51 26.23 0.00 -5.72 28.88 0.00 -20.00 0.00 58.71 GLCpts, PPCK,
TAL 333 ADHEr, 5.83 0.51 26.21 0.00 -5.71 28.86 0.00 -20.00 0.00
58.68 GLUDy, MDH, TAL 334 ADHEr, 5.82 0.47 26.84 0.00 -5.98 29.28
0.00 -20.00 0.00 59.61 MDH, PPCK, PYK 335 ADHEr, 5.82 0.47 26.84
0.00 -5.98 29.28 0.00 -20.00 0.00 59.61 FUM, PPCK, PYK 336 ADHEr,
5.82 0.52 26.09 0.00 -5.70 28.79 0.00 -20.00 0.00 58.55 GLUDy,
PPCK, TAL 337 ADHEr, 5.81 0.49 26.51 0.00 -5.87 29.10 0.00 -20.00
0.00 59.12 FUM, GLUDy, SUCD4 338 ADHEr, 5.80 0.53 25.90 0.00 -5.67
28.67 0.00 -20.00 0.00 58.33 MDH, SUCD4, TAL 339 ADHEr, 5.80 0.50
26.45 0.00 -5.86 29.06 0.00 -20.00 0.00 59.05 GLCpts, ME2, SUCD4
340 ADHEr, ME2, 5.79 0.57 25.28 0.00 -5.46 28.24 0.00 -20.00 0.00
57.53 RPE, SUCD4 341 ADHEr, 5.79 0.54 25.79 0.00 -5.66 28.61 0.00
-20.00 0.00 58.22 FUM, PPCK, TAL 342 ADHEr, 5.79 0.54 25.79 0.00
-5.66 28.61 0.00 -20.00 0.00 58.22 MDH, PPCK, TAL 343 ADHEr, 5.78
0.51 26.35 0.00 -5.85 29.00 0.00 -20.00 0.00 58.95 GLUDy, PRO1z,
SUCD4 344 ADHEr, 5.78 0.57 25.18 0.00 -5.45 28.18 0.00 -20.00 0.00
57.43 FUM, GLUDy, RPE 345 ADHEr, 5.78 0.57 25.17 0.00 -5.45 28.17
0.00 -20.00 0.00 57.41 GLUDy, RPE, SUCD4 346 ADHEr, 5.78 0.57 25.15
0.00 -5.44 28.15 0.00 -20.00 0.00 57.39 GLCpts, RPE, SUCD4 347
ADHEr, 5.78 0.51 26.30 0.00 -5.85 28.97 0.00 -20.00 0.00 58.89 FUM,
GLUDy, ME2 348 ADHEr, 5.78 0.51 26.30 0.00 -5.85 28.96 0.00 -20.00
0.00 58.88 GLUDy, ME2, SUCD4 349 ADHEr, 5.77 0.52 26.22 0.00 -5.84
28.91 0.00 -20.00 0.00 58.80 FUM, GLCpts, GLUDy 350 ADHEr, 5.76
0.52 26.17 0.00 -5.83 28.88 0.00 -20.00 0.00 58.74 GLCpts, GLUDy,
SUCD4 351 ADHEr, 5.74 0.53 25.99 0.00 -5.81 28.78 0.00 -20.00 0.00
58.55 FUM, ME2, SUCD4 352 ADHEr, ME2, 5.74 0.57 25.38 0.00 -5.60
28.34 0.00 -20.00 0.00 57.75 SUCD4, TAL 353 ADHEr, 5.72 0.58 25.26
0.00 -5.59 28.27 0.00 -20.00 0.00 57.62 FUM, GLUDy, TAL 354 ADHEr,
5.72 0.58 25.25 0.00 -5.58 28.26 0.00 -20.00 0.00 57.61 GLUDy,
SUCD4, TAL 355 ADHEr, 5.72 0.58 25.21 0.00 -5.58 28.24 0.00 -20.00
0.00 57.57 GLCpts, SUCD4, TAL 356 ADHEr, ME2, 5.71 0.95 0.00 0.00
6.29 15.52 0.00 -20.00 0.00 40.16 PGL, THD2 357 ADHEr, 5.71 0.95
0.00 0.00 6.29 15.52 0.00 -20.00 0.00 40.16 G6PDHy, ME2, THD2 358
ADHEr, 5.71 0.62 24.48 0.00 -5.35 27.73 0.00 -20.00 0.00 56.63 FUM,
RPE, SUCD4 359 ADHEr, 5.69 0.63 24.34 0.00 -5.32 27.64 0.00 -20.00
0.00 56.47 HEX1, RPE, SUCD4 360 ADHEr, 5.68 0.57 25.51 0.00 -5.76
28.48 0.00 -20.00 0.00 58.02 CBMK2, GLU5K, PPCK 361 ADHEr, 5.68
0.57 25.51 0.00 -5.76 28.48 0.00 -20.00 0.00 58.02 CBMK2, G5SD,
PPCK 362 ADHEr, 5.68 0.57 25.51 0.00 -5.76 28.48 0.00 -20.00 0.00
58.02 ASNS2, CBMK2, PPCK 363 ADHEr, 5.68 0.57 25.50 0.00 -5.75
28.47 0.00 -20.00 0.00 58.00 CBMK2, PPCK, SO4t2 364 ADHEr, 5.67
0.57 25.42 0.00 -5.75 28.42 0.00 -20.00 0.00 57.92 GLUDy, HEX1,
SUCD4 365 ADHEr, 5.64 0.63 24.56 0.00 -5.49 27.83 0.00 -20.00 0.00
56.84 FUM, SUCD4, TAL 366 ADHEr, 5.63 0.63 24.44 0.00 -5.48 27.75
0.00 -20.00 0.00 56.70 HEX1, SUCD4, TAL 367 ADHEr, 5.62 0.60 25.02
0.00 -5.70 28.17 0.00 -20.00 0.00 57.47 FUM, HEX1, SUCD4 368 ADHEr,
5.58 0.70 23.35 0.00 -5.18 27.02 0.00 -20.00 0.00 55.35 FUM, HEX1,
RPE 369 ADHEr, 5.58 0.62 24.73 0.00 -5.67 27.99 0.00 -20.00 0.00
57.15 CBMK2, FUM, SUCD4 370 ADHEr, 5.54 0.72 14.12 0.00 3.77 0.00
0.00 -20.00 0.00 37.22 HEX1, PFLi, RPE 371 ADHEr, 5.51 0.70 23.47
0.00 -5.35 27.15 0.00 -20.00 0.00 55.62 FUM, HEX1, TAL 372 ADHEr,
5.45 0.70 23.69 0.00 -5.54 27.34 0.00 -20.00 0.00 56.00 CBMK2, FUM,
HEX1 373 ADHEr, 5.40 0.72 14.11 0.00 3.45 0.00 0.00 -20.00 0.00
37.64 HEX1, PFLi, TAL 374 ADHEr, 5.25 0.72 14.10 0.00 3.09 0.00
0.00 -20.00 0.09 38.10 GLYCL, HEX1, PFLi 375 ADHEr, 5.14 0.46 27.54
0.00 -4.79 29.95 0.00 -20.00 0.00 60.78 ATPS4r, GLUDy, PGI 376
ADHEr, PFLi, 5.08 0.43 16.39 0.00 1.66 0.00 0.00 -20.00 0.00 43.14
PGDH, PGI 377 ADHEr, PFLi, 5.05 0.43 16.21 0.00 1.57 0.00 0.00
-20.00 0.00 43.21 PGI, TAL 378 ADHEr, 5.03 0.52 26.76 0.00 -4.73
29.48 0.00 -20.00 0.00 59.94 ATPS4r, PFK, RPE 379 ADHEr, 5.03 0.52
26.76 0.00 -4.73 29.48 0.00 -20.00 0.00 59.94 ATPS4r, FBA, RPE 380
ADHEr, 5.03 0.52 26.76 0.00 -4.73 29.48 0.00 -20.00 0.00 59.94
ATPS4r, RPE, TPI 381 ADHEr, PFLi, 5.02 0.43 16.04 0.00 1.47 0.00
0.00 -20.00 0.00 43.27 PGI, RPE 382 ADHEr, 5.02 0.47 27.70 0.00
-5.08 30.15 0.00 -20.00 0.00 61.18 ATPS4r, GLUDy, TPI 383 ADHEr,
5.02 0.47 27.70 0.00 -5.08 30.15 0.00 -20.00 0.00 61.18 ATPS4r,
FBA, GLUDy 384 ADHEr, 5.02 0.47 27.70 0.00 -5.08 30.15 0.00 -20.00
0.00 61.18 ATPS4r, GLUDy, PFK 385 ADHEr, FBA, 5.01 0.44 15.95 0.00
1.43 0.00 0.00 -20.00 0.00 43.31 PFLi, PGI 386 ADHEr, PFK, 5.01
0.44 15.95 0.00 1.43 0.00 0.00 -20.00 0.00 43.31 PFLi, PGI 387
ADHEr, PFLi, 5.01 0.44 15.95 0.00 1.43 0.00 0.00 -20.00 0.00 43.31
PGI, TPI 388 ADHEr, PFLi, 4.99 0.44 15.97 0.00 1.38 0.00 0.00
-20.00 0.00 43.37 RPE, TPI 389 ADHEr, PFK, 4.99 0.44 15.97 0.00
1.38 0.00 0.00 -20.00 0.00 43.37 PFLi, RPE 390 ADHEr, FBA, 4.99
0.44 15.97 0.00 1.38 0.00 0.00 -20.00 0.00 43.37 PFLi, RPE 391
ADHEr, 4.98 0.52 26.82 0.00 -4.85 29.56 0.00 -20.00 0.00 60.10
ATPS4r, PFK, TAL 392 ADHEr, 4.98 0.52 26.82 0.00 -4.85 29.56 0.00
-20.00 0.00 60.10 ATPS4r, FBA, TAL 393 ADHEr, 4.98 0.52 26.82 0.00
-4.85 29.56 0.00 -20.00 0.00 60.10 ATPS4r, TAL, TPI 394 ADHEr, FBA,
4.94 0.44 16.00 0.00 1.28 0.00 0.00 -20.00 0.00 43.49 PFLi, TAL 395
ADHEr, PFLi, 4.94 0.44 16.00 0.00 1.28 0.00 0.00 -20.00 0.00 43.49
TAL, TPI 396 ADHEr, PFK, 4.94 0.44 16.00 0.00 1.28 0.00 0.00 -20.00
0.00 43.49 PFLi, TAL 397 ADHEr, 4.90 0.74 22.16 0.00 -6.47 26.02
0.00 -20.00 0.09 56.37 GLYCL, HEX1, THD2 398 ADHEr, 4.89 0.39 16.45
0.00 1.06 0.00 0.00 -20.00 0.00 44.29 GLUDy, PFK, PFLi 399 ADHEr,
4.89 0.39 16.45 0.00 1.06 0.00 0.00 -20.00 0.00 44.29 GLUDy, PFLi,
TPI 400 ADHEr, FBA, 4.89 0.39 16.45 0.00 1.06 0.00 0.00 -20.00 0.00
44.29 GLUDy, PFLi 401 ADHEr, EDA, 4.83 0.50 0.00 0.00 -3.17 0.00
0.00 -20.00 0.00 45.11 PFLi, PGI 402 ADHEr, 4.72 0.72 23.93 0.00
-4.31 27.69 0.00 -20.00 0.00 56.73 ATPS4r, GLUDy, RPE 403 ADHEr,
4.65 0.72 24.01 0.00 -4.47 27.80 0.00 -20.00 0.00 56.96 ATPS4r,
GLUDy, TAL 404 ADHEr, 4.60 0.80 22.80 0.00 -4.14 26.97 0.00 -20.00
0.00 55.45 ATPS4r, CBMK2, RPE 405 ADHEr, 4.57 0.72 24.19 0.00 -4.67
27.97 0.00 -20.00 0.00 57.30 ATPS4r, CBMK2, GLUDy 406 ADHEr, 4.51
0.80 22.88 0.00 -4.32 27.09 0.00 -20.00 0.00 55.69 ATPS4r, CBMK2,
TAL 407 ADHEr, 4.42 0.81 22.95 0.00 -4.53 27.20 0.00 -20.00 0.00
55.93 ASNS2,
ATPS4r, GLU5K 408 ADHEr, 4.42 0.81 22.95 0.00 -4.53 27.20 0.00
-20.00 0.00 55.93 ASNS2, ATPS4r, G5SD 409 ADHEr, 3.00 0.50 3.27
0.00 -6.43 0.00 0.00 -20.00 0.00 50.40 ACKr, PFLi, PGI 410 ADHEr,
2.76 1.03 3.32 0.00 14.07 0.00 0.00 -20.00 0.00 25.81 ACKr, AKGD,
FRD2 411 ADHEr, 1.91 1.03 2.47 0.00 12.55 0.00 0.00 -20.00 0.00
27.45 ACKr, FRD2, SUCOAS 412 ADHEr, 1.40 0.71 14.98 0.00 -12.86
18.72 0.00 -20.00 0.00 62.31 FUM, G6PDHy, TAL 413 ADHEr, 1.40 0.71
14.98 0.00 -12.86 18.72 0.00 -20.00 0.00 62.31 FUM, PGDH, TAL 414
ADHEr, 1.40 0.71 14.98 0.00 -12.86 18.72 0.00 -20.00 0.00 62.31
FUM, PGL, TAL 415 ADHEr, 1.36 0.47 17.83 0.00 -14.86 20.31 0.00
-20.00 0.00 68.38 FUM, ME2, TPI 416 ADHEr, 1.36 0.47 17.83 0.00
-14.86 20.31 0.00 -20.00 0.00 68.38 FUM, ME2, PFK 417 ADHEr, FBA,
1.36 0.47 17.83 0.00 -14.86 20.31 0.00 -20.00 0.00 68.38 FUM, ME2
418 ADHEr, 1.22 0.24 0.00 0.00 -33.89 3.68 33.86 -20.00 0.00 73.09
FRD2, GLUDy, LDH_D 419 ADHEr, 1.15 0.40 0.00 0.00 15.41 4.39 0.00
-20.00 0.00 22.97 FRD2, LDH_D, THD2 420 ADHEr, 1.01 0.73 14.02 0.00
-13.53 17.85 0.00 -20.00 0.00 62.73 G6PDHy, HEX1, TAL 421 ADHEr,
1.01 0.73 14.02 0.00 -13.53 17.85 0.00 -20.00 0.00 62.73 HEX1,
PGDH, TAL 422 ADHEr, 1.01 0.73 14.02 0.00 -13.53 17.85 0.00 -20.00
0.00 62.73 HEX1, PGL, TAL 423 ADHEr, 0.89 0.65 14.71 0.00 -14.28
18.10 0.00 -20.00 0.00 64.72 MDH, PGDH, TAL 424 ADHEr, 0.89 0.65
14.71 0.00 -14.28 18.10 0.00 -20.00 0.00 64.72 G6PDHy, MDH, TAL 425
ADHEr, 0.89 0.65 14.71 0.00 -14.28 18.10 0.00 -20.00 0.00 64.72
MDH, PGL, TAL 426 ADHEr, 0.43 0.47 15.73 0.00 -16.28 18.18 0.00
-20.00 0.00 69.35 PGDH, TAL, TPI 427 ADHEr, 0.43 0.47 15.73 0.00
-16.28 18.18 0.00 -20.00 0.00 69.35 FBA, PGDH, TAL 428 ADHEr, 0.43
0.47 15.73 0.00 -16.28 18.18 0.00 -20.00 0.00 69.35 PFK, PGDH, TAL
429 ADHEr, 0.41 0.49 15.57 0.00 -16.70 18.12 0.00 -20.00 0.06 69.95
GLYCL, TAL, TPI 430 ADHEr, 0.40 0.49 15.57 0.00 -16.69 18.11 0.00
-20.00 0.00 69.95 TAL, THD5, TPI 431 ADHEr, 0.40 0.49 15.57 0.00
-16.69 18.11 0.00 -20.00 0.00 69.95 LDH_D, TAL, TPI 432 ADHEr,
16.17 0.05 8.39 0.00 11.88 24.76 0.00 -20.00 0.00 33.47 ASPT, EDA,
MDH, PGI 433 ADHEr, 15.11 0.23 0.00 0.00 23.25 9.12 0.00 -20.00
0.00 13.86 ATPS4r, FRD2, LDH_D, ME2 434 ADHEr, 14.76 0.16 16.11
0.00 22.13 0.00 0.00 -20.00 0.00 17.24 EDA, PFLi, PGI, PPCK 435
ADHEr, 15.02 0.23 0.00 0.00 23.19 9.11 0.00 -20.00 0.00 13.93
ATPS4r, FRD2, LDH_D, MDH 436 ADHEr, 14.65 0.19 15.67 0.00 21.97
0.00 0.00 -20.00 0.00 17.05 EDA, PFLi, PGI, SUCD4 437 ADHEr, 14.65
0.19 15.67 0.00 21.97 0.00 0.00 -20.00 0.00 17.05 EDA, NADH6, PFLi,
PGI 438 ADHEr, 14.47 0.09 17.44 0.00 19.97 3.46 0.00 -20.00 0.00
21.55 EDA, NADH6, PGI, PPCK 439 ADHEr, 14.38 0.36 13.01 0.00 21.55
0.00 0.00 -20.00 0.00 15.56 ASPT, LDH_D, MDH, PFLi 440 ADHEr, 14.31
0.31 14.25 0.00 21.45 0.00 0.00 -20.00 0.00 16.44 GLUDy, HEX1,
PFLi, PGI 441 ADHEr, 14.23 0.27 14.38 0.00 22.22 0.00 0.00 -20.00
0.00 16.72 ACKr, GLCpts, NADH6, PGI 442 ADHEr, 14.13 0.25 15.36
0.00 19.91 2.53 0.00 -20.00 0.00 19.65 GLUDy, HEX1, NADH6, PGI 443
ADHEr, 14.07 0.27 15.02 0.00 19.91 2.38 0.00 -20.00 0.00 19.34 EDA,
GLUDy, NADH6, PGI 444 ADHEr, 14.04 0.19 14.14 0.00 21.04 0.00 0.00
-20.00 0.00 17.69 ACKr, PFLi, PGI, SUCD4 445 ADHEr, 14.04 0.19
14.14 0.00 22.14 0.00 0.00 -20.00 0.00 16.60 ACKr, NADH6, PFLi, PGI
446 ADHEr, 14.03 0.30 14.19 0.00 22.02 0.00 0.00 -20.00 0.00 16.65
ACKr, GLUDy, NADH6, PGI 447 ADHEr, 14.02 0.28 14.76 0.00 21.90 0.00
0.00 -20.00 0.00 17.17 EDA, GLCpts, NADH6, PGI 448 ADHEr, 13.94
0.34 14.12 0.00 21.89 0.00 0.00 -20.00 0.00 16.54 ACKr, CBMK2,
NADH6, PGI 449 ADHEr, 13.86 0.22 14.56 0.00 21.78 0.00 0.00 -20.00
0.00 17.62 ATPS4r, FDH2, NADH6, PGI 450 ADHEr, 13.86 0.22 14.56
0.00 21.78 0.00 0.00 -20.00 0.00 17.62 ATPS4r, NADH6, PFLi, PGI 451
ADHEr, 13.80 0.27 16.36 0.00 20.68 0.00 0.00 -20.00 0.00 18.31
ATPS4r, GLCpts, NADH6, PFLi 452 ADHEr, 13.69 0.43 13.35 0.00 20.51
0.00 0.00 -20.00 0.00 16.43 ATPS4r, MDH, NADH6, PGL 453 ADHEr,
13.69 0.43 13.35 0.00 20.51 0.00 0.00 -20.00 0.00 16.43 ATPS4r,
G6PDHy, MDH, NADH6 454 ADHEr, 13.68 0.36 13.87 0.00 18.43 4.12 0.00
-20.00 0.00 20.56 ACKr, FUM, GLUDy, LDH_D 455 ADHEr, 13.66 0.25
15.79 0.00 19.61 3.75 0.00 -20.00 0.00 21.32 ATPS4r, NADH6, PGI,
SUCD4 456 ADHEr, 13.56 0.37 13.76 0.00 17.71 5.22 0.00 -20.00 0.00
21.60 ACKr, GLUDy, LDH_D, SUCD4 457 ADHEr, 13.53 0.44 10.63 0.00
14.18 12.16 0.00 -20.00 0.00 25.92 ATPS4r, G6PDHy, MDH, THD2 458
ADHEr, 13.53 0.44 10.63 0.00 14.18 12.16 0.00 -20.00 0.00 25.92
ATPS4r, MDH, PGL, THD2 459 ADHEr, 13.44 0.26 11.28 0.00 7.24 25.83
0.00 -20.00 0.00 38.92 ASPT, G6PDHy, MDH, PYK 460 ADHEr, 13.44 0.26
11.28 0.00 7.24 25.83 0.00 -20.00 0.00 38.92 ASPT, EDA, MDH, PYK
461 ADHEr, 13.44 0.26 11.28 0.00 7.24 25.83 0.00 -20.00 0.00 38.92
ASPT, MDH, PGL, PYK 462 ADHEr, 13.35 0.25 4.81 0.00 13.43 19.59
0.00 -20.00 0.00 28.60 FRD2, LDH_D, MDH, SUCOAS 463 ADHEr, 13.27
0.26 4.78 0.00 13.14 19.51 0.00 -20.00 0.00 28.92 ASPT, LDH_D, MDH,
SUCOAS 464 ADHEr, 13.22 0.26 0.00 0.00 17.54 14.32 0.00 -20.00 0.00
21.06 ACt6, LDH_D, MDH, SUCD4 465 ADHEr, 13.17 0.24 16.40 0.00
17.06 7.36 0.00 -20.00 0.00 25.48 ATPS4r, GLUDy, PGI, SUCD4 466
ADHEr, 13.15 0.27 5.76 0.00 12.63 20.09 0.00 -20.00 0.00 29.76
ASPT, FUM, LDH_D, MDH 467 ADHEr, 13.15 0.27 5.76 0.00 12.63 20.09
0.00 -20.00 0.00 29.76 ASPT, LDH_D, MALS, MDH 468 ADHEr, 13.15 0.27
5.76 0.00 12.63 20.09 0.00 -20.00 0.00 29.76 ASPT, ICL, LDH_D, MDH
469 ADHEr, 13.07 0.51 0.00 0.00 23.45 0.00 0.00 -20.00 0.00 13.57
ACt6, LDH_D, MDH, NADH6 470 ADHEr, 12.91 0.12 12.29 0.00 -0.02
38.75 0.00 -20.00 0.00 51.90 FRD2, GLUDy, LDH_D, PPCK 471 ADHEr,
12.89 0.13 12.25 0.00 -0.02 38.70 0.00 -20.00 0.00 51.85 FRD2,
LDH_D, PPCK, THD2
472 ADHEr, 12.73 0.47 12.99 0.00 16.72 6.70 0.00 -20.00 0.00 23.06
ACKr, ATPS4r, LDH_D, SUCD4 473 ADHEr, 12.62 0.11 12.68 0.00 0.33
39.20 0.00 -20.00 0.00 52.67 ACKr, ACS, PPC, PPCK 474 ADHEr, 12.60
0.16 11.81 0.00 0.47 38.84 0.00 -20.00 0.00 51.79 GLUDy, LDH_D,
PPC, PPCK 475 ADHEr, 12.60 0.48 14.92 0.00 19.87 0.00 0.00 -20.00
0.00 18.32 ATPS4r, FDH2, NADH6, SULabc 476 ADHEr, 12.60 0.16 11.72
0.00 0.49 38.81 0.00 -20.00 0.00 51.71 LDH_D, PPC, PPCK, THD2 477
ADHEr, 12.57 0.29 9.83 0.00 18.83 0.00 0.00 -20.00 0.00 18.28 ASPT,
ATPS4r, GLCpts, MDH 478 ADHEr, 12.37 0.68 7.32 0.00 24.57 2.87 0.00
-20.00 0.00 15.06 G6PDHy, MDH, NADH6, THD2 479 ADHEr, 12.37 0.68
7.32 0.00 24.57 2.87 0.00 -20.00 0.00 15.06 MDH, NADH6, PGL, THD2
480 ADHEr, 12.36 0.41 12.58 0.00 28.51 0.00 0.00 -20.00 0.00 15.47
ACKr, FBA, GLUDy, NADH6 481 ADHEr, 12.36 0.41 12.58 0.00 28.51 0.00
0.00 -20.00 0.00 15.47 ACKr, GLUDy, NADH6, PFK 482 ADHEr, 12.36
0.41 12.58 0.00 28.51 0.00 0.00 -20.00 0.00 15.47 ACKr, GLUDy,
NADH6, TPI 483 ADHEr, 12.33 0.49 15.46 0.00 19.46 0.00 0.00 -20.00
0.00 18.94 ATPS4r, MTHFC, NADH6, PFLi 484 ADHEr, 12.33 0.49 15.46
0.00 19.46 0.00 0.00 -20.00 0.00 18.94 ATPS4r, FTHFD, NADH6, PFLi
485 ADHEr, 12.30 0.34 0.00 0.00 12.96 0.00 0.00 -20.00 0.00 24.29
ATPS4r, G6PDHy, GLCpts, MDH 486 ADHEr, 12.30 0.34 0.00 0.00 12.96
0.00 0.00 -20.00 0.00 24.29 ATPS4r, GLCpts, MDH, PGL 487 ADHEr,
12.15 0.44 12.39 0.00 28.20 0.00 0.00 -20.00 0.00 15.54 ACKr, FBA,
GLCpts, NADH6 488 ADHEr, 12.15 0.44 12.39 0.00 28.20 0.00 0.00
-20.00 0.00 15.54 ACKr, GLCpts, NADH6, TPI 489 ADHEr, 12.15 0.44
12.39 0.00 28.20 0.00 0.00 -20.00 0.00 15.54 ACKr, GLCpts, NADH6,
PFK 490 ADHEr, 12.15 0.40 12.37 0.00 6.33 23.73 0.00 -20.00 0.00
38.94 ACKr, LDH_D, MDH, SUCD4 491 ADHEr, 12.13 0.22 12.25 0.00
37.66 0.00 0.00 -20.00 0.00 13.85 ACKr, AKGD, ATPS4r, FBA 492
ADHEr, 12.13 0.22 12.25 0.00 37.66 0.00 0.00 -20.00 0.00 13.85
ACKr, AKGD, ATPS4r, PFK 493 ADHEr, 12.13 0.22 12.25 0.00 12.10
25.56 0.00 -20.00 0.00 39.40 ACKr, AKGD, ATPS4r, TPI 494 ADHEr,
12.09 0.07 20.13 0.00 7.88 20.50 0.00 -20.00 0.00 41.14 EDA, PGI,
PPCK, SUCD4 495 ADHEr, 12.09 0.23 12.21 0.00 12.27 25.58 0.00
-20.00 0.00 39.40 ACKr, ATPS4r, FBA, SUCOAS 496 ADHEr, 12.09 0.23
12.21 0.00 12.27 25.58 0.00 -20.00 0.00 39.40 ACKr, ATPS4r, PFK,
SUCOAS 497 ADHEr, 12.09 0.23 12.21 0.00 37.86 0.00 0.00 -20.00 0.00
13.82 ACKr, ATPS4r, SUCOAS, TPI 498 ADHEr, 12.08 0.26 3.82 0.00
8.14 17.37 4.03 -20.00 0.00 33.19 FRD2, LDH_D, ME2, SUCOAS 499
ADHEr, 12.06 0.46 12.31 0.00 28.06 0.00 0.00 -20.00 0.00 15.57
ACKr, CBMK2, FBA, NADH6 500 ADHEr, 12.06 0.46 12.31 0.00 28.06 0.00
0.00 -20.00 0.00 15.57 ACKr, CBMK2, NADH6, PFK 501 ADHEr, 12.06
0.46 12.31 0.00 28.06 0.00 0.00 -20.00 0.00 15.57 ACKr, CBMK2,
NADH6, TPI 502 ADHEr, 12.05 0.46 12.30 0.00 28.05 0.00 0.00 -20.00
0.00 15.58 ACKr, NADH6, RPE, TPI 503 ADHEr, 12.05 0.46 12.30 0.00
28.05 0.00 0.00 -20.00 0.00 15.58 ACKr, NADH6, PFK, RPE 504 ADHEr,
12.05 0.46 12.30 0.00 28.05 0.00 0.00 -20.00 0.00 15.58 ACKr, FBA,
NADH6, RPE 505 ADHEr, 12.05 0.46 12.30 0.00 28.04 0.00 0.00 -20.00
0.00 15.58 ACKr, ASNS2, FBA, NADH6 H2O ILE LAC NH4 NO3 PHE PI SO4
SUC THR VAL 1 8.96 0.00 0.00 -6.26 -2.00 0.00 -0.78 -0.13 0.23 0.00
0.00 2 -12.07 0.00 0.00 -0.24 -2.00 0.00 -0.03 0.00 0.00 0.00 0.00
3 -12.07 0.00 0.00 -0.24 -2.00 0.00 -0.03 0.00 0.00 0.00 0.00 4
-0.90 0.00 0.00 -4.96 0.00 0.00 -0.61 -0.10 0.00 0.00 0.00 5 0.68
0.00 0.00 -5.60 0.00 0.00 -0.69 -0.11 0.00 0.00 0.00 6 6.08 0.00
0.00 -7.09 -5.00 0.00 -0.88 -0.14 0.00 0.00 0.00 7 21.51 0.00 0.00
-4.52 0.00 0.00 -0.56 -0.09 24.86 0.00 0.00 8 10.90 0.00 0.00 -6.44
0.00 0.00 -0.80 -0.13 12.16 0.00 0.00 9 12.29 0.00 0.00 -6.62 0.00
0.00 -0.82 -0.13 13.54 0.00 0.00 10 10.81 0.00 0.00 -5.83 0.00 0.00
-0.72 -0.12 14.32 0.00 0.00 11 8.25 0.00 0.00 -4.23 0.00 0.00 -0.52
-0.09 16.46 0.00 0.00 12 8.25 0.00 0.00 -4.23 0.00 0.00 -0.52 -0.09
16.46 0.00 0.00 13 8.25 0.00 0.00 -4.23 0.00 0.00 -0.52 -0.09 16.46
0.00 0.00 14 4.02 0.00 0.00 -3.39 0.00 0.00 -0.42 -0.07 0.00 0.00
0.00 15 14.07 0.00 0.00 -4.31 0.00 0.00 -0.53 -0.09 0.00 0.00 0.00
16 14.87 0.00 0.00 -4.72 0.00 0.00 -0.58 -0.10 0.00 0.00 0.00 17
30.54 0.00 0.00 -7.41 -20.00 0.00 -0.92 -0.15 0.00 0.00 0.00 18
15.46 0.00 0.00 -5.03 0.00 0.00 -0.62 -0.10 0.00 0.00 0.00 19 3.71
0.00 0.00 -0.73 -10.00 0.00 -0.09 -0.01 0.00 0.00 0.00 20 3.71 0.00
0.00 -0.73 -10.00 0.00 -0.09 -0.01 0.00 0.00 0.00 21 -1.88 0.00
0.00 -4.57 0.00 0.00 -0.57 -0.09 0.00 0.00 0.00 22 -0.60 0.00 0.00
-3.07 0.00 0.00 -0.38 -0.06 0.00 0.00 0.00 23 -0.71 0.00 0.00 -3.11
0.00 0.00 -0.38 -0.06 0.00 0.00 0.00 24 -0.71 0.00 0.00 -3.11 0.00
0.00 -0.38 -0.06 0.00 0.00 0.00 25 -0.71 0.00 0.00 -3.11 0.00 0.00
-0.38 -0.06 0.00 0.00 0.00 26 2.51 0.00 0.00 -4.48 0.00 0.00 -0.55
-0.09 0.00 0.00 0.00 27 4.00 0.00 0.00 -5.12 0.00 0.00 -0.63 -0.10
0.00 0.00 0.00 28 5.55 0.00 0.00 -5.78 0.00 0.00 -0.72 -0.12 0.00
0.00 0.00 29 0.13 0.00 0.00 -4.83 0.00 0.00 -0.60 -0.10 0.00 0.00
0.00 30 13.24 0.00 0.00 -6.66 -5.00 0.00 -0.82 -0.13 0.23 0.00 0.00
31 12.86 0.00 0.00 -6.69 -5.00 0.00 -0.83 -0.14 0.00 0.00 0.00 32
-6.65 0.00 0.00 -2.62 0.00 0.00 -0.32 -0.05 0.00 0.00 0.00 33 -6.10
0.00 0.00 -2.84 0.00 0.00 -0.35 -0.06 0.00 0.00 0.00 34 1.88 0.00
0.00 -5.25 -2.00 0.00 -0.65 -0.11 0.00 0.00 0.00 35 -6.68 0.00 0.00
-2.66 0.00 0.00 -0.33 -0.05 0.00 0.00 0.00 36 -6.68 0.00 0.00 -2.66
0.00 0.00 -0.33 -0.05 0.00 0.00 0.00 37 -6.68 0.00 0.00 -2.66 0.00
0.00 -0.33 -0.05 0.00 0.00 0.00 38 -6.39 0.00 0.00 -2.77 0.00 0.00
-0.34 -0.06 0.00 0.00 0.00 39 -6.39 0.00 0.00 -2.77 0.00 0.00 -0.34
-0.06 0.00 0.00 0.00 40 -6.39 0.00 0.00 -2.77 0.00 0.00 -0.34 -0.06
0.00 0.00 0.00 41 8.45 0.00 0.00 -5.39 0.00 0.00 -0.67 -0.11 6.20
0.00 0.00 42 -6.13 0.00 0.00 -2.88 0.00 0.00 -0.36 -0.06 0.00 0.00
0.00 43 -6.13 0.00 0.00 -2.88 0.00 0.00 -0.36 -0.06 0.00 0.00 0.00
44 -6.13 0.00 0.00 -2.88 0.00 0.00 -0.36 -0.06 0.00 0.00 0.00 45
19.66 0.00 0.00 -6.02 0.00 0.00 -0.75 -0.12 7.94 0.00 0.00 46 -3.22
0.00 0.00 -4.04 0.00 0.00 -0.50 -0.08 0.00 0.00 0.00 47 -0.76 0.00
0.00 -4.95 0.00 0.00 -0.61 -0.10 0.00 0.00 0.00 48 -2.34 0.00 0.00
-4.39 0.00 0.00 -0.54 -0.09 0.00 0.00 0.00 49 -2.29 0.00 0.00 -4.41
0.00 0.00 -0.55 -0.09 0.00 0.00 0.00 50 -2.13 0.00 0.00 -4.47 0.00
0.00 -0.55 -0.09 0.00 0.00 0.00 51 -1.81 0.00 0.00 -4.60 0.00 0.00
-0.57 -0.09 0.00 0.00 0.00 52 -0.83 0.00 0.00 -4.95 0.00 0.00 -0.61
-0.10 0.00 0.00 0.00 53 -1.66 0.00 0.00 -4.66 0.00 0.00 -0.58 -0.09
0.00 0.00 0.00 54 -1.66 0.00 0.00 -4.66 0.00 0.00 -0.58 -0.09 0.00
0.00 0.00 55 0.74 0.00 0.00 -5.54 0.00 0.00 -0.69 -0.11 0.00 0.00
0.00 56 -1.01 0.00 0.00 -4.92 0.00 0.00 -0.61 -0.10 0.00 0.00 0.00
57 -0.80 0.00 0.00 -5.00 0.00 0.00 -0.62 -0.10 0.00 0.00 0.00 58
-0.78 0.00 0.00 -5.01 0.00 0.00 -0.62 -0.10 0.00 0.00 0.00 59 -0.69
0.00 0.00 -5.05 0.00 0.00 -0.62 -0.10 0.00 0.00 0.00 60 0.71 0.00
0.00 -5.56 0.00 0.00 -0.69 -0.11 0.00 0.00 0.00 61 0.30 0.00 0.00
-5.44 0.00 0.00 -0.67 -0.11 0.00 0.00 0.00 62 0.47 0.00 0.00 -5.51
0.00 0.00 -0.68 -0.11 0.00 0.00 0.00 63 0.56 0.00 0.00 -5.55 0.00
0.00 -0.69 -0.11 0.00 0.00 0.00 64 1.94 0.00 0.00 -6.10 0.00 0.00
-0.75 -0.12 0.00 0.00 0.00 65 20.54 0.00 0.00 -6.25 0.00 0.00 -0.77
-0.13 9.44 0.00 0.00 66 -0.22 0.00 0.00 -4.49 -5.00 0.00 -0.56
-0.09 0.00 0.00 0.00 67 -0.25 0.00 0.00 -4.56 -5.00 0.00 -0.56
-0.09 0.00 0.00 0.00 68 -0.25 0.00 0.00 -4.56 -5.00 0.00 -0.56
-0.09 0.00 0.00 0.00 69 -0.25 0.00 0.00 -4.56 -5.00 0.00 -0.56
-0.09 0.00 0.00 0.00 70 16.61 0.00 0.00 -3.82 0.00 0.00 -0.47 -0.08
12.23 0.00 0.00 71 16.61 0.00 0.00 -3.82 0.00 0.00 -0.47 -0.08
12.23 0.00 0.00 72 16.61 0.00 0.00 -3.82 0.00 0.00 -0.47 -0.08
12.23 0.00 0.00 73 3.70 0.00 0.00 -6.40 0.00 0.00 -0.79 -0.13 1.51
0.00 0.00 74 6.06 0.00 0.00 -6.97 -5.00 0.00 -0.86 -0.14 0.00 0.00
0.00 75 4.14 0.00 0.00 -6.31 -5.00 0.00 -0.78 -0.13 0.00 0.00 0.00
76 6.07 0.00 0.00 -7.03 -5.00 0.00 -0.87 -0.14 0.00 0.00 0.00 77
5.91 0.00 0.00 -7.02 -5.00 0.00 -0.87 -0.14 0.00 0.00 0.00 78 20.66
0.00 0.00 -4.37 0.00 0.00 -0.54 -0.09 21.30 0.00 0.00 79 22.80 0.00
0.00 -4.46 0.00 0.00 -0.55 -0.09 24.28 0.00 0.00 80 5.91 0.00 0.00
-4.11 0.00 0.00 -0.51 -0.08 13.43 0.00 0.00 81 5.91 0.00 0.00 -4.11
0.00 0.00 -0.51 -0.08 13.43 0.00 0.00 82 5.91 0.00 0.00 -4.11 0.00
0.00 -0.51 -0.08 13.43 0.00 0.00 83 12.13 0.00 0.00 -6.59 0.00 0.00
-0.82 -0.13 13.08 0.00 0.00 84 10.68 0.00 0.00 -5.80 0.00 0.00
-0.72 -0.12 13.90 0.00 0.00 85 12.21 0.00 0.00 -6.61 0.00 0.00
-0.82 -0.13 13.30 0.00 0.00 86 10.74 0.00 0.00 -5.81 0.00 0.00
-0.72 -0.12 14.10 0.00 0.00 87 8.23 0.00 0.00 -4.19 0.00 0.00 -0.52
-0.08 16.35 0.00 0.00 88 8.23 0.00 0.00 -4.19 0.00 0.00 -0.52 -0.08
16.35 0.00 0.00 89 8.23 0.00 0.00 -4.19 0.00 0.00 -0.52 -0.08 16.35
0.00 0.00 90 8.24 0.00 0.00 -4.21 0.00 0.00 -0.52 -0.09 16.41 0.00
0.00 91 8.24 0.00 0.00 -4.21 0.00 0.00 -0.52 -0.09 16.41 0.00 0.00
92 13.38 0.00 0.00 -2.97 0.00 0.00 -0.37 -0.06 0.00 0.00 0.00
93 10.91 0.00 0.00 -2.40 0.00 0.00 -0.30 -0.05 0.00 0.00 0.00 94
11.52 0.00 0.00 -2.66 0.00 0.00 -0.33 -0.05 0.00 0.00 0.00 95 14.23
0.00 0.00 -2.97 -2.00 0.00 -0.37 -0.06 0.00 0.00 0.00 96 6.78 2.14
0.00 -4.35 0.00 0.00 -0.27 -0.04 0.00 0.00 0.00 97 9.08 0.00 0.00
-2.36 -2.00 0.00 -0.29 -0.05 0.00 0.00 0.00 98 16.09 0.00 0.00
-4.23 -2.00 0.00 -0.52 -0.09 0.00 0.00 0.00 99 24.49 0.00 0.00
-4.01 -20.00 0.00 -0.50 -0.08 0.00 0.00 0.00 100 24.49 0.00 0.00
-4.01 -20.00 0.00 -0.50 -0.08 0.00 0.00 0.00 101 24.49 0.00 0.00
-4.01 -20.00 0.00 -0.50 -0.08 0.00 0.00 0.00 102 10.90 1.81 0.00
-4.09 0.00 0.00 -0.28 -0.05 0.00 0.00 0.00 103 0.36 0.00 0.00 -1.93
0.00 0.00 -0.24 -0.04 0.00 0.00 0.00 104 0.38 0.00 0.00 -1.94 0.00
0.00 -0.24 -0.04 0.00 0.00 0.00 105 1.01 0.00 0.00 -2.19 0.00 0.00
-0.27 -0.04 0.00 0.00 0.00 106 1.07 0.00 0.00 -2.21 0.00 0.00 -0.27
-0.04 0.00 0.00 0.00 107 2.80 0.00 0.00 -2.91 0.00 0.00 -0.36 -0.06
0.00 0.00 0.00 108 3.17 0.00 0.00 -3.05 0.00 0.00 -0.38 -0.06 0.00
0.00 0.00 109 2.41 0.00 0.00 -4.15 0.00 0.00 -0.51 -0.08 0.00 0.00
0.00 110 2.41 0.00 0.00 -4.15 0.00 0.00 -0.51 -0.08 0.00 0.00 0.00
111 9.09 0.00 0.00 -1.73 0.00 0.00 -0.21 -0.03 0.00 0.00 0.00 112
9.11 0.00 0.00 -1.75 0.00 0.00 -0.22 -0.04 0.00 0.00 0.00 113 9.11
0.00 0.00 -1.75 0.00 0.00 -0.22 -0.04 0.00 0.00 0.00 114 9.11 0.00
0.00 -1.75 0.00 0.00 -0.22 -0.04 0.00 0.00 0.00 115 26.42 0.00 0.00
-6.41 -15.00 0.00 -0.79 -0.13 0.00 0.00 0.00 116 9.59 0.00 0.00
-1.99 0.00 0.00 -0.25 -0.04 0.00 0.00 0.00 117 9.59 0.00 0.00 -1.99
0.00 0.00 -0.25 -0.04 0.00 0.00 0.00 118 9.62 0.00 0.00 -2.01 0.00
0.00 -0.25 -0.04 0.00 0.00 0.00 119 9.62 0.00 0.00 -2.01 0.00 0.00
-0.25 -0.04 0.00 0.00 0.00 120 9.62 0.00 0.00 -2.01 0.00 0.00 -0.25
-0.04 0.00 0.00 0.00 121 9.62 0.00 0.00 -2.01 0.00 0.00 -0.25 -0.04
0.00 0.00 0.00 122 9.62 0.00 0.00 -2.01 0.00 0.00 -0.25 -0.04 0.00
0.00 0.00 123 9.62 0.00 0.00 -2.01 0.00 0.00 -0.25 -0.04 0.00 0.00
0.00 124 9.67 0.00 0.00 -2.04 0.00 0.00 -0.25 -0.04 0.00 0.00 0.00
125 9.67 0.00 0.00 -2.04 0.00 0.00 -0.25 -0.04 0.00 0.00 0.00 126
9.67 0.00 0.00 -2.04 0.00 0.00 -0.25 -0.04 0.00 0.00 0.00 127 14.16
0.00 0.00 -4.23 0.00 0.00 -0.52 -0.09 0.00 0.00 0.00 128 28.93 0.00
0.00 -6.51 -20.00 0.00 -0.81 -0.13 0.00 0.00 0.00 129 29.25 0.00
0.00 -6.68 -20.00 0.00 -0.83 -0.14 0.00 0.00 0.00 130 12.00 0.00
0.00 -4.99 -15.00 0.00 -0.62 -0.10 0.00 0.00 0.00 131 14.83 0.00
1.08 -4.62 0.00 0.00 -0.57 -0.09 0.00 0.00 0.00 132 12.93 0.00 0.00
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-4.03 0.00 0.00 -0.50 -0.08 0.00 0.00 0.00 137 13.59 0.00 0.00
-4.06 0.00 0.00 -0.50 -0.08 0.00 0.00 0.00 138 14.08 0.00 0.00
-4.30 0.00 0.00 -0.53 -0.09 0.00 0.00 0.00 139 14.07 0.00 0.00
-4.30 0.00 0.00 -0.53 -0.09 0.00 0.00 0.00 140 13.88 0.00 0.00
-4.21 0.00 0.00 -0.52 -0.09 0.00 0.00 0.00 141 13.99 0.00 0.00
-4.27 0.00 0.00 -0.53 -0.09 0.00 0.00 0.00 142 16.33 0.00 0.00
-4.91 -2.00 0.00 -0.61 -0.10 0.00 0.00 0.00 143 14.82 0.00 0.00
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-7.33 -20.00 0.00 -0.91 -0.15 0.00 0.00 0.00 145 14.84 0.00 0.00
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-7.34 -20.00 0.00 -0.91 -0.15 0.00 0.00 0.00 147 30.45 0.00 0.00
-7.36 -20.00 0.00 -0.91 -0.15 0.00 0.00 0.00 148 30.47 0.00 0.00
-7.37 -20.00 0.00 -0.91 -0.15 0.00 0.00 0.00 149 30.48 0.00 0.00
-7.38 -20.00 0.00 -0.91 -0.15 0.00 0.00 0.00 150 14.78 0.00 0.00
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-7.38 -20.00 0.00 -0.91 -0.15 0.00 0.00 0.00 152 30.51 0.00 0.00
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0.00 0.00 -0.59 -0.10 0.00 0.00 0.00 160 8.00 0.00 0.00 -4.76 0.00
0.00 -0.59 -0.10 0.00 0.00 0.00 161 14.08 0.00 0.00 -5.43 0.00 0.00
-0.67 -0.11 0.00 0.00 0.00 162 -3.19 0.00 0.00 -2.28 0.00 0.00
-0.28 -0.05 0.00 0.00 0.00 163 0.03 0.00 0.00 -4.13 0.00 0.00 -0.51
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0.00 0.00 0.00 165 -1.24 0.00 0.00 -4.34 0.00 0.00 -0.54 -0.09 0.00
0.00 0.00 166 17.57 0.00 0.00 -5.62 -5.00 0.00 -0.70 -0.11 0.00
0.00 0.00 167 -8.18 0.00 0.00 -2.02 0.00 0.00 -0.25 -0.04 0.00 0.00
0.00 168 -8.17 0.00 0.00 -2.06 0.00 0.00 -0.25 -0.04 0.00 0.00 0.00
169 -8.17 0.00 0.00 -2.06 0.00 0.00 -0.25 -0.04 0.00 0.00 0.00 170
-8.17 0.00 0.00 -2.06 0.00 0.00 -0.25 -0.04 0.00 0.00 0.00 171 0.48
0.00 0.00 -2.25 0.00 0.00 -0.28 -0.05 0.00 0.00 0.00 172 0.48 0.00
0.00 -2.25 0.00 0.00 -0.28 -0.05 0.00 0.00 0.00 173 0.48 0.00 0.00
-2.25 0.00 0.00 -0.28 -0.05 0.00 0.00 0.00 174 11.87 0.00 0.00
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0.00 0.00 -0.53 -0.09 0.00 0.00 0.00 177 0.51 0.00 0.00 -3.99 0.00
0.00 -0.49 -0.08 0.00 0.00 0.00 178 1.11 0.00 0.00 -4.22 0.00 0.00
-0.52 -0.09 0.00 0.00 0.00 179 1.11 0.00 0.00 -4.22 0.00 0.00 -0.52
-0.09 0.00 0.00 0.00 180 3.75 0.00 0.00 -0.73 -10.00 0.00 -0.09
-0.01 0.00 0.00 0.00 181 3.75 0.00 0.00 -0.73 -10.00 0.00 -0.09
-0.01 0.00 0.00 0.00 182 3.73 0.00 0.00 -0.73 -10.00 0.00 -0.09
-0.01 0.00 0.00 0.00 183 3.73 0.00 0.00 -0.73 -10.00 0.00 -0.09
-0.01 0.00 0.00 0.00 184 -3.46 0.00 0.00 -3.94 0.00 0.00 -0.49
-0.08 0.00 0.00 0.00 185 -1.79 0.00 0.00 -4.54 0.00 0.00 -0.56
-0.09 0.00 0.00 0.00 186 -3.11 0.00 0.00 -4.08 0.00 0.00 -0.51
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-0.15 0.00 0.00 0.00 188 0.98 0.00 0.00 -4.53 0.00 0.00 -0.56 -0.09
0.00 0.00 0.00 189 -1.83 0.00 0.00 -4.56 0.00 0.00 -0.56 -0.09 0.00
0.00 0.00 190 -3.12 0.00 0.00 -2.02 0.00 0.00 -0.25 -0.04 0.00 0.00
0.00 191 0.18 0.00 0.00 -4.28 0.00 0.00 -0.53 -0.09 0.00 0.00 0.00
192 0.18 0.00 0.00 -4.28 0.00 0.00 -0.53 -0.09 0.00 0.00 0.00 193
-0.44 0.00 0.00 -4.05 0.00 0.00 -0.50 -0.08 0.00 0.00 0.00 194
-1.99 0.00 0.00 -4.53 0.00 0.00 -0.56 -0.09 0.00 0.00 0.00 195 7.01
0.00 0.00 -3.62 -5.00 0.00 -0.45 -0.07 0.00 0.00 0.00 196 7.01 0.00
0.00 -3.62 -5.00 0.00 -0.45 -0.07 0.00 0.00 0.00 197 7.01 0.00 0.00
-3.62 -5.00 0.00 -0.45 -0.07 0.00 0.00 0.00 198 -3.17 0.00 0.00
-2.06 0.00 0.00 -0.25 -0.04 0.00 0.00 0.00 199 -3.17 0.00 0.00
-2.06 0.00 0.00 -0.25 -0.04 0.00 0.00 0.00 200 -3.17 0.00 0.00
-2.06 0.00 0.00 -0.25 -0.04 0.00 0.00 0.00 201 19.49 0.00 0.00
-6.01 0.00 0.00 -0.74 -0.12 6.13 0.00 0.00 202 -2.76 0.00 0.00
-2.23 0.00 0.00 -0.28 -0.05 0.00 0.00 0.00 203 -2.76 0.00 0.00
-2.23 0.00 0.00 -0.28 -0.05 0.00 0.00 0.00 204 -2.76 0.00 0.00
-2.23 0.00 0.00 -0.28 -0.05 0.00 0.00 0.00 205 18.41 0.00 0.00
-4.99 0.00 0.00 -0.62 -0.10 8.31 0.00 0.00 206 18.41 0.00 0.00
-4.99 0.00 0.00 -0.62 -0.10 8.31 0.00 0.00 207 -1.39 0.00 0.00
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-2.77 0.00 0.00 -0.34 -0.06 0.00 0.00 0.00 216 -1.50 0.00 0.00
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0.00 0.00 -0.71 -0.12 0.00 0.00 0.00 218 -0.66 0.00 0.00 -3.09 0.00
0.00 -0.38 -0.06 0.00 0.00 0.00 219 -0.66 0.00 0.00 -3.09 0.00 0.00
-0.38 -0.06 0.00 0.00 0.00 220 -0.66 0.00 0.00 -3.09 0.00 0.00
-0.38 -0.06 0.00 0.00 0.00 221 4.01 0.00 0.00 -5.35 0.00 0.00 -0.66
-0.11 1.04 0.00 0.00 222 1.07 0.00 0.00 -3.87 0.00 0.00 -0.48 -0.08
0.00 0.00 0.00 223 1.14 0.00 0.00 -3.90 0.00 0.00 -0.48 -0.08 0.00
0.00 0.00 224 2.70 0.00 0.00 -4.46 0.00 0.00 -0.55 -0.09 0.00 0.00
0.00 225 1.46 0.00 0.00 -4.03 0.00 0.00 -0.50 -0.08 0.00 0.00 0.00
226 1.61 0.00 0.00 -4.10 0.00 0.00 -0.51 -0.08 0.00 0.00 0.00 227
1.61 0.00 0.00 -4.10 0.00 0.00 -0.51 -0.08 0.00 0.00 0.00 228 27.21
0.00 0.00 -13.29 -2.00 0.00 -0.35 -0.06 0.00 0.00 10.48 229 2.61
0.00 0.00 -4.47 0.00 0.00 -0.55 -0.09 0.00 0.00 0.00 230 2.48 0.00
0.00 -4.47 0.00 0.00 -0.55 -0.09 0.00 0.00 0.00 231 2.59 0.00 0.00
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0.00 -0.63 -0.10 0.00 0.00 0.00 234 27.21 0.00 0.00 -7.98 -15.00
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0.00 0.00 0.00 238 5.65 0.00 0.00 -5.75 0.00 0.00 -0.71 -0.12 0.00
0.00 0.00 239 17.83 0.00 0.00 -5.22 0.00 0.00 -0.65 -0.11 7.01 0.00
0.00 240 5.22 0.00 0.00 -5.64 0.00 0.00 -0.70 -0.11 0.00 0.00 0.00
241 5.43 0.00 0.00 -5.73 0.00 0.00 -0.71 -0.12 0.00 0.00 0.00 242
-1.43 0.00 0.00 -4.27 0.00 0.00 -0.53 -0.09 0.00 0.00 0.00 243
-1.17 0.00 0.00 -4.36 0.00 0.00 -0.54 -0.09 0.00 0.00 0.00 244
12.72 0.00 0.00 -6.63 -5.00 0.00 -0.82 -0.13 0.00 0.00 0.00 245
18.04 0.00 0.00 -5.25 0.00 0.00 -0.65 -0.11 7.45 0.00 0.00 246 8.28
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0.00 0.00 -1.95 0.00 0.00 -0.24 -0.04 0.00 0.00 0.00 248 18.24 0.00
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-0.08 0.00 0.00 0.00 501 24.41 0.00 0.00 -3.97 -20.00 0.00 -0.49
-0.08 0.00 0.00 0.00 502 24.45 0.00 0.00 -3.99 -20.00 0.00 -0.49
-0.08 0.00 0.00 0.00 503 24.45 0.00 0.00 -3.99 -20.00 0.00 -0.49
-0.08 0.00 0.00 0.00 504 24.45 0.00 0.00 -3.99 -20.00 0.00 -0.49
-0.08 0.00 0.00 0.00 505 24.46 0.00 0.00 -4.00 -20.00 0.00 -0.49
-0.08 0.00 0.00 0.00
TABLE-US-00008 TABLE 8 Corresponding genes to be knocked out to
prevent a particular reaction from occurring in E. coli. Genes
Encoding the Enzyme(s) Reaction Catalyzing Each Abbreviation
Reaction Stoichiometry* Reaction& ACKr [c]: ac + atp <==>
actp + adp (b3115 or b2296 or b1849) ACS [c]: ac + atp + coa -->
accoa + amp + ppi b4069 ACt6 ac[p] + h[p] <==> ac[c] + h[c]
Non-gene associated ADHEr [c]: etoh + nad <==> acald + h +
nadh (b0356 or b1478 or b1241) [c]: acald + coa + nad <==>
accoa + h + nadh (b1241 or b0351) AKGD [c]: akg + coa + nad -->
co2 + nadh + succoa (b0116 and b0726 and b0727) ASNS2 [c]: asp-L +
atp + nh4 --> amp + asn-L + h + ppi b3744 ASPT [c]: asp-L -->
fum + nh4 b4139 ATPS4r adp[c] + (4) h[p] + pi[c] <==> atp[c]
+ (3) h[c] + h2o[c] (((b3736 and b3737 and b3738) and (b3731 and
b3732 and b3733 and b3734 and b3735)) or ((b3736 and b3737 and
b3738) and (b3731 and b3732 and b3733 and b3734 and b3735) and
b3739)) CBMK2 [c]: atp + co2 + nh4 <==> adp + cbp + (2) h
(b0521 or b0323 or b2874) EDA [c]: 2ddg6p --> g3p + pyr b1850
ENO [c]: 2pg <==> h2o + pep b2779 FBA [c]: fdp <==>
dhap + g3p (b2097 or b2925 or b1773) FBP [c]: fdp + h2o --> f6p
+ pi (b4232 or b3925) FDH2 for[p] + (2) h[c] + q8[c] --> co2[c]
+ h[p] + q8h2[c] ((b3892 and b3893 for[p] + (2) h[c] + mqn8[c]
--> co2[c] + h[p] + mql8[c] and b3894) or (b1474 and b1475 and
b1476)) FRD2 [c]: fum + mql8 --> mqn8 + succ (b4151 and b4152
[c]: 2dmmql8 + fum --> 2dmmq8 + succ and b4153 and b4154) FTHFD
[c]: 10fthf + h2o --> for + h + thf b1232 FUM [c]: fum + h2o
<==> mal-L (b1612 or b4122 or b1611) G5SD [c]: glu5p + h +
nadph --> glu5sa + nadp + pi b0243 G6PDHy [c]: g6p + nadp
<==> 6pgl + h + nadph b1852 GLCpts glc-D[p] + pep[c] -->
g6p[c] + pyr[c] ((b2417 and b1101 and b2415 and b2416) or (b1817
and b1818 and b1819 and b2415 and b2416) or (b2417 and b1621 and
b2415 and b2416)) GLU5K [c]: atp + glu-L --> adp + glu5p b0242
GLUDy [c]: glu-L + h2o + nadp <==> akg + h + nadph + nh4
b1761 GLYCL [c]: gly + nad + thf --> co2 + mlthf + nadh + nh4
(b2904 and b2903 and b2905 and b0116) HEX1 [c]: atp + glc-D -->
adp + g6p + h b2388 ICL [c]: icit --> glx + succ b4015 LDH_D
[c]: lac-D + nad <==> h + nadh + pyr (b2133 or b1380) MALS
[c]: accoa + glx + h2o --> coa + h + mal-L (b4014 or b2976) MDH
[c]: mal-L + nad <==> h + nadh + oaa b3236 ME2 [c]: mal-L +
nadp --> co2 + nadph + pyr b2463 MTHFC [c]: h2o + methf
<==> 10fthf + h b0529 NADH12 [c]: h + mqn8 + nadh --> mql8
+ nad b1109 [c]: h + nadh + q8 --> nad + q8h2 [c]: 2dmmq8 + h +
nadh --> 2dmmql8 + nad NADH6 (4) h[c] + nadh[c] + q8[c] -->
(3) h[p] + nad[c] + q8h2[c] (b2276 and b2277 (4) h[c] + mqn8[c] +
nadh[c] --> (3) h[p] + mql8[c] + and b2278 and b2279 nad[c] and
b2280 and b2281 2dmmq8[c] + (4) h[c] + nadh[c] --> 2dmmql8[c] +
(3) and b2282 and b2283 h[p] + nad[c] and b2284 and b2285 and b2286
and b2287 and b2288) PFK [c]: atp + f6p --> adp + fdp + h (b3916
or b1723) PFLi [c]: coa + pyr --> accoa + for (((b0902 and
b0903) and b2579) or (b0902 and b0903) or (b0902 and b3114) or
(b3951 and b3952)) PGDH [c]: 6pgc + nadp --> co2 + nadph +
ru5p-D b2029 PGI [c]: g6p <==> f6p b4025 PGL [c]: 6pgl + h2o
--> 6pgc + h b0767 PGM [c]: 2pg <==> 3pg (b3612 or b4395
or b0755) PPC [c]: co2 + h2o + pep --> h + oaa + pi b3956 PPCK
[c]: atp + oaa --> adp + co2 + pep b3403 PRO1z [c]: fad + pro-L
--> 1pyr5c + fadh2 + h b1014 PYK [c]: adp + h + pep --> atp +
pyr b1854 or b1676) PYRt2 h[p] + pyr[p] <==> h[c] + pyr[c]
Non-gene associated RPE [c]: ru5p-D <==> xu5p-D (b4301 or
b3386) SO4t2 so4[e] <==> so4[p] (b0241 or b0929 or b1377 or
b2215) SUCD4 [c]: q8 + succ --> fum + q8h2 (b0721 and b0722 and
b0723 and b0724) SUCOAS [c]: atp + coa + succ <==> adp + pi +
succoa (b0728 and b0729) SULabc atp[c] + h2o[c] + so4[p] -->
adp[c] + h[c] + pi[c] + ((b2422 and b2425 so4[c] and b2424 and
b2423) or (b0763 and b0764 and b0765) or (b2422 and b2424 and b2423
and b3917)) TAL [c]: g3p + s7p <==> e4p + f6p (b2464 or
b0008) THD2 (2) h[p] + nadh[c] + nadp[c] --> (2) h[c] + nad[c] +
(b1602 and b1603) nadph[c] THD5 [c]: nad + nadph --> nadh + nadp
(b3962 or (b1602 and b1603)) TPI [c]: dhap <==> g3p b3919
TABLE-US-00009 TABLE 9 Metabolite names corresponding to
abbreviations used in Table 8. Metabolite Abbreviation Metabolite
Name 10fthf 10-Formyltetrahydrofolate 1pyr5c
1-Pyrroline-5-carboxylate 2ddg6p 2-Dehydro-3-deoxy-D-gluconate
6-phosphate 2dmmq8 2-Demethylmenaquinone 8 2dmmql8
2-Demethylmenaquinol 8 2pg D-Glycerate 2-phosphate 3pg
3-Phospho-D-glycerate 6pgc 6-Phospho-D-gluconate 6pgl
6-phospho-D-glucono-1,5-lactone ac Acetate acald Acetaldehyde accoa
Acetyl-CoA actp Acetyl phosphate adp ADP akg 2-Oxoglutarate amp AMP
asn-L L-Asparagine asp-L L-Aspartate atp ATP cbp Carbamoyl
phosphate co2 CO2 coa Coenzyme A dhap Dihydroxyacetone phosphate
e4p D-Erythrose 4-phosphate etoh Ethanol f6p D-Fructose 6-phosphate
fad Flavin adenine dinucleotide oxidized fadh2 Flavin adenine
dinucleotide reduced fdp D-Fructose 1,6-bisphosphate for Formate
fum Fumarate g3p Glyceraldehyde 3-phosphate g6p D-Glucose
6-phosphate glc-D D-Glucose glu5p L-Glutamate 5-phosphate glu5sa
L-Glutamate 5-semialdehyde glu-L L-Glutamate glx Glyoxylate gly
Glycine h H+ h2o H2O icit Isocitrate lac-D D-Lactate mal-L L-Malate
methf 5,10-Methenyltetrahydrofolate mlthf
5,10-Methylenetetrahydrofolate mql8 Menaquinol 8 mqn8 Menaquinone 8
nad Nicotinamide adenine dinucleotide nadh Nicotinamide adenine
dinucleotide - reduced nadp Nicotinamide adenine dinucleotide
phosphate nadph Nicotinamide adenine dinucleotide phosphate -
reduced nh4 Ammonium oaa Oxaloacetate pep Phosphoenolpyruvate pi
Phosphate ppi Diphosphate pro-L L-Proline pyr Pyruvate q8
Ubiquinone-8 q8h2 Ubiquinol-8 ru5p-D D-Ribulose 5-phosphate s7p
Sedoheptulose 7-phosphate so4 Sulfate succ Succinate succoa
Succinyl-CoA thf 5,6,7,8-Tetrahydrofolate xu5p-D D-Xylulose
5-phosphate
[0175] A number of criteria were applied to select the most
practical sets of genes to target for removal. First, the designs
were limited to include only knockouts that would not significantly
(that is, >5%) reduce the maximum theoretical yield of BDO under
anaerobic conditions with or without the presence of nitrate as an
electron acceptor. Such knockouts would create an artificial
ceiling on any future metabolic engineering efforts and are thus
undesirable. To this end, a series of linear programming (LP)
problems were solved that maximized the BDO yield for the wild-type
E. coli metabolic network assuming every reaction was individually
deleted from the network. As used herein, reference to the
wild-type E. coli network assumes that the BDO pathway is
available. The term "wild-type" is thus a surrogate name for the
undeleted E. coli network. Reactions whose deletion negatively
affects the maximum BDO yield assuming PEP carboxykinase to be
irreversible or reversible are shown in Tables 10 and 11,
respectively. Table 10 shows reactions which, when deleted, reduce
the maximum theoretical BDO yield under anaerobic conditions with
or without the presence of nitrate, assuming that PEP carboxykinase
cannot be used to produce oxaloacetate. Strain AB3 contains
deletions in ADHE, LDH_D, and PFLi. `Inf` indicates that the
non-growth associated energetic requirements cannot be
satisfied.
TABLE-US-00010 TABLE 10 Reactions which, when deleted, reduce the
maximum theoretical BDO yield under anaerobic conditions with or
without the presence of nitrate, assuming that PEP carboxykinase
cannot be used to produce oxaloacetate. AB3 MDH AB3 MDH WT,
Anaerobic WT, Nitrate AB3, Anaerobic AB3, Nitrate ASPT, Anaerobic
ASPT, Nitrate MAXIMUMMASS YIELD 0.477 g/g 0.528 g/g 0.477 g/g 0.528
g/g 0.477 g/g 0.528 g/g % of % of % of % of % of % of BDO Max BDO
Max BDO Max BDO Max BDO Max BDO Max Abbreviation Reaction Name
Yield Yield Yield Yield Yield Yield Yield Yield Yield Yield Yield
Yield 4HBACT 4-hydroxybutyrate 0.00 0% 0.00 0% 0.00 0% 0.00 0% 0.00
0% 0.00 0% acetyl-CoA transferase 4HBDH 4-hydroxybutyrate 0.00 0%
0.00 0% 0.00 0% 0.00 0% 0.00 0% 0.00 0% dehydrogenase 4HBTAL
4-hydroxybutyraldehyde 0.00 0% 0.00 0% 0.00 0% 0.00 0% 0.00 0% 0.00
0% DDH dehydrogenase ACKr acetate kinase 0.44 93% 0.50 95% 0.44 93%
0.50 95% 0.44 93% 0.50 95% ACONT aconitase 0.41 86% 0.48 91% 0.41
86% 0.48 91% 0.32 67% 0.43 81% ACt6 acetate transport in/out 0.42
89% 0.53 100% 0.40 83% 0.53 100% 0.39 83% 0.53 100% via proton
symport ATPS4r ATP synthase (four 0.45 95% 0.45 86% 0.45 95% 0.45
86% 0.45 95% 0.45 86% protons for one ATP) BTDP2 1,4 butanediol
0.00 0% 0.00 0% 0.00 0% 0.00 0% 0.00 0% 0.00 0% dehydrogenase BTDt1
1,4-butanediol transport 0.00 0% 0.00 0% 0.00 0% 0.00 0% 0.00 0%
0.00 0% (Diffusion) CO2t CO2 transport out via 0.36 75% 0.36 68%
0.27 57% 0.27 52% 0.26 54% 0.26 49% diffusion CS citrate synthase
0.41 86% 0.48 91% 0.41 86% 0.48 91% 0.32 67% 0.43 81% ENO enolase
0.03 7% 0.46 88% Inf Inf 0.46 88% Inf Inf 0.45 85% FBA
fructose-bisphosphate 0.47 98% 0.53 100% 0.47 98% 0.53 100% 0.46
96% 0.52 99% aldolase FRD3 fumarate reductase 0.47 99% 0.53 100%
0.47 99% 0.53 100% 0.47 99% 0.53 100% FUM fumarase 0.48 100% 0.53
100% 0.48 100% 0.53 100% 0.40 84% 0.52 99% GAPD glyceraldehyde-3-
Inf Inf 0.44 82% Inf Inf 0.44 82% Inf Inf 0.42 79% phosphate
dehydrogenase (NAD) GLCpts D-glucose transport via 0.45 94% 0.52
99% 0.45 94% 0.52 99% 0.45 94% 0.52 99% PEP:Pyr PTS H2Ot5 H2O
tmnsport via 0.44 91% 0.47 88% 0.39 81% 0.39 73% 0.36 76% 0.37 70%
diffusion ICDHy isocitrate dehydrogenase 0.48 100% 0.53 100% 0.48
100% 0.53 100% 0.46 96% 0.51 97% (NADP) ICL Isocitrate lyase 0.48
100% 0.53 100% 0.48 100% 0.53 100% 0.37 78% 0.52 99% MALS malate
synthase 0.48 100% 0.53 100% 0.48 100% 0.53 100% 0.40 84% 0.52 99%
NADH6 NADH dehydrogenase 0.48 100% 0.53 100% 0.48 100% 0.53 100%
0.48 100% 0.53 100% (ubiquinone-8 & 3.5 protons) NADH8 NADH
dehydrogenase 0.47 99% 0.53 100% 0.47 99% 0.53 100% 0.47 99% 0.53
100% (demethylmenaquinone- 8 & 2.8 protons) NO3R1 Nitrate
reductase 0.48 100% 0.52 99% 0.48 100% 0.52 99% 0.48 100% 0.52 99%
(Ubiquinol-8) NO3t7 nitmte transport in via 0.48 100% 0.48 90% 0.48
100% 0.48 90% 0.48 100% 0.48 90% nitrite antiport PDH pyruvate
dehydrogenase 0.43 89% 0.51 97% 0.29 60% 0.48 91% 0.22 46% 0.47 90%
PFK phosphofructokinase 0.47 98% 0.53 100% 0.47 98% 0.53 100% 0.46
96% 0.52 99% PGI glucose-6-phosphate 0.43 90% 0.52 98% 0.43 90%
0.52 98% 0.24 51% 0.52 98% isomerase PGK phosphoglycerate kinase
Inf Inf 0.44 82% Inf Inf 0.44 82% Inf Inf 0.42 79% PGM
phosphoglycerate mutase 0.03 7% 0.46 88% Inf Inf 0.46 88% Inf Inf
0.45 85% PPC phosphoenolpyruvate 0.40 84% 0.52 98% 0.14 30% 0.52
98% 0.00 0% 0.00 0% carboxylase PTAr phosphotransacetylase 0.44 93%
0.50 95% 0.44 93% 0.50 95% 0.44 93% 0.50 95% SSALcoax CoA-dependant
0.40 83% 0.52 98% 0.31 64% 0.52 98% 0.19 39% 0.52 98% succinate
semialdehyde dehydrogenase TPI triose-phosphate 0.35 74% 0.50 95%
0.35 74% 0.50 95% 0.28 58% 0.49 93% isomerase
[0176] Table 11 shows reactions which, when deleted, reduce the
maximum theoretical BDO yield under anaerobic conditions with or
without the presence of nitrate, assuming that PEP carboxykinase
can be used to produce oxaloacetate. `Inf` indicates that the
non-growth associated energetic requirements cannot be
satisfied.
TABLE-US-00011 TABLE 11 Reactions which, when deleted, reduce the
maximum theoretical BDO yield under anaerobic conditions with or
without the presence of nitrate, assuming that PEP carboxykinase
can be used to produce oxaloacetate. AB3 MDH AB3 MDH WT, Anaerobic
WT, Nitrate AB3, Anaerobic AB3, Nitrate ASPT, Anaerobic ASPT,
Nitrate MAXIMUM MASS YIELD 0.545 g/g 0.545 g/g 0.545 g/g 0.545 g/g
0.545 g/g 0.545 g/g % of % of % of % of % of % of BDO Max BDO Max
BDO Max BDO Max BDO Max BDO Max Abbreviation Reaction Name Yield
Yield Yield Yield Yield Yield Yield Yield Yield Yield Yield Yield
4HBACT 4-hydroxybutyrate 0.00 0% 0.00 0% 0.00 0% 0.00 0% 0.00 0%
0.00 0% acetyl-CoA transferase 4HBDH 4-hydroxybutyrate 0.00 0% 0.00
0% 0.00 0% 0.00 0% 0.00 0% 0.00 0% dehydrogenase 4HBTAL
4-hydroxybutyralde- 0.00 0% 0.00 0% 0.00 0% 0.00 0% 0.00 0% 0.00 0%
DDH hyde dehydrogenase ACKr acetate kinase 0.50 92% 0.53 97% 0.50
92% 0.53 97% 0.48 89% 0.53 96% ACONT aconitase 0.47 86% 0.51 94%
0.47 86% 0.51 94% 0.36 66% 0.45 83% BTDP2 1,4 butanediol 0.00 0%
0.00 0% 0.00 0% 0.00 0% 0.00 0% 0.00 0% dehydrogenase BTDt1
1,4-butanediol 0.00 0% 0.00 0% 0.00 0% 0.00 0% 0.00 0% 0.00 0%
transport (Diffusion) CO2t CO2 transport out 0.36 67% 0.36 67% 0.33
61% 0.33 61% 0.29 54% 0.30 54% via diffusion CS citrate synthase
0.47 86% 0.51 94% 0.47 86% 0.51 94% 0.36 66% 0.45 83% ENO enolase
0.07 14% 0.46 85% Inf Inf 0.46 85% 0.00 0% 0.46 85% GAPD
glyceraldehyde-3- Inf Inf 0.44 80% Inf Inf 0.44 80% 0.00 0% 0.43
80% phosphate dehydrogenase (NAD) H2Ot5 H2O transport via 0.50 92%
0.50 92% 0.46 84% 0.46 84% 0.44 81% 0.44 81% diffusion ICDHy
isocitrate 0.50 92% 0.53 97% 0.50 92% 0.53 97% 0.50 92% 0.53 97%
dehydrogenase (NADP) PDH pyruvate 0.53 97% 0.54 99% 0.39 71% 0.51
94% 0.31 57% 0.50 92% dehydrogenase PGI glucose-6-phosphate 0.54
98% 0.54 100% 0.54 98% 0.54 100% 0.40 73% 0.53 98% isomerase PGK
phosphoglycerate Inf Inf 0.44 80% Inf Inf 0.44 80% 0.00 0% 0.43 80%
kinase PGM phosphoglycerate 0.07 14% 0.46 85% Inf Inf 0.46 85% 0.00
0% 0.46 85% mutase PPCK Phosphoenol- 0.48 88% 0.53 97% 0.48 88%
0.53 97% 0.48 88% 0.53 97% pyruvate carboxykinase PTAr
Phosphotrans- 0.50 92% 0.53 97% 0.50 92% 0.53 97% 0.48 89% 0.53 96%
acetylase SSALcoax CoA-dependant 0.52 95% 0.54 98% 0.48 89% 0.54
98% 0.46 84% 0.54 98% succinate semialdehyde dehydrogenase TPI
triose-phosphate 0.44 81% 0.52 96% 0.44 81% 0.52 96% 0.40 73% 0.52
95% isomerase
[0177] The above-described analysis led to three critical
observations. One critical observation was that acetate kinase and
phosphotransacetylase are required to achieve the maximum BDO
yields under all conditions by regenerating acetyl-CoA from the
acetate produced by 4-hydroxybutyrate:acetyl-CoA transferase. This
finding strongly suggests that eliminating acetate formation by
deleting ackA-pta may not be a viable option. Thus a successful
strain will likely have to provide an intracellular environment
where converting acetate to acetyl-CoA is beneficial and
thermodynamically feasible. Otherwise, a set of enzymes capable of
performing the required BDO reductions without passing through a
CoA derivative would have to be found or the co-production of 1 mol
of acetate per mol of BDO would have to be accepted.
[0178] A second critical observation was that the TCA cycle enzymes
citrate synthase (CS), aconitase (ACONT), and isocitrate
dehydrogenase (ICDHy) are required to achieve the maximum BDO
yields under all conditions. This indicates that the reverse TCA
cycle flux from oxaloacetate to succinate to succinyl-CoA must be
complemented to some extent by CS, ACONT, and ICDHy for maximum
production.
[0179] A third critical observation was that supplanting PEP
carboxylase with PEP carboxykinase in E. coli can positively impact
the BDO program. The maximum BDO yield under anaerobic conditions
with and without the presence of nitrate is 3% and 12% lower,
respectively, if PEP carboxylase carries out the PEP to
oxaloacetate conversion as compared to if PEP carboxykinase carries
out the conversion. Furthermore, under anaerobic conditions without
nitrate addition, PEP carboxykinase can lessen the requirement for
pyruvate dehydrogenase activity for maximum BDO production.
Specifically, the maximum BDO yield drops 11% if this typically
aerobic enzyme has no activity if PEP carboxykinase is assumed
irreversible as compared to a 3% reduction if PEP carboxykinase can
catalyze the production of oxaloacetate. An alternative to pyruvate
dehydrogenase activity can be utilized by coupling non-native
formate dehydrogenase, capable of catalyzing the reduction of
formate to carbon dioxide, to pyruvate formate lyase activity.
[0180] The next two criteria applied to evaluate the OptKnock
designs were the number of required knockouts and the predicted BDO
yield at maximum growth. Analysis of the one, two, and three
reaction deletion strategies in Table 6 (that is, PEP carboxykinase
assumed irreversible) revealed that so few knockouts were
insufficient to prevent high acetate yields. The predicted
acetate/BDO ratios for all one, two, or three deletion designs, was
at least 1.5. Even allowing for four deletions led to only two
designs, #99 and #100, with predicted BDO yields above 0.35 g/g,
and those designs suggest the removal of the glycolysis gene, pgi,
which encodes phosphoglucoisomerase. Given the anticipated
importance of glycolysis in the fermentation of E. coli, it was
decided to pursue such high-risk designs only as a last resort.
Furthermore, the suggested deletions lower the maximum theoretical
yield in designs #99 and #100 by 8% and 15%, respectively. The
highest producing four deletion strategy that did not negatively
impact the maximum theoretical yield was design #129, which had a
predicted BDO yield at maximum growth of only 0.26 g/g.
[0181] Only one five deletion design in Table 6 satisfies all
criteria for a successful design. This knockout strategy involves
the removal of ADHEr (alcohol dehydrogenase), PFLi (pyruvate
formate lyase), LDH_D (lactate dehydrogenase), MDH (malate
dehydrogenase), and ASPT (aspartate transaminase). The suggested
knockouts do not reduce the maximum theoretical BDO yield under any
of the conditions examined. A strain engineered with these
knockouts is predicted to achieve a BDO yield at maximum growth of
0.37 g/g assuming anaerobic conditions and PEP carboxykinase
irreversibility. This design has several desirable properties. Most
notably, it prevents the network from producing high yields of the
natural fermentation products, ethanol, formate, lactate, and
succinate. The prevention of homosuccinate production via the MDH
deletion as opposed to removing PEP carboxylase, fumarase, or
fumarate reductase, is particularly intriguing because it blocks
the energy-yielding fermentation pathway from oxaloacetate to
succinate that could arise if PEP carboxykinase is assumed
reversible without negatively impacting the maximum BDO yield. In
this design, succinate semialdehyde can be made via succinyl-CoA or
alpha-ketoglutarate. The succinyl-CoA is formed from succinate via
succinyl-CoA synthetase. The succinate can be formed from both the
reverse TCA cycle reactions (PEP carboxylase, PEP carboxykinase,
fumarase, fumarate reductase) and the glyoxylate shunt (malate
synthase, isocitrate lyase).
[0182] The BDO versus biomass solution boundaries for the ADHEr,
PFLi, LDH_D, MDH, ASPT knockout strategy are shown in FIGS. 7A and
7B, assuming PEP carboxykinase irreversibility or reversibility,
respectively. Note that the solution boundaries are obtained using
a genome-scale model of E. coli metabolism as opposed to the
reduced model because these calculations are not CPU-intensive. The
solution boundaries reveal that the growth coupling of BDO is
robust with respect to the assumption of PEP carboxykinase
reversibility. However, the deletion of the proton-pumping
transhydrogenase (THD2) or glutamate dehydrogenase (GLUDy) may be
necessary to achieve an obligatory coupling of cell growth with BDO
production. The only negative aspect of the design is that the MDH
and ASPT deletions drop the maximum ATP yield of BDO production
slightly (.about.20%) if PEP carboxykinase reversibility is
assumed. This causes the optimal growth solution of the design
strategy to drop below the black BDO vs. biomass line of the
wild-type network in FIG. 7B. However, this finding suggests the
possibility of engineering an optimal balance of MDH activity where
enough is present to ensure efficient BDO production while also
being limited enough to prevent succinate from becoming the major
fermentation product. Note that succinate is predicted to be the
major fermentation product of the wild-type network if PEP
carboxykinase reversibility is assumed.
[0183] Tables 10 and 11 list reactions whose deletion negatively
impacts the maximum BDO yield in an intermediate strain, referred
to as AB3, which lacks ADHEr, LDH_D, and PFLi as well as a strain
lacking ASPT and MDH in addition to the AB3 deletions. Note that
the suggested deletions place a very high importance on obtaining
pyruvate dehydrogenase, citrate synthase, and aconitase activity
under completely anaerobic conditions. If sufficient pyruvate
dehydrogenase activity cannot be attained, an alternative is to
leave PFLi intact and supplement its activity with a non-native
formate dehydrogenase that can capture one reducing equivalent
while converting formate to carbon dioxide.
[0184] FIG. 8 and Table 12 depict the flux ranges that the E. coli
network can attain while reaching either the maximum BDO yield
(cases 1-4) or the maximum biomass yield (case 5) under anaerobic
conditions. Cases 1 and 2 assume that no gene deletions have taken
place. Case 2 exhibits tighter flux ranges than case 1 due to the
fact that an additional constraint enforcing the maximum ATP yield
at the maximum BDO yield is imposed. Cases 3 and 4 are analogous to
cases 1 and 2 except that the fluxes encoding the reactions for
ADHEr, ASPT, LDH_D, MDH, and PFLi have been set to zero. The flux
ranges assuming biomass yield maximization in the presence of the
ADHEr, ASPT, LDH_D, MDH, and PFLi knockouts are shown in case
5.
[0185] Table 12 shows achievable ranges of central metabolic fluxes
under anaerobic conditions assuming PEP carboxykinase to be
reversible. Bold flux values were set as constraints on the system.
Five cases are considered: Case 1, maximum BDO yield of the
wild-type network; Case 2, maximum ATP yield assuming the maximum
BDO yield of the wild-type network; Case3, maximum BDO yield of the
network with fluxes through ADHEr, ASPT, LDH_D, MDH, and PFLi set
to zero; Case 4, maximum ATP yield assuming the maximum BDO yield
of the network with fluxes through ADHEr, ASPT, LDH_D, MDH, and
PFLi set to zero; and Case 5, maximum biomass yield of the network
with fluxes through ADHEr, ASPT, LDH_D, MDH, and PFLi set to
zero.
TABLE-US-00012 TABLE 12 Achievable ranges of central metabolic
fluxes under anaerobic conditions assuming PEP carboxykinase to be
reversible. Reactions that are assumed inactive in cases 3, 4, and
5 are indicated with bold font. Reaction CASE 1 CASE 2 CASE 3 CASE
4 CASE 5 Abbreviation MIN MAX MIN MAX MIN MAX MIN MAX MIN MAX
mmol/gDW/hr GLCpts 0.0 20.0 18.2 18.2 0.0 20.0 20.0 20.0 20.0 20.0
HEX1 0.0 20.0 1.8 1.8 0.0 20.0 0.0 0.0 0.0 0.0 G6PDHy 0.0 27.3 0.0
0.0 0.0 15.3 0.0 0.0 0.0 0.0 PGL 0.0 27.3 0.0 0.0 0.0 15.3 0.0 0.0
0.0 0.0 PGDH 0.0 27.3 0.0 0.0 0.0 15.3 0.0 0.0 0.0 0.0 RPE -6.4
18.2 0.0 0.0 -6.2 10.2 0.0 0.0 -0.2 -0.2 RPI -9.1 0.0 0.0 0.0 -6.2
0.0 0.0 0.0 -0.2 -0.2 TKT1 -3.2 9.1 0.0 0.0 -3.1 5.1 0.0 0.0 -0.1
-0.1 TKT2 -3.2 9.1 0.0 0.0 -3.1 5.1 0.0 0.0 -0.2 -0.2 TAL -3.2 9.1
0.0 0.0 -3.1 5.1 0.0 0.0 -0.1 -0.1 EDA 0.0 12.7 0.0 0.0 0.0 10.5
0.0 0.0 0.0 0.0 PGDHY 0.0 12.7 0.0 0.0 0.0 10.5 0.0 0.0 0.0 0.0 PGI
-7.3 20.0 20.0 20.0 4.7 20.0 20.0 20.0 20.0 20.0 FBP 0.0 15.9 0.0
0.0 0.0 12.7 0.0 0.0 0.0 0.0 PFK 7.3 35.9 20.0 20.0 9.5 32.7 20.0
20.0 19.7 19.7 FBA 7.3 20.0 20.0 20.0 9.5 20.0 20.0 20.0 19.7 19.7
TPI 7.3 20.0 20.0 20.0 9.5 20.0 20.0 20.0 19.7 19.7 GAPD 27.3 40.0
40.0 40.0 29.5 40.0 40.0 40.0 39.3 39.3 PGK 27.3 40.0 40.0 40.0
29.5 40.0 40.0 40.0 39.3 39.3 PGM 27.3 43.6 40.0 40.0 29.5 43.6
40.0 40.0 39.0 39.0 ENO 27.3 43.6 40.0 40.0 29.5 43.6 40.0 40.0
39.0 39.0 PYK 0.0 29.5 0.0 0.0 0.0 29.5 1.8 1.8 10.7 10.7 PDH 0.0
34.5 14.5 18.2 12.0 34.5 21.8 21.8 30.1 30.1 PFLi 0.0 11.9 0.0 3.6
0.0 0.0 0.0 0.0 0.0 0.0 PPC 0.0 15.9 0.0 0.0 0.0 12.7 0.0 0.0 0.0
0.0 PPCK 5.5 51.4 21.8 21.8 5.5 30.9 18.2 18.2 8.1 8.1 CS 9.1 34.1
18.2 18.2 13.1 32.7 18.2 18.2 7.5 7.5 CITL 0.0 15.9 0.0 0.0 0.0
12.7 0.0 0.0 0.0 0.0 ACONT 9.1 21.8 18.2 18.2 13.1 21.8 18.2 18.2
7.5 7.5 ICDHy 1.8 21.4 18.2 18.2 1.8 21.3 14.5 14.5 0.2 0.2 AKGD
0.0 21.4 0.0 18.2 0.0 21.3 0.0 14.5 0.0 0.0 SUCOAS 0.5 20.0 3.6 3.6
0.6 20.0 7.3 7.3 14.9 14.9 FRD 0.0 12.7 3.6 3.6 0.0 8.7 3.6 3.6 7.5
7.5 FUM -55.5 12.7 -14.5 3.6 -5.1 8.7 3.6 3.6 7.2 7.2 MDH -50.9
33.2 -14.5 3.6 0.0 0.0 0.0 0.0 0.0 0.0 ICL 0.0 18.2 0.0 0.0 0.0
18.2 3.6 3.6 7.2 7.2 MALS 0.0 16.4 0.0 0.0 0.0 16.4 3.6 3.6 7.2 7.2
ME 0.0 29.5 0.0 0.0 0.0 12.7 0.0 0.0 0.0 0.0 ASPTA 0.0 66.7 0.0
18.2 0.0 7.5 0.0 0.0 0.6 0.6 ASPT 0.0 66.7 0.0 18.2 0.0 0.0 0.0 0.0
0.0 0.0 LDH_D 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 ADHEr 0.0 0.0
0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 PTAr 5.9 37.7 21.8 21.8 9.1 34.5
21.8 21.8 0.1 0.1 ACKr 5.9 37.7 21.8 21.8 9.1 34.5 21.8 21.8 0.1
0.1 GLUDc 0.0 21.4 0.0 18.2 0.0 21.3 0.0 14.5 0.0 0.0 ABTA 0.0 21.4
0.0 18.2 0.0 21.2 0.0 14.5 0.0 0.0 SSAL_coa 0.5 37.7 3.6 21.8 0.6
34.5 7.3 21.8 14.8 14.8 ATPM 0.0 15.9 15.9 15.9 0.0 12.7 12.7 12.7
7.6 7.6 BDOsyn 21.8 21.8 21.8 21.8 21.8 21.8 21.8 21.8 14.8 14.8
1/hr BIOMASS 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.21 0.21
[0186] The first two four deletion designs (#92 and #93) listed in
Table 7 (that is, PEP carboxykinase assumed reversible) were
considered next due to their relatively high predicted BDO yields.
Design #92 (ADHEr, HEX1, PFLi, and PGI) would need additional
knockouts that eliminate succinate and lactate production and the
maximum BDO yield of design #93 (ADHEr, EDA, NADH6, PGI) was only
90% of the theoretical maximum of the wild-type network. Both
designs called for the removal of PGI which, as mentioned above,
was ruled undesirable due to the anticipated importance of
glycolysis on fermentation. Design #98 (ADHEr, ATPS4r, FDH2, NADH6)
was the first four deletion design to require the removal of ATP
synthase without additionally requiring the PGI knockout. However,
this design would also require the deletion of genes to prevent
lactate and succinate production in order to raise the predicted
BDO yield at maximum growth. Upon further analysis, it was found
that nearly all promising designs in Table 7 could be improved by
ensuring that the deletions (i.e, ADHEr, LDH_D, MDH, ASPT, and
PFLi) were also implemented if not already specified.
[0187] Efforts were next focused on identifying reaction deletions
that could supplement the core set (that is, ADHEr, LDH_D, MDH,
ASPT, PFLi). Table 13 shows known E. coli genes responsible for
catalyzing the reactions targeted for removal. The designation
"[c]" refers to cytosolic. The genes encoding these reactions along
with the reactions of the core set are provided in Table 13. The
effect of the additional deletions on the BDO versus biomass
solution boundaries is shown in FIG. 9. Notably, the coupling
between BDO and biomass production becomes more and more pronounced
as additional deletions are accumulated. The predicted BDO yield at
maximum growth after accumulating all deletions is 0.46 g/g.
Lastly, none of the deletions negatively impact the maximum
theoretical BDO yield.
TABLE-US-00013 TABLE 13 Known E. coli genes responsible for
catalyzing the reactions targeted for removal. Genes Encoding the
Enzyme(s) Reaction Catalyzing Each Abbreviation Reaction
Stoichiometry Reaction& ADHEr [c]: etoh + nad <==> acald
+ h + nadh (b0356 or b1478 or [c]: acald + coa + nad <==>
accoa + h + nadh b1241) (b1241 or b0351) PFLi [c]: coa + pyr -->
accoa + for (((b0902 and b0903) and b2579) or (b0902 and b0903) or
(b0902 and b3114) or (b3951 and b3952)) MDH [c]: mal-L + nad
<==> h + nadh + oaa b3236 ASPT [c]: asp-L --> fum + nh4
b4139 LDH_D [c]: lac-D + nad <==> h + nadh + pyr (b2133 or
b1380) DHAPT [c]: dha + pep --> dhap + pyr (b1200 and b1199 and
b1198 and b2415 and b2416) DRPA [c]: 2dr5p --> acald + g3p b4381
PYK [c]: adp + h + pep --> atp + pyr (b1854 or b1676) EDD [c]:
6pgc --> 2ddg6p + h2o b1851 GLYCLTDx [c]: glx + h + nadh -->
glyclt + nad (b3553 or b1033) GLYCLTDy [c]: glx + h + nadph -->
glyclt + nadp MCITS [c]: h2o + oaa + ppcoa --> 2mcit + coa + h
b0333 INSK [c]: atp + ins --> adp + h + imp b0477
[0188] In the results shown in Table 13, OptKnock identifies
reactions to be eliminated from an organism to enhance biochemical
production. Any combination (that is, at least one and at most all)
of the listed gene deletions could conceivably have the desired
effect of ensuring that the corresponding reaction is
non-functional in E. coli. The most practical experimental strategy
for eliminating the reactions targeted for removal must be
determined on a case-by-case basis.
Example VI
Generation of Engineered Strains
[0189] In order to validate the computational predictions of
Example V, the strains are constructed, evolved, and tested.
Escherichia coli K-12 MG1655 serves as the wild-type strain into
which the deletions are introduced. The strains are constructed by
incorporating in-frame deletions using homologous recombination via
the X Red recombinase system of (Datsenko and Wanner, Proc. Natl.
Acad. Sci. USA 97:6640-6645 (2000)). The approach involves
replacing a chromosomal sequence (that is, the gene targeted for
removal) with a selectable antibiotic resistance gene, which itself
is later removed. The knockouts are integrated one by one into the
recipient strain. No drug resistance markers or scars will remain
after each deletion, allowing accumulation of multiple mutations in
each target strain. The deletion technology completely removes the
gene targeted for removal so as to substantially reduce the
possibility of the constructed mutants reverting back to the
wild-type. During the initial stages of strain development,
non-native genes enabling BDO production are expressed in a
synthetic operon behind an inducible promoter on a medium- or
high-copy plasmid; for example the PBAD promoter which is induced
by arabinose, on a plasmid of the pBAD series (Guzman et al., J.
Bacteriol. 177:4121-4130 (1995)). This promoter is known to be very
easily titratable, allowing expression to be fine tuned over a
1000-fold range of arabinose concentrations. If BDO production is
successful, these genes are then be integrated into the chromosome
to promote stability.
[0190] The engineered strains are characterized by measuring growth
rate, substrate uptake rate, and product/byproduct secretion rate.
These strains are initially anticipated to exhibit suboptimal
growth rates until their metabolic networks have adjusted to their
missing functionalities. To enable this adjustment, the strains are
adaptively evolved. By subjecting the strains to adaptive
evolution, cellular growth rate becomes the primary selection
pressure and the mutant cells are compelled to reallocate their
metabolic fluxes in order to enhance their rates of growth. This
reprogramming of metabolism has been recently demonstrated for
several E. coli mutants that had been adaptively evolved on various
substrates to reach the growth rates predicted a priori by an in
silico model (Fong and Palsson, Nat. Genet. 36:1056-1058 (2004)).
Should the OptKnock predictions prove successful, the growth
improvements brought about by adaptive evolution are accompanied by
enhanced rates of BDO production. Adaptive evolution is performed
in triplicate (running in parallel) due to differences in the
evolutionary patterns witnessed previously in E. coli (Fong and
Palsson, supra, 2004; Fong et al., J. Bacteriol. 185:6400-6408
(2003); Ibarra et al., Nature 420:186-189 (2002)) that could
potentially result in one strain having superior production
qualities over the others. Evolutions iw run for a period of 2-6
weeks, depending on the rate of growth improvement obtained. In
general, evolutions q43 stopped once a stable growth phenotype is
obtained.
[0191] Following the adaptive evolution process, the new strains
are again characterized by measuring growth rate, substrate uptake
rate, and product/byproduct secretion rate. These results are
compared to the OptKnock predictions by plotting actual growth and
production yields along side the production described above. The
most successful OptKnock design/evolution combinations are chosen
to pursue further, and are characterized in lab-scale batch and
continuous fermentations. The growth-coupled biochemical production
concept behind the OptKnock approach salso results in the
generation of genetically stable overproducers. Thus, the cultures
are maintained in continuous mode for one month to evaluate
long-term stability. Periodic samples are taken to ensure that
yield and productivity are maintained throughout the process.
Example VII
OptKnock Strains for Production of BDO
[0192] As described in Examples V and VI, the application of the
OptKnock methodology has been applied for generating promising
deletion targets to generate BDO producing strains. OptKnock
identifies reactions to be eliminated from an organism to couple
the biochemical production and biomass yields. The designs provide
a list of the metabolic reactions to be targeted for removal by
OptKnock. The E. coli genes known to encode the enzymes that
catalyze each reaction were also provided to describe which genetic
modifications must be implemented to realize the predicted
growth-coupled production phenotypes. Obviously, if new discoveries
reveal that additional genes in the E. coli genome can confer one
or more of the reaction functionalities targeted for removal in a
given design, then these genes should be removed as well as the
ones described herein. Note that preventing the activity of only a
subset (that is, at least one and at most all) of the reactions in
each of the designs may sometimes be sufficient to confer a
growth-coupled producing phenotype. For example, if a design calls
for the removal of a particular reaction whose activity in vivo is
not sufficient to uncouple growth from BDO production, then the
genes encoding the enzymes that catalyze this reaction can be left
intact. In addition, any combination (that is, at least one and at
most all) of the listed gene deletions for a given reaction could
conceivably have the desired effect of ensuring that the reaction
is non-functional in E. coli.
[0193] Multiple deletion strategies are listed in Table 6 and 7 for
enhancing the coupling between 1,4-butanediol production and E.
coli growth assuming PEP carboxykinase to be irreversible and
reversible, respectively. One design (that is, ADHEr, ASPT, MDH,
LDH_D, PFLi) emerged as the most promising upon satisfying multiple
criteria. The suggested deletions 1) led to a high predicted BDO
yield at maximum growth, 2) required a reasonable number of
knockouts, 3) had no detrimental effect on the maximum theoretical
BDO yield, 4) brought about a tight coupling of BDO production with
cell growth, and 5) was robust with respect to the
irreversibility/reversibility of PEP carboxykinase. The following
list specifies the minimal set of required gene deletions predicted
to render BDO the major fermentation product of E. coli:
[0194] adhE (b1421), ldhA (b1380).
[0195] pflAB (b0902, b0903) is not included in the minimal set
because its deletion forces a reliance on pyruvate dehyrogenase to
provide sufficient acetyl-CoA for cell growth and one reducing
equivalent from pyruvate. As pyruvate dehydrogenase activity is low
under anaerobic conditions and inhibited by high NADH
concentrations, a plausible alternative to the pflAB deletion is to
add a non-native formate dehydrogenase to E. coli that can capture
the reducing power that is otherwise lost via formate secretion.
Nevertheless, adding pflAB to the minimal deletion set yields:
[0196] adhE (b1421), ldhA (b1380), pflAB (b0902, b0903).
[0197] mdh (b3236) is not included in the minimal set because there
are multiple deletions capable of preventing succinate from
becoming the major fermentation product of E. coli as opposed to
BDO. Examples include the genes encoding fumarase and/or fumarate
reductase. However, eliminating malate dehydrogenase appears to be
the most logical choice to attenuate succinate production as it
leaves intact a pathway for the conversion of the glyoxylate shunt
product, malate, to BDO. Adding the malate dehydrogenase deletion
to the minimal set above yields:
[0198] adhE (b1421), ldhA (b1380), pflAB (b0902, b0903), mdh
(b3236).
[0199] The gene, mqo, which encodes a
malate:quinone-oxidoreductase, is believed to catalyze the
oxidation of malate to oxaloactate (van der Rest et al., J.
Bacteriol. 182:6892-6899 (2000)). However, if it is shown to also
catalyze the formation of malate from oxaloacetate, its removal
will be necessary to ensure that it does not circumvent the mdh
deletion. This leads to the deletion set:
[0200] adhE (b1421), ldhA (b1380), pflAB (b0902, b0903), mdh
(b3236), mqo (b2210).
[0201] aspA is left out of the minimal set as it is questionable
whether or not aspartate deaminase can carry enough flux to
circumvent the malate dehydrogenase deletion. However, if this
scenario is indeed possible, then the minimal list of required
deletions becomes:
[0202] adhE (b1421), ldhA (b1380), pflAB (b0902, b0903), mdh
(b3236), aspA (b4139).
[0203] For the calculations above, NADH and NADPH-dependent malic
enzymes of E. coli were assumed to operate irreversibly catalyzing
only the conversion of malate to carbon dioxide and pyruvate. If
these enzymes can also catalyze the formation of malate from
pyruvate and carbon dioxide, the genes encoding one or both malic
enzymes will have to be removed to prevent succinate from becoming
the major fermentation product. This leads to the following sets of
deletions:
[0204] adhE (b1421), mdh (b3236), ldhA (b1380), pflAB (b0902,
b0903), sfcA (b1479)
[0205] adhE (b1421), mdh (b3236), ldhA (b1380), pflAB (b0902,
b0903), maeB (b2463)
[0206] adhE (b1421), mdh (b3236), ldhA (b1380), pflAB (b0902,
b0903), sfcA (b1479), maeB (b2463)
[0207] The minimal set of deletions can be supplemented with
additional deletions aimed at tightening the coupling of BDO
production to cell growth. These sets of the deletions are listed
below.
[0208] adhE (b1421), ldhA (b1380), pflAB (b0902, b0903), mdh
(b3236), pntAB (b1602, b1603)
[0209] adhE (b1421), ldhA (b1380), pflAB (b0902, b0903), mdh
(b3236), gdhA (b1761)
[0210] adhE (b1421), ldhA (b1380), pflAB (b0902, b0903), mdh
(b3236), pykA (b1854), pykF (b1676), dhaKLM (b1198, b1199, b1200),
deoC (b4381), edd (b1851), yiaE (b3553), ycdW(b1033)
[0211] adhE (b1421), ldhA (b1380), pflAB (b0902, b0903), mdh
(b3236), pykA (b1854), pykF (b1676), dhaKLM (b1198, b1199, b1200),
deoC (b4381), edd (b1851), yiaE (b3553), ycdW (b1033), prpC
(b0333), gsk (b0477)
[0212] Strains possessing the deletions listed in this Example can
be supplemented with additional deletions if it is found that the
suggested deletions do not reduce the activity of their
corresponding reactions to the extent required to attain growth
coupled BDO production or if native E. coli genes, through adaptive
evolution or mutagenesis, attain mutations conferring activities
capable of circumventing the proposed design strategies.
[0213] Throughout this application various publications have been
referenced within parentheses. The disclosures of these
publications in their entireties are hereby incorporated by
reference in this application in order to more fully describe the
state of the art to which this invention pertains. Although the
invention has been described with reference to the disclosed
embodiments, those skilled in the art will readily appreciate that
the specific examples and studies detailed above are only
illustrative of the invention. It should be understood that various
modifications can be made without departing from the spirit of the
invention. Accordingly, the invention is limited only by the
following claims.
* * * * *