U.S. patent application number 15/141002 was filed with the patent office on 2017-01-26 for microorganisms for the production of aniline.
The applicant listed for this patent is Genomatica, Inc.. Invention is credited to Priti Pharkya.
Application Number | 20170022508 15/141002 |
Document ID | / |
Family ID | 43898755 |
Filed Date | 2017-01-26 |
United States Patent
Application |
20170022508 |
Kind Code |
A1 |
Pharkya; Priti |
January 26, 2017 |
MICROORGANISMS FOR THE PRODUCTION OF ANILINE
Abstract
A non-naturally occurring microbial organism having an aniline
pathway includes at least one exogenous nucleic acid encoding an
aniline pathway enzyme expressed in a sufficient amount to produce
aniline. The aniline pathway includes (1) an aminodeoxychorismate
synthase, an aminodeoxychorismate lyase, and a 4-aminobenzoate
carboxylyase or (2) an anthranilate synthase and an anthranilate
decarboxylase. A method for producing aniline, includes culturing
these non-naturally occurring microbial organisms under conditions
and for a sufficient period of time to produce aniline.
Inventors: |
Pharkya; Priti; (San Diego,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genomatica, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
43898755 |
Appl. No.: |
15/141002 |
Filed: |
April 28, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14664033 |
Mar 20, 2015 |
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15141002 |
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12910671 |
Oct 22, 2010 |
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14664033 |
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61254630 |
Oct 23, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/1096 20130101;
C12Y 206/01085 20130101; C12Y 401/01024 20130101; C12Y 401/03038
20130101; C12N 15/52 20130101; C12N 15/70 20130101; C12P 13/001
20130101; C12N 9/88 20130101 |
International
Class: |
C12N 15/52 20060101
C12N015/52; C12P 13/00 20060101 C12P013/00; C12N 9/88 20060101
C12N009/88; C12N 15/70 20060101 C12N015/70; C12N 9/10 20060101
C12N009/10 |
Claims
1-46. (canceled)
47. A non-naturally occurring Escherichia coli, comprising an
aniline pathway, said aniline pathway comprising an anthranilate
synthase, a 3-dehydroquinate synthase, a 3-dehydroquinate
dehydratase, a shikimate dehydrogenase or a quinate/shikimate
dehydrogenase, a shikimate kinase, a
3-phosphoshikimate-1-carboxyvinyltransferase, a chorismate synthase
and an anthranilate decarboxylase, wherein said non-naturally
occurring Escherichia coli, comprises at least two exogenous
nucleic acids, the two exogenous nucleic acids encoding enzymes
selected from the group consisting of the 3-dehydroquinate
synthase, the anthranilate synthase, the 3-dehydroquinate
dehydratase, the shikimate dehydrogenase or the quinate/shikimate
dehydrogenase, the shikimate kinase, the
3-phosphoshikimate-1-carboxyvinyltransferase, and the chorismate
synthase, wherein an endogenous nucleic acid encodes the
anthranilate decarboxylase, and wherein the aniline pathway enzymes
are expressed in a sufficient amount to produce aniline.
48. The non-naturally occurring Escherichia coli of claim 47,
wherein the aniline pathway further comprises a
3-deoxy-D-arabino-heptulosonic acid-7-phosphate (DAHP)
synthase.
49. The non-naturally occurring Escherichia coli of claim 47,
wherein said microbial organism comprises three exogenous nucleic
acids each encoding an aniline pathway enzyme.
50. The non-naturally occurring Escherichia coli of claim 49,
wherein said three exogenous nucleic acids encode the DAHP
synthase, the anthranilate synthase and the 3-dehydroquinate
synthase.
51. The non-naturally occurring Escherichia coli of claim 47,
wherein said microbial organism comprises four exogenous nucleic
acids each encoding an aniline pathway enzyme.
52. The non-naturally occurring Escherichia coli of claim 51
wherein said four exogenous nucleic acids encode the
3-dehydroquinate synthase, the DAHP synthase, the anthranilate
synthase and the chorismate synthase.
53. The non-naturally occurring Escherichia coli of claim 47,
wherein said at least one exogenous nucleic acid is a heterologous
nucleic acid.
54. An anaerobic culture medium comprising the microbial organism
of claim 47.
55. The non-naturally occurring Escherichia coli of claim 47,
wherein said microbial organism comprises five exogenous nucleic
acids each encoding an aniline pathway enzyme.
56. The non-naturally occurring Escherichia coli of claim 55,
wherein said five exogenous nucleic acids encoding the anthranilate
synthase, the DAHP synthase, the 3-dehydroquinate synthase, the
3-dehydroquinate dehydratase, the shikimate dehydrogenase or the
quinate/shikimate dehydrogenase.
57. The non-naturally occurring Escherichia coli of claim 47,
wherein said microbial organism comprises six exogenous nucleic
acids each encoding an aniline pathway enzyme.
58. The non-naturally occurring Escherichia coli of claim 57,
wherein said six exogenous nucleic acids encoding the anthranilate
synthase, the DAHP synthase, the 3-dehydroquinate synthase, the
3-dehydroquinate dehydratase, the shikimate dehydrogenase or the
quinate/shikimate dehydrogenase and a shikimate kinase.
59. The non-naturally occurring Escherichia coli of claim 47,
wherein said microbial organism comprises seven exogenous nucleic
acids each encoding an aniline pathway enzyme.
60. The non-naturally occurring Escherichia coli of claim 59,
wherein said seven exogenous nucleic acids encoding the
anthranilate synthase, the DAHP synthase, the 3-dehydroquinate
synthase, the 3-dehydroquinate dehydratase, the shikimate
dehydrogenase or the quinate/shikimate dehydrogenase, the shikimate
kinase, the 3-phosphoshikimate-1carboxyvinyltransferase.
61. The non-naturally occurring Escherichia coli of claim 47,
wherein said microbial organism comprises eight exogenous nucleic
acids each encoding an aniline pathway enzyme.
62. The non-naturally occurring Escherichia coli of claim 61,
wherein said eight exogenous nucleic acids encoding the
anthranilate synthase, the DAHP synthase, the 3-dehydroquinate
synthase, the 3-dehydroquinate dehydratase, the shikimate
dehydrogenase or the quinate/shikimate dehydrogenase the shikimate
kinase, the 3-phosphoshikimate-1-carboxyvinyltransferase, and the
chorismate synthase.
Description
[0001] This application claims the benefit of priority of U.S.
Provisional Application No. 61/254,630, filed Oct. 23, 2009, the
entire contents of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] This invention relates generally to in silica design of
organisms and engineering of organisms, more particularly to
organisms having aniline biosynthesis capability.
[0003] Aniline is an organic compound with the formula
C.sub.6H.sub.7N and is a precursor to numerous complex chemicals.
Aniline is usually produced industrially in two steps from benzene.
First, benzene is nitrated using a concentrated mixture of nitric
acid and sulfuric acid at 50 to 60.degree. C., to provide
nitrobenzene. In the second step, nitrobenzene is hydrogenated,
typically at 600.degree. C. in presence of a nickel catalyst to
give aniline. In an alternative process, aniline is prepared from
phenol and ammonia as described in U.S. Pat. No. 3,965,182. The
phenol, in tum, is derived from the cumene process.
[0004] The main application of aniline is in the manufacture of
polyurethane. Aniline also has value in the production of
dyestuffs. In addition to its use as a precursor to dyestuffs, it
is a starting-product for the manufacture of many drugs, such as
paracetamol (acetaminophen, Tylenol). Currently, the largest market
for aniline is preparation of methylene diphenyl diisocyanate
(MDI), some 85% of aniline serving this market. Other uses include
rubber processing chemicals (9%), herbicides (2%), and dyes and
pigments (2%).
[0005] When polymerized, aniline can be used as a type of nanowire
for use as a semiconducting electrode bridge in, for example,
nano-scale devices such as biosensors. These polyaniline nanowires
can be doped in order to achieve certain semiconducting
properties.
[0006] It is desirable to develop a method for production of
aniline by alternative means that substitute renewable for
petroleum-based feedstocks, while also using less energy- and
capital-intensive processes. The present invention satisfies this
need and provides related advantages as well.
SUMMARY OF THE INVENTION
[0007] In some aspects, embodiments disclosed herein relate to a
non-naturally occurring microbial organism having an aniline
pathway that includes at least one exogenous nucleic acid encoding
an aniline pathway enzyme expressed in a sufficient amount to
produce aniline. The aniline pathway includes an
aminodeoxychorismate synthase, an aminodeoxychorismate lyase, and a
4-aminobenzoate carboxylyase.
[0008] In some aspects, embodiments disclosed herein relate to a
method for producing aniline, that includes culturing a
non-naturally occurring microbial organism having an aniline
pathway that includes at least one exogenous nucleic acid encoding
an aniline pathway enzyme expressed in a sufficient amount to
produce aniline, under conditions and for a sufficient period of
time to produce aniline. The aniline pathway includes an
aminodeoxychorismate synthase, an aminodeoxychorismate lyase, and a
4-aminobenzoate carboxylyase.
[0009] In some aspects, embodiments disclosed herein relate to a
non-naturally occurring microbial organism having an aniline
pathway that includes at least one exogenous nucleic acid encoding
an aniline pathway enzyme expressed in a sufficient amount to
produce aniline. The aniline pathway includes an anthranilate
synthase and an anthranilate decarboxylase.
[0010] In some aspects, embodiments disclosed herein relate to a
method for producing aniline, that includes culturing a
non-naturally occurring microbial organism having an aniline
pathway that includes at least one exogenous nucleic acid encoding
an aniline pathway enzyme expressed in a sufficient amount to
produce aniline, under conditions and for a sufficient period of
time to produce aniline. The aniline pathway includes an
anthranilate synthase and an anthranilate decarboxylase.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 shows the metabolic pathway to chorismate. E4P is
erythrose-4-phosphate, PEP is phosphoenolpyruvate, DAHP is
3-deoxy-D-arabino-heptulosonic acid-7-phosphate.
[0012] FIG. 2 shows metabolic pathways for the production of
aniline. E4P is erythrose-4-phosphate, PEP is phosphoenolpyruvate,
DAHP is 3-deoxy-D-arabino-heptulosonic acid-7-phosphate.
DETAILED DESCRIPTION OF THE INVENTION
[0013] This invention is directed, in part, to the design and
production of cells and microbial organisms incorporating
biosynthetic pathways for the production of aniline. Enzymes useful
for the production of aniline from the central metabolism
precursors erythrose-4-phosphate (E4P) and phosphoenolpyruvate
(PEP), via multiple routes, are depicted in FIG. 2. Such organisms
can utilize renewable feedstocks, providing an alternative to
petroleum based aniline production. The maximum theoretical yield
of aniline from glucose as the carbon source is 0.857 mole/mole
glucose based on the equation 1 below.
7C.sub.6H.sub.12O.sub.6+6NH.sub.3.fwdarw.6C.sub.6H.sub.5NH.sub.2+6CO.sub-
.2+30H.sub.2O equation 1
[0014] Engineering these pathways into a microorganism involves
cloning an appropriate set of genes encoding a set of enzymes into
a production host described herein, optimizing fermentation
conditions, and assaying product formation following fermentation.
To engineer a production host for the production of aniline, one or
more exogenous DNA sequence(s) can be expressed in a microorganism.
In addition, the microorganism can have endogenous gene(s)
functionally disrupted, deleted or overexpressed.
[0015] In some embodiments, the invention provides a non-naturally
occurring microbial organism having an aniline pathway that
includes at least one exogenous nucleic acid encoding an aniline
pathway enzyme expressed in a sufficient amount to produce aniline.
The aniline pathway includes an aminodeoxychorismate synthase, an
aminodeoxychorismate lyase, and a 4-aminobenzoate carboxylyase, as
depicted in FIG. 2.
[0016] In some embodiments, the invention provides a non-naturally
occurring microbial organism having an aniline pathway that
includes at least one exogenous nucleic acid encoding an aniline
pathway enzyme expressed in a sufficient amount to produce aniline.
The aniline pathway includes an anthranilate synthase and an
anthranilate decarboxylase, as depicted in FIG. 2.
[0017] In some embodiments, the invention provides a method for
producing aniline that includes culturing a non-naturally occurring
microbial organism having an aniline pathway. The pathway includes
at least one exogenous nucleic acid encoding an aniline pathway
enzyme expressed in a sufficient amount to produce aniline, under
conditions and for a sufficient period of time to produce aniline.
In some embodiments, the aniline pathway includes an
aminodeoxychorismate synthase, an aminodeoxychorismate lyase, and a
4-aminobenzoate carboxylyase. In other embodiments the aniline
pathway includes an anthranilate synthase and an anthranilate
decarboxylase.
[0018] 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 organism's genetic material. Such modifications 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 or proteins within an
aniline biosynthetic pathway.
[0019] A metabolic modification refers to a biochemical reaction
that is altered from its naturally occurring state. Therefore,
non-naturally occurring microorganisms can have genetic
modifications to nucleic acids encoding metabolic polypeptides or,
functional fragments thereof. Exemplary metabolic modifications are
disclosed herein.
[0020] 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.
[0021] 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.
[0022] 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.
[0023] "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. The molecule can be introduced,
for example, by introduction of an encoding nucleic acid into the
host genetic material such as by integration into a host chromosome
or as non-chromosomal genetic material such as a plasmid.
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 acid 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.
[0024] It is understood that when more than one exogenous nucleic
acid is included in a microbial organism that the more than one
exogenous nucleic acid refers to the referenced encoding nucleic
acid or biosynthetic activity, as discussed above. It is further
understood, as disclosed herein, that such more than one exogenous
nucleic acids can be introduced into the host microbial organism on
separate nucleic acid molecules, on polycistronic nucleic acid
molecules, or a combination thereof, and still be considered as
more than one exogenous nucleic acid. For example, as disclosed
herein a microbial organism can be engineered to express two or
more exogenous nucleic acids encoding a desired pathway enzyme or
protein. In the case where two exogenous nucleic acids encoding a
desired activity are introduced into a host microbial organism, it
is understood that the two exogenous nucleic acids can be
introduced as a single nucleic acid, for example, on a single
plasmid, on separate plasmids, can be integrated into the host
chromosome at a single site or multiple sites, and still be
considered as two exogenous nucleic acids. Similarly, it is
understood that more than two exogenous nucleic acids can be
introduced into a host organism in any desired combination, for
example, on a single plasmid, on separate plasmids, can be
integrated into the host chromosome at a single site or multiple
sites, and still be considered as two or more exogenous nucleic
acids, for example three exogenous nucleic acids. Thus, the number
of referenced exogenous nucleic acids or biosynthetic activities
refers to the number of encoding nucleic acids or the number of
biosynthetic activities, not the number of separate nucleic acids
introduced into the host organism.
[0025] The non-naturally occurring microbial 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.
[0026] Those skilled in the art will understand that the genetic
alterations, including metabolic modifications exemplified herein,
are described with reference to a suitable host organism such as E.
coli and their corresponding metabolic reactions or a suitable
source organism for desired genetic material such as genes for a
desired metabolic pathway. 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.
[0027] 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.
[0028] 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 production of a biochemical product, those skilled in the
art will understand that the orthologous gene harboring the
metabolic activity to be introduced or 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.
[0029] 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.
[0030] 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 product compared to a gene
encoding the function sought to be substituted. Therefore, a
nonorthologous gene includes, for example, a paralog or an
unrelated gene.
[0031] Therefore, in identifying and constructing the non-naturally
occurring microbial organisms of the invention having aniline
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.
[0032] 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.
[0033] 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.
[0034] In some embodiments, the invention provides a non-naturally
occurring microbial organism, comprising a microbial organism
having an aniline pathway comprising at least one exogenous nucleic
acid encoding an aniline pathway enzyme expressed in a sufficient
amount to produce aniline, said aniline pathway comprising an
aminodeoxychorismate synthase, an aminodeoxychorismate lyase, and a
4-aminobenzoate carboxylyase. In some embodiments, such a
non-naturally occurring microbial organism can further include a
DAHP synthase and in still further embodiments, the non-naturally
occurring microbial organism can further include a 3-dehydroquinate
synthase.
[0035] In some embodiments, the non-naturally occurring microbial
organism includes two exogenous nucleic acids each encoding an
aniline pathway enzyme, while in other embodiments the
non-naturally occurring microbial organism includes three exogenous
nucleic acids each encoding an aniline pathway enzyme. For example,
in some embodiments, the non-naturally occurring microbial organism
can include three exogenous nucleic acids encoding an
aminodeoxychorismate synthase, an aminodeoxychorismate lyase, and a
4-aminobenzoate carboxylyase.
[0036] In some embodiments, the non-naturally occurring microbial
organism can include four exogenous nucleic acids each encoding an
aniline pathway enzyme. For example, a non-naturally occurring
microbial organism having four exogenous nucleic acids can encode a
DAHP synthase, an aminodeoxychorismate synthase, an
aminodeoxychorismate lyase, and a 4-aminobenzoate carboxylyase. The
DAHP synthase, which can be endogenous to the non-naturally
occurring microbial organism, can be overexpressed, for example, by
insertion of additional copies of the gene and/or through the use
of exogenous regulatory genes and removing feedback regulation by
the aromatic amino acids.
[0037] In still further embodiments, the non-naturally occurring
microbial organism can include five exogenous nucleic acids each
encoding an aniline pathway enzyme. For example, the non-naturally
occurring microbial organism having five exogenous nucleic acids
can encode a 3-dehydroquinate synthase, a DAHP synthase, an
aminodeoxychorismate synthase, an aminodeoxychorismate lyase, and a
4-aminobenzoate carboxylase. The 3-dehydroquinate synthase, which
can be endogenous to the non-naturally occurring microbial
organism, can also be overexpressed, for example, by insertion of
additional copies of the gene and/or through the use of exogenous
regulatory genes.
[0038] Moreover, any one or more of the other enzymes that are in a
pathway en route to chorismate, which can be endogenous in some
embodiments, can be overexpressed to increase the production of
chorismate. These include, for example, a 3-dehydroquinate
dehydratase (EC 4.2.1.10), a shikimate dehydrogenase (1.1.1.25), a
quinate/shikimate dehydrogenase (1.1.1.282), a shikimate kinase
(2.7.1.71), a 3-phosphoshikimate-1-carboxyvinyltransferase
(2.5.1.19), and a chorismate synthase (4.2.3.5). These enzymes
constitute the pathway for making chorismate from DAHP in
prokaryotes and most eukaryotes. An alternative pathway for
formation of 3-dehydroquinate (the steps from 3-dehydroquinate to
chorismate are the same in all organisms, including arachea)
includes the following enzymatic steps: triosephosphate isomerase,
frustose-1,6-bisphosphate aldolase,
2-amino-3,7-dideoxy-D-threo-hept-6-ulosonate synthase, and
dehydroquinate synthase.
[0039] In some embodiments, the non-naturally occurring microbial
organisms described above can have at least one exogenous nucleic
acid which is a heterologous nucleic acid. Moreover, the
non-naturally occurring microbial organisms described above, can be
provided in a substantially anaerobic culture medium.
[0040] In some embodiments, the present invention also provides a
non-naturally occurring microbial organism having an aniline
pathway that includes at least one exogenous nucleic acid encoding
an aniline pathway enzyme expressed in a sufficient amount to
produce aniline, in which the aniline pathway includes an
anthranilate synthase and an anthranilate decarboxylase. Such a
non-naturally occurring microbial organism can further include a
DAHP synthase, as described above. In some embodiments, such a
non-naturally occurring microbial organism can further include a
3-dehydroquinate synthase.
[0041] In some embodiments, this non-naturally occurring microbial
includes two exogenous nucleic acids each encoding an aniline
pathway enzyme. For example, the two exogenous nucleic acids can
encode an anthranilate synthase and an anthranilate decarboxylase.
In some embodiments the microbial organism can include three
exogenous nucleic acids each encoding an aniline pathway enzyme.
For example, the three exogenous nucleic acids can encode a DAHP
synthase, an anthranilate synthase and an anthranilate
decarboxylase. In some embodiments, the microbial organism includes
four exogenous nucleic acids each encoding an aniline pathway
enzyme. For example, the four exogenous nucleic acids can encode a
3-dehydroquinate synthase, a DAHP synthase, an anthranilate
synthase and an anthranilate decarboxylase.
[0042] As described above, any one or more of the other enzymes
that are in a pathway en route to chorismate, which can be
endogenous in some embodiments, can be overexpressed to increase
the production of chorismate. These include, for example, a
3-dehydroquinate dehydratase, a shikimate dehydrogenase, a
quinate/shikimate dehydrogenase, a shikimate kinase, a
3-phosphoshikimate-1-carboxyvinyltransferase, and a chorismate
synthase.
[0043] In some embodiments, such non-naturally occurring microbial
organisms described above can include at least one exogenous
nucleic acid is a heterologous nucleic acid. In some embodiments,
such non-naturally occurring microbial organisms are in a
substantially anaerobic culture medium.
[0044] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having an aniline
pathway, wherein the non-naturally occurring microbial organism
comprises at least one exogenous nucleic acid encoding an enzyme or
protein that converts a substrate to a product selected from the
group consisting of chorismate to 4-amino-4-deoxychorismate,
4-amino-4-deoxychorismate to p-aminobenzoate, and p-aminobenzoate
to aniline.
[0045] In an additional embodiment, the invention provides a
non-naturally occurring microbial organism having an aniline
pathway, wherein the non-naturally occurring microbial organism
comprises at least one exogenous nucleic acid encoding an enzyme or
protein that converts a substrate to a product selected from the
group consisting of chorismate to anthranilate, and anthranilate to
aniline.
[0046] One skilled in the art will understand that these are merely
exemplary and that any of the substrate-product pairs disclosed
herein suitable to produce a desired product and for which an
appropriate activity is available for the conversion of the
substrate to the product can be readily determined by one skilled
in the art based on the teachings herein. Thus, the invention
provides a non-naturally occurring microbial organism containing at
least one exogenous nucleic acid encoding an enzyme or protein,
where the enzyme or protein converts the substrates and products of
an aniline pathway, such as that shown in FIG. 1.
[0047] While generally described herein as a microbial organism
that contains an aniline pathway, it is understood that the
invention additionally provides a non-naturally occurring microbial
organism comprising at least one exogenous nucleic acid encoding an
aniline pathway enzyme expressed in a sufficient amount to produce
an intermediate of an aniline pathway. For example, as disclosed
herein, an aniline pathway is exemplified in FIG. 1. Therefore, in
addition to a microbial organism containing an aniline pathway that
produces aniline, the invention provides a non-naturally occurring
microbial organism comprising at least one exogenous nucleic acid
encoding an aniline pathway enzyme, where the microbial organism
produces an aniline pathway intermediate, for example, DAHP,
chorismate, anthranilate, 4-amino-4-deoxychorismate, or
p-aminobenzoate.
[0048] It is understood that any of the pathways disclosed herein,
as described in the Examples and exemplified in the Figures,
including the pathways of FIG. 1, can be utilized to generate a
non-naturally occurring microbial organism that produces any
pathway intermediate or product, as desired. As disclosed herein,
such a microbial organism that produces an intermediate can be used
in combination with another microbial organism expressing
downstream pathway enzymes to produce a desired product. However,
it is understood that a non-naturally occurring microbial organism
that produces an aniline pathway intermediate can be utilized to
produce the intermediate as a desired product.
[0049] 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, or a protein associated
with, 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 reference to any of
these metabolic constituents also references the gene or genes
encoding the enzymes that catalyze or proteins involved in 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 or a protein associated with the reaction as
well as the reactants and products of the reaction.
[0050] As shown in FIG. 1, the first step of an aniline pathway is
an aldol-type condensation that combines one molecule of E4P and
one molecule of PEP to form the intermediate,
3-deoxy-D-arabino-heptulosonic acid 7-phosphonate (DAHP). This
enzyme is referred to as DAHP synthase, or equivalently,
2-dehydro-3-deoxyphosphoheptonate aldolase. This reaction (EC
#2.5.1.54) is the first committed step in the shikimate pathway and
is required for the biosynthesis of aromatic amino acids, folates,
quinones and other secondary metabolites in bacteria, fungi and
plants. DAHP synthases have been categorized into AroAI and AroAII
classes (Wu et al., J. Biol. Chem. 281:4042-4048 (2006)). The
former class comprises of mainly microbial proteins while the
latter is comprised of primarily plant proteins.
[0051] In Escherichia coli, the function is catalyzed by three
genes: aroFGH. Each of these encodes for an isozyme and is feedback
regulated by a different aromatic amino acid. In contrast, some
other organisms, such as Bacillus subtilis and Porphyromonas
gingivalis are bifunctional enzymes. The aroA gene encodes for DAHP
synthase activity and aroQ gene encodes for chorismate mutase
activity in B. subtilis. However, these activities can be separated
by domain truncation ((Wu et al., J. Biol. Chem. 281:4042-4048
(2006)). The B. subtilis enzyme is sensitive to the downstream
intermediates, chorismate and prephanate. The DAHP synthase from
Corynebacterium glutamicum is feedback sensitive to both,
phenylalanine and tyrosine (Wu et al., J. Biol. Chem.
278:27525-27531 (2003)).
[0052] These enzymes are metalloenzymes and their mechanisms of
regulation are well-understood by those skilled in the art. The
crystal structures of the E. coli and S. cerevisiae DAHP synthases
have been solved and reveal structures consisting of
(.beta./.alpha.).sub.8 barrel. There are several enzymes that
however, don't have regulatory domains and belong to organisms such
as Pyrococcus furiosus and Nostoc sp. Exemplary genes are
summarized below in Table 1.
TABLE-US-00001 TABLE 1 Gene GI number GenBank ID Organism aroF
16130522 NP_417092.1 Escherichia coli K12 MG1655 aroG 16128722
NP_415275.1 Escherichia coli K12 MG1655 aroH 16129660 NP_416219.1
Escherichia coli K12 MG1655 aroA 16080027 NP_390853.1 Bacillus
subtilis aroA 34396967 AAQ66031.1 Porphyromonas gingivalis W83 Aro4
6319726 NP_009808.1 Saccharomyces cerevisiae aroG 1168513 P44303.1
Haemophilus influenza aroF 16765985 NP_461600.1 Salmonella
typhimurium aroG 21903376 P35170.2 Corynebacterium glutamicum
PF1690 18893851 AAL81814.1 Pyrococcus furiosus alr3050 17132144
BAB74749 Nostoc sp
[0053] Chorismate is a known intermediate for aromatic amino acid
biosynthesis in Gram positive, Gram-negative bacteria and in
archaea. It is also a precursor for the production of folic acid,
ubiquinone, menaquinone and enterocholein in some microorganisms.
DAHP can be converted into 3-dehydroquinate and that can be
subsequently converted into chorismate via multiple well-known
steps. In E. coli, DAHP can be converted into 3-dehydroquinate by
3-dehydroquinate synthase. The synthase in E. coli is understood to
catalyze an oxidation, a .beta.-elimination, an intramolecular
aldol condensation and a reduction (Frost et al., Biochemistry
23:4470-4475 (1984); Maitra et al., J. Biol. Chem. 253:5426-5430
(1978)). The enzyme requires catalytic amounts of NAD.sup.+ and
Co.sup.2+ (Maitra et al., J. Biol. Chem. 253:5426-5430 (1978)).
Enzymes useful for the production of chorismate include, for
example, a 3-dehydroquinate dehydratase, a shikimate dehydrogenase,
a quinate/shikimate dehydrogenase, a shikimate kinase, a
3-phosphoshikimate-1-carboxyvinyltransferase, and a chorismate
synthase, as described above
[0054] The conversion of chorismate into 4-amino-4-deoxychorismate
can be accomplished by aminodeoxychorismate synthase (EC#2.6.1.85),
also referred to as chorismate L-glutamine aminotransferase. In E.
coli, the function is catalyzed by two genes, pabA and pabB. The
pabA polypeptide is a conditional glutaminase which requires a 1:1
complex with pabB for activity. The pabB enzyme uses the nascent
ammonia released by this reaction to transform chorismate to
4-amino-4-deoxychorismate (in the presence of Mg.sup.2+). The pabB
reaction is fully reversible. In the absence of pabA, pabB utilizes
NH.sub.3 at significantly reduced rates (Roux and Walsh,
Biochemistry 32:3763-3768 (1993); Roux and Walsh, Biochemistry
31:6904-6910 (1992)).
[0055] A similar enzyme complex formed by pabA and pabB, catalyzes
the conversion of chorismate into 4-amino-4-dexoychorismate in
Streptomyces venezuelae (Brown et al., Microbiology 142(pt 6):
1345-1355 (1996)). This organism is known to have more than one set
of pabAB genes (Chang et al., Microbiology 147:2113-2126 (2001)).
The gene with the aforementioned function has been identified in
Arabidopsis thaliana and Solanum lycopersicum also. The protein
sequences of the PabA and PabB genes of E. coli were used to
isolate the cDNA encoding the aminodeoxychorismate synthase (ADCS)
in Arabidopsis thaliana (Basset et al., Proc. Natl. Acad. Sci.
U.S.A. 101:1496-1501 (2004)). The enzyme was recombinantly
expressed in E. coli demonstrating the formation of
4-amino-4-deoxychorismate. No feedback inhibition of the enzyme has
been reported for either p-aminobenzoate or folate. The
corresponding genes, along with their GenBank ids are listed below
in Table 2:
TABLE-US-00002 TABLE 2 pabAB 710438 AAB30312.1 Streptomyces
venezuelae pabA 16131239 NP_417819.1 Escherichia coli K12 MG1655
pabB 16129766 NP_416326.1 Escherichia coli K12 MG1655 pabA
152972254 YP_001337400.1 Klebsiella pneumoniae pabB 152970875
YP_001335984.1 Klebsiella pneumoniae pabA 118467576 YP_884448.1
Mycobacterium smegmatis pabB 118473035 YP_889684.1 Mycobacterium
smegmatis
[0056] In several organisms, the gene encoding for
aminodeoxychorismate lyase (EC#4.1.3.38) is typically coupled with
pabB and pabA to catalyze the conversion of aminodexoychorismate
into p-aminobenzoate, with the release of a pyruvate molecule. In
both E. coli (Green et al., J. Bacteriol. 174:5317-5323 (1992);
Green and Nichols, J. Biol. Chem. 266:12971-12975 (1991)) and S.
venezuelae, pabC catalyzes this reaction. Recently,
4-amino-4-deoxychorismate lyase was functionally characterized in
two more species of Streptomcyes, namely FR-008 and griseus (Zhang
et al., Microbiology 155:2450-2459 (2009)). Aminodeoxychorismate
synthase and aminodeoxychorismate lyase are typically part of
folate biosynthesis in most organisms and facilitate the conversion
of chorismate into para-aminobenzoate. Aminodeoxychorismate lyase
is a pyridoxal-phosphate dependent protein. A putative enzyme has
been found in A. thaliana ((Basset et al., Proc. Natl. Acad. Sci.
U.S.A. 101:1496-1501 (2004)) and B. subtilis (Schadt et al., J. Am.
Chem. Soc. 131:3481-3493 (2009); Slock et al., J. Bacteriol.
172:7211-7226 (1990)), as part of the folate operon. Some exemplary
genes are shown below in Table 3:
TABLE-US-00003 TABLE 3 pabC 16129059 NP_415614.1 Escherichia coli
K12 MG1655 pabC 16077144 NP_387957.1 Bacillus subtilis pabC
29828105 NP_822739.1 Streptomyces avermitilis pabC-1 224831591
AAQ82550.2 Streptomyces sp. FR-008 pabC-2 219879202 ACL50980.1
Streptomyces sp. FR-008
[0057] Anthranilate synthase (EC: 4.1.3.27), also known by the
systematic name chorismate pyruvate-lyase (amino-accepting:
anthranilate-forming) or by the synonym glutamine amidotransferase,
is the first step in the tryptophan synthesis pathway from
chorismate. The formation of anthranilate is accompanied by the
transfer of an amine group from glutamine and leading to the
formation of glutamate. Pyruvate is also released during the
reaction. In E. coli, this reaction is catalyzed by a tetrameric
enzyme complex comprised of two monomers of TrpD and two monomers
of TrpE. TrpE on its own can carry out an alternate version of this
reaction, using ammonium sulfate rather than glutamine as an amino
donor (Ito et al., Acta. Pathol. Jpn. 19:55-67 (1969): Ito and
Yanofsky, J. Bacteriol. 97:734-742 (1969)). However, TrpD increases
the affinity of TrpE for glutamine over TrpE alone. The enzyme is
feedback regulated by tryptophan. This feedback regulation is also
observed for the enzyme complex in the hyperthermophilic Sulfolobus
solfataricus. The enzyme complex from this organism has been
expressed in E. coli (Tutino et al., Biochem. Biophys. Res. Commun.
230:306-310 (1997)). The thermodynamics of the reaction catalyzed
by anthranilate synthase has been described in Salmonella
typhimurium (Byrnes et al., Biophys. Chem. 84:45-64 (2000)). The
subunits of the enzyme complex have also been described in
Thermotoga maritima (Kim et al., J. Mol. Biol. 231:960-981 (1993)).
A summary of these genes is shown below in Table 4.
TABLE-US-00004 TABLE 4 TrpD 16129224 NP_415779.1 Escherichia coli
K12 MG1655 TrpE 16129225 NP_415780.1 Escherichia coli K12 MG1655
TrpE 15897780 NP_342385.1 Sulfolobus solfataricus TrpGD 15897781
NP_342386.1 Sulfolobus solfataricus trpD 16765068 NP_460683.1
Salmonella typhimurium trpE 16765067 NP_460682.1 Salmonella
typhimurium trpE 15642916 NP_227957.1 Thermotoga maritima trpGD
15642915 NP_227956.1 Thermotoga maritima
[0058] The decarboxylation of p-aminobenzoate and anthranilate can
be catalyzed by an aminobenzoate carboxylyase (McCullough et al.,
J. Am. Chem. Soc. 79:628-630 (1957)). It has been indicated that
the cell free enzyme obtained from E. coli 0111:B4 was capable of
decarboxylating both of these molecules. The activity of the enzyme
was found to be dependent on pyridoxal phosphate and iron (III).
The conversion of p-aminobenzoate to aniline in some extracts of
Mycobacteria has been described (Sloane et al., J. Biol. Chem.
193:453-458 (1951)). New strains have been identified that are
capable of degrading aniline anaerobically (Kahng et al., FEMS
Microbiol. Lett. 190:215-221 (2000); Schnell et al., Arch.
Microbiol. 152:556-563 (1989)). These strains first carboxylate
aniline to 4-aminobenzoate. In the strain. Desulfobacterium
anilini, the rate of aniline degradation is dependent on the
presence of CO.sub.2 in the medium. GC analysis of aniline culture
supernatant of strain HY99 under anaerobic, denitrifying conditions
showed the presence of 4-aminobenzoate (Kahng et al., FEMS
Microbiol. Lett. 190:215-221 (2000)).
[0059] Numerous other studies have been conducted on
decarboxylation of aromatic compounds, primarily hydroxyl
aromatics. For example, a 4-hydroxybenzoate decarboxylase has been
identified from the facultative anaerobe. Enterobacter cloacae
(Matsui et al., Arch. Microbiol. 186:21-29 (2006)). The
corresponding gene has been sequenced. The enzyme has been tested
for activity on multiple substrates and was shown to be induced by
both 4-hydroxybenzoic acid and 4-aminobenzoic acid. Another
decarboxylase has been reported in Clostridium theromaceticum that
can remove CO.sub.2 from p-hydroxy benzoate (Hsu et al., J.
Bacteriol. 172:5901-5907 (1990)). The enzyme has broad substrate
specificity and can act on p-hydroxy benzoate with varied
functional group substituents at the meta-position. These include
hydroxyl, chloro, fluoro, and methoxy groups. The enzyme was not
repressed by glucose or other external energy sources. Klebsiella
aerogens was also reported to be able to carry out non-oxidative
decarboxylation of para-hydroxy benzoate, 2,5-dihydroxybenzoate,
3,4-dihydroxybenzoate and 3,4,5-trihydroxybenzoate (Grant et al.,
Antonie Van Leeuwenhoek 35:325-343 (1969)). A reversible
4-hydroxybenzoate decarboxylase was purified from Clostridium
hydroxybenzoicum (now called Sedimentibacter hydroxybenzoicus).
This enzyme is encoded by three clustered genes, shdB, C and D. The
enzyme can act on both 4-hydroxybenzoate and 3,4-dihydroxybenzoate.
The enzyme activity was not affected by metal ions or other
cofactor (He et al., Eur. J. Biochem. 229:77-82 (1995)). Bacillus
subtilis was recently demonstrated to have a hydroxyarylic acid
decarboxylase activity. Three genes bcdB, C, and D were cloned in
E. coli and showed activity on 4-hydroxybenzoate and vanillate
(Lupa et al., Can. J. Microbiol. 54:75-81 (2008)). These
decarboxylases have been reported in several other organisms (Lupa
et al., Genomics 86:342-351 (2005)) and gene candidates for some of
these are listed below in Table 5.
TABLE-US-00005 TABLE 5 shdB 67462197 AAY67850.1 Sedimentibacter
hydroxybenzoicus shdC 5739200 AAD50377.1 Sedimentibacter
hydroxybenzoicus shdD 67462198 AAY67851.1 Sedimentibacter
hydroxybenzoicus 110331749 BAE97712.1 Enterobacter cloacae bsdB
13124411 P94404.1 Bacillus subtilis bsdC 6686207 P94405.1 Bacillus
subtilis bsdD 239977069 C0H3U9.1 Bacillus subtilis STM292 16766227
NP_461842.1 Salmonella typhimurium LT2 STM2922 16766228 NP_461843.1
Salmonella typhimurium LT2 STM2923 16766229 NP_461844.1 Salmonella
typhimurium LT2 kpdB 206580833 YP_002236894.1 Klebsiella pneumoniae
342 kpdC 206576360 YP_002236895.1 Klebsiella pneumoniae 342 kpdD
206579343 YP_002236896.1 Klebsiella pneumoniae 342 pad1 15832847
NP_311620.1 Escherichia coli O157 yclC 15832846 NP_311619.1
Escherichia coli O157 yclD 15832845 NP_311618.1 Escherichia coli
O157
[0060] 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.
[0061] The non-naturally occurring microbial organisms of the
invention can be produced by introducing expressible nucleic acids
encoding one or more of the enzymes or proteins participating in
one or more aniline biosynthetic pathways. Depending on the host
microbial organism chosen for biosynthesis, nucleic acids for some
or all of a particular aniline biosynthetic pathway can be
expressed. For example, if a chosen host is deficient in one or
more enzymes or proteins for a desired biosynthetic pathway, then
expressible nucleic acids for the deficient enzyme(s) or protein(s)
are introduced into the host for subsequent exogenous expression.
Alternatively, if the chosen host exhibits endogenous expression of
some pathway genes, but is deficient in others, then an encoding
nucleic acid is needed for the deficient enzyme(s) or protein(s) to
achieve aniline biosynthesis. Thus, a non-naturally occurring
microbial organism of the invention can be produced by introducing
exogenous enzyme or protein activities to obtain a desired
biosynthetic pathway or a desired biosynthetic pathway can be
obtained by introducing one or more exogenous enzyme or protein
activities that, together with one or more endogenous enzymes or
proteins, produces a desired product such as aniline.
[0062] 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 Escherichia coli, Klebsiella
oxytoca, Anaerobiospirillum succiniciproducens, Actinobacillus
succinogenes, Mannheimia succiniciproducens, Rhizobium etli.
Bacillus subtilis, Corynebacterium glutamicum, Gluconobacter
oxydans, Zvmomonas 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, Kluyveromvces lactis, Kluyveromyces
marxianus, Aspergillus terreus, Aspergillus niger, Pichia pastoris,
Rhizopus arrhizus, Rhizopus oryzae, and the like. E. coli is a
particularly useful host organism since it is a well characterized
microbial organism suitable for genetic engineering. Other
particularly useful host organisms include yeast such as
Saccharomyces cerevisiae. It is understood that any suitable
microbial host organism can be used to introduce metabolic and/or
genetic modifications to produce a desired product.
[0063] Depending on the aniline biosynthetic pathway constituents
of a selected host microbial organism, the non-naturally occurring
microbial organisms of the invention will include at least one
exogenously expressed aniline pathway-encoding nucleic acid and up
to all encoding nucleic acids for one or more aniline biosynthetic
pathways. For example, aniline biosynthesis can be established in a
host deficient in a pathway enzyme or protein through exogenous
expression of the corresponding encoding nucleic acid. In a host
deficient in all enzymes or proteins of an aniline pathway,
exogenous expression of all enzyme or proteins in the pathway can
be included, although it is understood that all enzymes or proteins
of a pathway can be expressed even if the host contains at least
one of the pathway enzymes or proteins. For example, exogenous
expression of all enzymes or proteins in a pathway for production
of aniline can be included, such as a 3-dehydroquinate synthase, a
DAHP synthase, an aminodeoxychorismate synthase, an
aminodeoxychorismate lyase, and a 4-aminobenzoate carboxylyase or a
3-dehydroquinate synthase, a DAHP synthase, an anthranilate
synthase and an anthranilate decarboxylase.
[0064] 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, at least,
parallel the aniline 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, up
to all nucleic acids encoding the enzymes or proteins constituting
an aniline biosynthetic pathway disclosed herein. In some
embodiments, the non-naturally occurring microbial organisms also
can include other genetic modifications that facilitate or optimize
aniline 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
aniline pathway precursors such as chorismate, anthranilate,
4-amino-4-deoxychorismate, and p-aminobenzoate.
[0065] Generally, a host microbial organism is selected such that
it produces the precursor of an aniline pathway, either as a
naturally produced molecule or as an engineered product that either
provides de novo production of a desired precursor or increased
production of a precursor naturally produced by the host microbial
organism. For example, chorismate is produced naturally in a host
organism such as E. coli. A host organism can be engineered to
increase production of a precursor, as disclosed herein. In
addition, a microbial organism that has been engineered to produce
a desired precursor can be used as a host organism and further
engineered to express enzymes or proteins of an aniline
pathway.
[0066] In some embodiments, a non-naturally occurring microbial
organism of the invention is generated from a host that contains
the enzymatic capability to synthesize aniline. In this specific
embodiment it can be useful to increase the synthesis or
accumulation of an aniline pathway product to, for example, drive
aniline pathway reactions toward aniline production. Increased
synthesis or accumulation can be accomplished by, for example,
overexpression of nucleic acids encoding one or more of the
above-described aniline pathway enzymes or proteins. Over
expression of the enzyme or enzymes and/or protein or proteins of
the aniline pathway 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
occurring microbial organisms of the invention, for example,
producing aniline, through overexpression of one, two, three, four,
five, up to all nucleic acids encoding aniline biosynthetic pathway
enzymes or proteins. 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 aniline
biosynthetic pathway.
[0067] 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.
[0068] It is understood that, in methods of the invention, any of
the one or more exogenous nucleic acids can be introduced into a
microbial organism to produce a non-naturally occurring microbial
organism of the invention. The nucleic acids can be introduced so
as to confer, for example, an aniline biosynthetic pathway onto the
microbial organism. Alternatively, encoding nucleic acids can be
introduced to produce an intermediate microbial organism having the
biosynthetic capability to catalyze some of the required reactions
to confer aniline biosynthetic capability. For example, a
non-naturally occurring microbial organism having an aniline
biosynthetic pathway can comprise at least two exogenous nucleic
acids encoding desired enzymes or proteins, such as the combination
of aminodoxychorismate synthase and aminodeoxychorismate lyase, or
aminodeoxychorismate lyase and 4-aminobenzoate carboxylase, or
aminodeoxychorismate synthase and 4-amionbenzoate carboxylase, and
the like. Thus, it is understood that any combination of two or
more enzymes or proteins 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 or proteins of a biosynthetic pathway can be included in a
non-naturally occurring microbial organism of the invention, and so
forth, as desired, so long as the combination of enzymes and/or
proteins of the desired biosynthetic pathway results in production
of the corresponding desired product. Similarly, any combination of
four, or more enzymes or proteins of a biosynthetic pathway as
disclosed herein can be included in a non-naturally occurring
microbial organism of the invention, as desired, so long as the
combination of enzymes and/or proteins of the desired biosynthetic
pathway results in production of the corresponding desired
product.
[0069] In addition to the biosynthesis of aniline 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 aniline other than use of
the aniline producers is through addition of another microbial
organism capable of converting an aniline pathway intermediate to
aniline. One such procedure includes, for example, the fermentation
of a microbial organism that produces an aniline pathway
intermediate. The aniline pathway intermediate can then be used as
a substrate for a second microbial organism that converts the
aniline pathway intermediate to aniline. The aniline pathway
intermediate can be added directly to another culture of the second
organism or the original culture of the aniline pathway
intermediate 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 be utilized to
produce the final product without intermediate purification
steps.
[0070] 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,
aniline. 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 aniline can be accomplished by
constructing a microbial organism that contains biosynthetic
pathways for conversion of one pathway intermediate to another
pathway intermediate or the product. Alternatively, aniline also
can be biosynthetically produced from microbial organisms through
co-culture or co-fermentation using two organisms in the same
vessel, where the first microbial organism produces an aniline
intermediate and the second microbial organism converts the
intermediate to aniline.
[0071] 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 aniline.
[0072] Sources of encoding nucleic acids for an aniline pathway
enzyme or protein 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. Exemplary species for
such sources include, for example, Escherichia coli,
Sedimentibacter hydroxybenzoicus, and Bacillus subtilis, as well as
other exemplary species disclosed herein or available as source
organisms for corresponding genes. 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 aniline 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 allowing biosynthesis of aniline described herein with
reference to a particular organism such as E. coli 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.
[0073] In some instances, such as when an alternative aniline
biosynthetic pathway exists in an unrelated species, aniline
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 gene 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 aniline.
[0074] Methods for constructing and testing the expression levels
of a non-naturally occurring aniline-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); and
Ausubel et al., Current Protocols in Molecular Biology, John Wiley
and Sons, Baltimore, Md. (1999).
[0075] Exogenous nucleic acid sequences involved in a pathway for
production of aniline can be introduced stably or transiently into
a host cell using techniques well known in the art including, but
not limited to, conjugation, electroporation, chemical
transformation, transduction, transfection, and ultrasound
transformation. For exogenous expression in E. coli or other
prokaryotic cells, some nucleic acid sequences in the genes or
cDNAs of eukaryotic nucleic acids can encode targeting signals such
as an N-terminal mitochondrial or other targeting signal, which can
be removed before transformation into prokaryotic host cells, if
desired. For example, removal of a mitochondrial leader sequence
led to increased expression in E. coli (Hoffmeister et al., J.
Biol. Chem. 280:4329-4338 (2005)). For exogenous expression in
yeast or other eukaryotic cells, genes can be expressed in the
cytosol without the addition of leader sequence, or can be targeted
to mitochondrion or other organelles, or targeted for secretion, by
the addition of a suitable targeting sequence such as a
mitochondrial targeting or secretion signal suitable for the host
cells. Thus, it is understood that appropriate modifications to a
nucleic acid sequence to remove or include a targeting sequence can
be incorporated into an exogenous nucleic acid sequence to impart
desirable properties. Furthermore, genes can be subjected to codon
optimization with techniques well known in the art to achieve
optimized expression of the proteins.
[0076] An expression vector or vectors can be constructed to
include one or more aniline biosynthetic pathway encoding nucleic
acids as exemplified herein operably linked to expression control
sequences functional in the host organism. Expression vectors
applicable for use in the microbial host organisms of the invention
include, for example, plasmids, phage vectors, viral vectors,
episomes and artificial chromosomes, including vectors and
selection sequences or markers operable for stable integration into
a host chromosome. Additionally, the expression vectors can include
one or more selectable marker genes and appropriate expression
control sequences. Selectable marker genes also can be included
that, for example, provide resistance to antibiotics or toxins,
complement auxotrophic deficiencies, or supply critical nutrients
not in the culture media. Expression control sequences can include
constitutive and inducible promoters, transcription enhancers,
transcription terminators, and the like which are well known in the
art. When two or more exogenous encoding nucleic acids are to be
co-expressed, both nucleic acids can be inserted, for example, into
a single expression vector or in separate expression vectors. For
single vector expression, the encoding nucleic acids can be
operationally linked to one common expression control sequence or
linked to different expression control sequences, such as one
inducible promoter and one constitutive promoter. The
transformation of exogenous nucleic acid sequences involved in a
metabolic or synthetic pathway can be confirmed using methods well
known in the art. Such methods include, for example, nucleic acid
analysis such as Northern blots or polymerase chain reaction (PCR)
amplification of mRNA, or immunoblotting for expression of gene
products, or other suitable analytical methods to test the
expression of an introduced nucleic acid sequence or its
corresponding gene product. It is understood by those skilled in
the art that the exogenous nucleic acid is expressed in a
sufficient amount to produce the desired product, and it is further
understood that expression levels can be optimized to obtain
sufficient expression using methods well known in the art and as
disclosed herein.
[0077] In some embodiments, the present invention provides a method
for producing aniline that includes culturing a non-naturally
occurring microbial organism having an aniline pathway in which at
least one exogenous nucleic acid encoding an aniline pathway enzyme
is expressed in a sufficient amount to produce aniline, under
conditions and for a sufficient period of time to produce aniline.
The aniline pathway includes an aminodeoxychorismate synthase, an
aminodeoxychorismate lyase, and a 4-aminobenzoate carboxylyase. In
some embodiments, the pathway further includes a DAHP synthase. In
some embodiments, the pathway further includes a 3-dehydroquinate
synthase. A method for producing aniline, includes culturing the
non-naturally occurring microbial organism under conditions and for
a sufficient period of time to produce aniline. Moreover, the
non-naturally occurring microbial organism can be cultured in a
substantially anaerobic culture medium.
[0078] Methods of the invention can include culturing a microbial
organism having two exogenous nucleic acids each encoding an
aniline pathway enzyme. In some embodiments, the cultured microbial
organism can include three exogenous nucleic acids each encoding an
aniline pathway enzyme. For example, the three exogenous nucleic
acids can encode an aminodeoxychorismate synthase, an
aminodeoxychorismate lyase, and a 4-aminobenzoate carboxylase. In
some embodiments, the cultured microbial organism can include four
exogenous nucleic acids each encoding an aniline pathway enzyme.
For example, the four exogenous nucleic acids can encode a DAHP
synthase, an aminodeoxychorismate synthase, an aminodeoxychorismate
lyase, and a 4-aminobenzoate carboxylyase.
[0079] In still further embodiments, the cultured microbial
organism can include five exogenous nucleic acids each encoding an
aniline pathway enzyme. For example, the five exogenous nucleic
acids can encode a 3-dehydroquinate synthase, a DAHP synthase, an
aminodeoxychorismate synthase, an aminodeoxychorismate lyase, and a
4-aminobenzoate carboxylyase.
[0080] Any of the cultured organisms described above can have at
least one exogenous nucleic acid that is a heterologous nucleic
acid.
[0081] In some embodiments, the present invention provides a method
for producing aniline, that includes culturing a non-naturally
occurring microbial organism having an aniline pathway in which at
least one exogenous nucleic acid encoding an aniline pathway enzyme
expressed in a sufficient amount to produce aniline, under
conditions and for a sufficient period of time to produce aniline.
In some embodiments, the aniline pathway includes an anthranilate
synthase and an anthranilate decarboxylase. In some embodiments,
such an organism can further include a DAHP synthase. In some
embodiments, such an organism can further include a
3-dehydroquinate synthase, 3-dehydroquinate dehydratase, a
shikimate dehydrogenase or a quinate/shikimate dehydrogenase, a
shikimate kinase, a 3-phosphoshikimate-1-carboxyvinyltransferase,
and a chorismate synthase. In some embodiments, the cultured
non-naturally occurring microbial organism is cultured in a
substantially anaerobic culture medium.
[0082] In some embodiments, the above cultured microbial organism
can include two exogenous nucleic acids each encoding an aniline
pathway enzyme. For example, the two exogenous nucleic acids can
encode an anthranilate synthase and an anthranilate decarboxylase.
In some embodiments, the cultured microbial organism can include
three exogenous nucleic acids each encoding an aniline pathway
enzyme. For example, the three exogenous nucleic acids encode a
DAHP synthase, an anthranilate synthase and an anthranilate
decarboxylase. In still further embodiments, the cultured microbial
organism can include four exogenous nucleic acids each encoding an
aniline pathway enzyme. For example, the four exogenous nucleic
acids encode a 3-dehydroquinate synthase, a DAHP synthase, an
anthranilate synthase and an anthranilate decarboxylase. Any of the
at least one exogenous nucleic acids can be provided as a
heterologous nucleic acid.
[0083] Suitable purification and/or assays to test for the
production of aniline can be performed using well known methods.
Suitable replicates such as triplicate cultures can be grown for
each engineered strain to be tested. For example, product and
byproduct formation in the engineered production host can be
monitored. The final product and intermediates, and other organic
compounds, can be analyzed by methods such as HPLC (High
Performance Liquid Chromatography), GC-MS (Gas Chromatography-Mass
Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy)
or other suitable analytical methods using routine procedures well
known in the art. The release of product in the fermentation broth
can also be tested with the culture supernatant. Byproducts and
residual glucose can be quantified by HPLC using, for example, a
refractive index detector for glucose and alcohols, and a UV
detector for organic acids (Lin et al., Biotechnol. Bioeng.
90:775-779 (2005)), or other suitable assay and detection methods
well known in the art. The individual enzyme or protein activities
from the exogenous DNA sequences can also be assayed using methods
well known in the art (McCullough et al., J. Am. Chem. Soc.
79:628-630 (1957)).
[0084] Aniline can be separated from other components in the
culture using a variety of methods well known in the art. Such
separation methods include, for example, extraction procedures as
well as methods that 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.
[0085] Any of the non-naturally occurring microbial organisms
described herein can be cultured to produce and/or secrete the
biosynthetic products of the invention. For example, the aniline
producers can be cultured for the biosynthetic production of
aniline.
[0086] For the production of aniline, the recombinant strains are
cultured in a medium with carbon source and other essential
nutrients. It is highly desirable to maintain anaerobic conditions
in the fermenter to reduce the cost of the overall process. Such
conditions can be obtained, for example, by first sparging the
medium with nitrogen and then sealing the flasks with a septum and
crimp-cap. For strains where growth is not observed anaerobically,
microaerobic conditions can be applied by perforating the septum
with a small hole for limited aeration. Exemplary anaerobic
conditions have been described previously and are well-known in the
art. Exemplary aerobic and anaerobic conditions are described, for
example, in U.S. patent application Ser. No. 11/891,602, filed Aug.
10, 2007. Fermentations can be performed in a batch, fed-batch or
continuous manner, as disclosed herein.
[0087] If desired, the pH of the medium can be maintained at a
desired pH, in particular neutral pH, such as a pH of around 7 by
addition of a base, such as NaOH or other bases, or acid, as needed
to maintain the culture medium at a desirable pH. The growth rate
can be determined by measuring optical density using a
spectrophotometer (600 nm), and the glucose uptake rate by
monitoring carbon source depletion over time.
[0088] The growth medium can include, for example, any carbohydrate
source which can supply a source of carbon to the non-naturally
occurring microorganism. Such sources include, for example, sugars
such as glucose, xylose, arabinose, galactose, mannose, fructose,
sucrose and starch. Other sources of carbohydrates include, for
example, renewable feedstocks and biomass. Exemplary types of
biomasses that can be used as feedstocks in the methods of the
invention include cellulosic biomass, hemicellulosic biomass and
lignin feedstocks or portions of feedstocks. Such biomass
feedstocks contain, for example, carbohydrate substrates useful as
carbon sources such as glucose, xylose, arabinose, galactose,
mannose, fructose and starch. Given the teachings and guidance
provided herein, those skilled in the art will understand that
renewable feedstocks and biomass other than those exemplified above
also can be used for culturing the microbial organisms of the
invention for the production of aniline.
[0089] In addition to renewable feedstocks such as those
exemplified above, the aniline microbial organisms of the invention
also can be modified for growth on syngas as its source of carbon.
In this specific embodiment, one or more proteins or enzymes are
expressed in the aniline producing organisms to provide a metabolic
pathway for utilization of syngas or other gaseous carbon
source.
[0090] Synthesis gas, also known as syngas or producer gas, is the
major product of gasification of coal and of carbonaceous materials
such as biomass materials, including agricultural crops and
residues. Syngas is a mixture primarily of H.sub.2 and CO and can
be obtained from the gasification of any organic feedstock,
including but not limited to coal, coal oil, natural gas, biomass,
and waste organic matter. Gasification is generally carried out
under a high fuel to oxygen ratio. Although largely H.sub.2 and CO,
syngas can also include CO.sub.2 and other gases in smaller
quantities. Thus, synthesis gas provides a cost effective source of
gaseous carbon such as CO and, additionally, CO.sub.2.
[0091] The Wood-Ljungdahl pathway catalyzes the conversion of CO
and H.sub.2 to acetyl-CoA and other products such as acetate.
Organisms capable of utilizing CO and syngas also generally have
the capability of utilizing CO.sub.2 and CO.sub.2/H.sub.2 mixtures
through the same basic set of enzymes and transformations
encompassed by the Wood-Ljungdahl pathway. H.sub.2-dependent
conversion of CO.sub.2 to acetate by microorganisms was recognized
long before it was revealed that CO also could be used by the same
organisms and that the same pathways were involved. Many acetogens
have been shown to grow in the presence of CO.sub.2 and produce
compounds such as acetate as long as hydrogen is present to supply
the necessary reducing equivalents (see for example, Drake,
Acetogenesis, pp. 3-60 Chapman and Hall, New York, (1994)). This
can be summarized by the following equation:
2CO.sub.2+4H.sub.2+nADP+nPi.fwdarw.CH.sub.3COOH+2H.sub.2O+nATP
[0092] Hence, non-naturally occurring microorganisms possessing the
Wood-Ljungdahl pathway can utilize CO.sub.2 and H.sub.2 mixtures as
well for the production of acetyl-CoA and other desired
products.
[0093] The Wood-Ljungdahl pathway is well known in the art and
consists of 12 reactions which can be separated into two branches:
(1) methyl branch and (2) carbonyl branch. The methyl branch
converts syngas to methyl-tetrahydrofolate (methyl-THF) whereas the
carbonyl branch converts methyl-THF to acetyl-CoA. The reactions in
the methyl branch are catalyzed in order by the following enzymes
or proteins: ferredoxin oxidoreductase, formate dehydrogenase,
formyltetrahydrofolate synthetase, methenyltetrahydrofolate
cyclodehydratase, methylenetetrahydrofolate dehydrogenase and
methylenetetrahydrofolate reductase. The reactions in the carbonyl
branch are catalyzed in order by the following enzymes or proteins:
methyltetrahydrofolate:corrinoid protein methyltransferase (for
example, AcsE), corrinoid iron-sulfur protein, nickel-protein
assembly protein (for example, AcsF), ferredoxin, acetyl-CoA
synthase, carbon monoxide dehydrogenase and nickel-protein assembly
protein (for example, CooC). Following the teachings and guidance
provided herein for introducing a sufficient number of encoding
nucleic acids to generate an aniline pathway, those skilled in the
art will understand that the same engineering design also can be
performed with respect to introducing at least the nucleic acids
encoding the Wood-Ljungdahl enzymes or proteins absent in the host
organism. Therefore, introduction of one or more encoding nucleic
acids into the microbial organisms of the invention such that the
modified organism contains the complete Wood-Ljungdahl pathway will
confer syngas utilization ability.
[0094] Accordingly, given the teachings and guidance provided
herein, those skilled in the art will understand that a
non-naturally occurring microbial organism can be produced that
secretes the biosynthesized compounds of the invention when grown
on a carbon source such as a carbohydrate. Such compounds include,
for example, aniline and any of the intermediate metabolites in the
aniline pathway. All that is required is to engineer in one or more
of the required enzyme or protein activities to achieve
biosynthesis of the desired compound or intermediate including, for
example, inclusion of some or all of the aniline biosynthetic
pathways. Accordingly, the invention provides a non-naturally
occurring microbial organism that produces and/or secretes aniline
when grown on a carbohydrate or other carbon source and produces
and/or secretes any of the intermediate metabolites shown in the
aniline pathway when grown on a carbohydrate or other carbon
source. The aniline producing microbial organisms of the invention
can initiate synthesis from an intermediate, for example,
chorismate, anthranilate, 4-amino-4-deoxychorismate, or
p-aminobenzoate.
[0095] The non-naturally occurring microbial organisms of the
invention are constructed using methods well known in the art as
exemplified herein to exogenously express at least one nucleic acid
encoding an aniline pathway enzyme or protein in sufficient amounts
to produce aniline. It is understood that the microbial organisms
of the invention are cultured under conditions sufficient to
produce aniline. Following the teachings and guidance provided
herein, the non-naturally occurring microbial organisms of the
invention can achieve biosynthesis of aniline resulting in
intracellular concentrations between about 0.1-200 mM or more.
Generally, the intracellular concentration of aniline is between
about 3-150 mM, particularly between about 5-125 mM and more
particularly between about 8-100 mM, including about 10 mM, 20 mM,
50 mM, 80 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.
[0096] 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 herein and are described, for example, in
U.S. publication 2009/0047719, filed Aug. 10, 2007. 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 aniline
producers can synthesize aniline at intracellular concentrations of
5-10 mM or more as well as all other concentrations exemplified
herein. It is understood that, even though the above description
refers to intracellular concentrations, aniline producing microbial
organisms can produce aniline intracellularly and/or secrete the
product into the culture medium.
[0097] In addition to the culturing and fermentation conditions
disclosed herein, growth condition for achieving biosynthesis of
aniline can include the addition of an osmoprotectant to the
culturing conditions. In certain embodiments, the non-naturally
occurring microbial organisms of the invention can be sustained,
cultured or fermented as described herein in the presence of an
osmoprotectant. Briefly, an osmoprotectant refers to a compound
that acts as an osmolyte and helps a microbial organism as
described herein survive osmotic stress. Osmoprotectants include,
but are not limited to, betaines, amino acids, and the sugar
trehalose. Non-limiting examples of such are glycine betaine,
praline betaine, dimethylthetin, dimethylslfonioproprionate,
3-dimethylsulfonio-2-methylproprionate, pipecolic acid,
dimethylsulfonioacetate, choline, L-carnitine and ectoine. In one
aspect, the osmoprotectant is glycine betaine. It is understood to
one of ordinary skill in the art that the amount and type of
osmoprotectant suitable for protecting a microbial organism
described herein from osmotic stress will depend on the microbial
organism used. The amount of osmoprotectant in the culturing
conditions can be, for example, no more than about 0.1 mM, no more
than about 0.5 mM, no more than about 1.0 mM, no more than about
1.5 mM, no more than about 2.0 mM, no more than about 2.5 mM, no
more than about 3.0 mM, no more than about 5.0 mM, no more than
about 7.0 mM, no more than about 10 mM, no more than about 50 mM,
no more than about 100 mM or no more than about 500 mM.
[0098] The culture conditions can include, for example, liquid
culture procedures as well as fermentation and other large scale
culture procedures. As described herein, particularly useful yields
of the biosynthetic products of the invention can be obtained under
anaerobic or substantially anaerobic culture conditions.
[0099] As described herein, one exemplary growth condition for
achieving biosynthesis of aniline 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 refer 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.
[0100] The culture conditions described herein can be scaled up and
grown continuously for manufacturing of aniline. 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. Fermentation procedures are
particularly useful for the biosynthetic production of commercial
quantities of aniline. Generally, and as with non-continuous
culture procedures, the continuous and/or near-continuous
production of aniline will include culturing a non-naturally
occurring aniline producing organism of the invention in sufficient
nutrients and medium to sustain and/or nearly sustain growth in an
exponential phase. Continuous culture under such conditions can
include, for example, growth for 1 day, 2, 3, 4, 5, 6 or 7 days or
more. Additionally, continuous culture can include longer time
periods of 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. It is further understood that the time of
culturing the microbial organism of the invention is for a
sufficient period of time to produce a sufficient amount of product
for a desired purpose.
[0101] Fermentation procedures are well known in the art. Briefly,
fermentation for the biosynthetic production of aniline 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 are well known in the
art.
[0102] In addition to the above fermentation procedures using the
aniline producers of the invention for continuous production of
substantial quantities of aniline, the aniline producers also can
be, for example, simultaneously subjected to chemical synthesis
procedures to convert the product to other compounds or the product
can be separated from the fermentation culture and sequentially
subjected to chemical conversion to convert the product to other
compounds, if desired.
[0103] In some embodiments, methods for producing aniline include a
step of isolating aniline from the fermentation broth. This can be
achieved by means of standard extraction, distillation, salt
crystallization techniques, and combinations of these techniques
and those described above. For a basic product such as aniline, a
salt crystallization can include the formation of an acid salt of a
Bronsted or Lewis acid. Exemplary acid salts include, without
limitation, acetate, aspartate, benzoate, bicarbonate, carbonate,
bisulfate, sulfate, chloride, bromide, benzene sulfonate, methyl
sulfonate, phosphate, biphosphate, lactate, maleate, malate,
malonate, fumarate, lactate, tartrate, borate, camsylate, citrate,
edisylate, esylate, formate, fumarate, gluceptate, glucuronate,
gluconate oxalate, palmitate, pamoate, saccharate, stearate,
succinate, tartrate, tosylate and trifluoroacetate salts.
[0104] To generate better producers, metabolic modeling can be
utilized to optimize growth conditions. Modeling can also be used
to design gene knockouts that additionally optimize utilization of
the pathway (see, 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 U.S. Pat.
No. 7,127,379). Modeling analysis allows reliable predictions of
the effects on cell growth of shifting the metabolism towards more
efficient production of aniline.
[0105] One computational method for identifying and designing
metabolic alterations favoring biosynthesis of a desired product is
the OptKnock computational framework (Burgard et al., Biotechnol.
Bioeng. 84:647-657 (2003)). OptKnock is a metabolic modeling and
simulation program that suggests gene deletion or disruption
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 a
desired product or used in connection with the non-naturally
occurring microbial organisms for further optimization of
biosynthesis of a desired product.
[0106] 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 allow
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.
publication 2002/0168654, filed Jan. 10, 2002, in International
Patent No. PCT/US02/00660, filed Jan. 10, 2002, and U.S.
publication 2009/0047719, filed Aug. 10, 2007.
[0107] Another computational method for identifying and designing
metabolic alterations favoring biosynthetic production of a product
is a metabolic modeling and simulation system termed SimPheny.RTM..
This computational method and system is described in, for example,
U.S. publication 2003/0233218, filed Jun. 14, 2002, and in
International Patent Application No. PCT/US03/18838, filed Jun. 13,
2003. 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.
[0108] These computational approaches are 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.
[0109] 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 a desired compound in host microbial
organisms. 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.
[0110] The methods described above will provide one set of
metabolic reactions to disrupt. Elimination of each reaction within
the set or metabolic modification can result in a desired product
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.
[0111] Once identified, the set of reactions that are to be
disrupted in order to achieve production of a desired product 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 coupling of a
product are desired or when genetic reversion is less likely to
occur.
[0112] 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 a
desired 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 obligatorily 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. The integer cut method is well known in the art and can
be found described in, for example, 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..
[0113] The methods exemplified herein allow the construction of
cells and organisms that biosynthetically produce a desired
product, including the obligatory coupling of production of a
target biochemical product to growth of the cell or organism
engineered to harbor the identified genetic alterations. Therefore,
the computational methods described herein allow the identification
and implementation of metabolic modifications that are identified
by an in silico method selected from OptKnock or SimPheny.RTM.. 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.
[0114] 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)).
[0115] An in silico stoichiometric model of E. coli metabolism can
be 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 can be applied to pinpoint gene deletions
leading to the growth-coupled production of a desired product.
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] As disclosed herein, a nucleic acid encoding a desired
activity of an aniline pathway can be introduced into a host
organism. In some cases, it can be desirable to modify an activity
of an aniline pathway enzyme or protein to increase production of
aniline. For example, known mutations that increase the activity of
a protein or enzyme can be introduced into an encoding nucleic acid
molecule. Additionally, optimization methods can be applied to
increase the activity of an enzyme or protein and/or decrease an
inhibitory activity, for example, decrease the activity of a
negative regulator.
[0117] One such optimization method is directed evolution. Directed
evolution is a powerful approach that involves the introduction of
mutations targeted to a specific gene in order to improve and/or
alter the properties of an enzyme. Improved and/or altered enzymes
can be identified through the development and implementation of
sensitive high-throughput screening assays that allow the automated
screening of many enzyme variants (for example, >10.sup.4).
Iterative rounds of mutagenesis and screening typically are
performed to afford an enzyme with optimized properties.
Computational algorithms that can help to identify areas of the
gene for mutagenesis also have been developed and can significantly
reduce the number of enzyme variants that need to be generated and
screened. Numerous directed evolution technologies have been
developed (for reviews, see Hibbert et al., Biomol. Eng 22:11-19
(2005); Huisman and Lalonde, In Biocatalysis in the pharmaceutical
and biotechnology industries pgs. 717-742 (2007), Patel (ed.), CRC
Press; Otten and Quax. Biomol. Eng 22:1-9 (2005).; and Sen et al.,
Appl Biochem. Biotechnol 143:212-223 (2007)) to be effective at
creating diverse variant libraries, and these methods have been
successfully applied to the improvement of a wide range of
properties across many enzyme classes. Enzyme characteristics that
have been improved and/or altered by directed evolution
technologies include, for example: selectivity/specificity, for
conversion of non-natural substrates; temperature stability, for
robust high temperature processing; pH stability, for bioprocessing
under lower or higher pH conditions; substrate or product
tolerance, so that high product titers can be achieved; binding
(K.sub.m), including broadening substrate binding to include
non-natural substrates; inhibition (K.sub.i), to remove inhibition
by products, substrates, or key intermediates; activity (kcat), to
increases enzymatic reaction rates to achieve desired flux;
expression levels, to increase protein yields and overall pathway
flux; oxygen stability, for operation of air sensitive enzymes
under aerobic conditions; and anaerobic activity, for operation of
an aerobic enzyme in the absence of oxygen.
[0118] A number of exemplary methods have been developed for the
mutagenesis and diversification of genes to target desired
properties of specific enzymes. Such methods are well known to
those skilled in the art. Any of these can be used to alter and/or
optimize the activity of an aniline pathway enzyme or protein. Such
methods include, but are not limited to EpPCR, which introduces
random point mutations by reducing the fidelity of DNA polymerase
in PCR reactions (Pritchard et al., J Theor. Biol. 234:497-509
(2005)); Error-prone Rolling Circle Amplification (epRCA), which is
similar to epPCR except a whole circular plasmid is used as the
template and random 6-mers with exonuclease resistant thiophosphate
linkages on the last 2 nucleotides are used to amplify the plasmid
followed by transformation into cells in which the plasmid is
re-circularized at tandem repeats (Fujii et al., Nucleic Acids Res.
32:e145 (2004); and Fujii et al., Nat. Protoc. 1:2493-2497 (2006));
DNA or Family Shuffling, which typically involves digestion of two
or more variant genes with nucleases such as Dnase I or EndoV to
generate a pool of random fragments that are reassembled by cycles
of annealing and extension in the presence of DNA polymerase to
create a library of chimeric genes (Stemmer. Proc Natl Acad Sci USA
91:10747-10751 (1994); and Stemmer, Nature 370:389-391 (1994));
Staggered Extension (StEP), which entails template priming followed
by repeated cycles of 2 step PCR with denaturation and very short
duration of annealing/extension (as short as 5 sec) (Zhao et al.,
Nat. Biotechnol. 16:258-261 (1998)); Random Priming Recombination
(RPR), in which random sequence primers are used to generate many
short DNA fragments complementary to different segments of the
template (Shao et al., Nucleic Acids Res 26:681-683 (1998)).
[0119] Additional methods include Heteroduplex Recombination, in
which linearized plasmid DNA is used to form heteroduplexes that
are repaired by mismatch repair (Volkov et al, Nucleic Acids Res.
27:e18 (1999); and Volkov et al., Methods Enzyvmol. 328:456-463
(2000)); Random Chimeragenesis on Transient Templates (RACHITT),
which employs Dnase I fragmentation and size fractionation of
single stranded DNA (ssDNA) (Coco et al., Nat. Biotechnol.
19:354-359 (2001)); Recombined Extension on Truncated templates
(RETT), which entails template switching of unidirectionally
growing strands from primers in the presence of unidirectional
ssDNA fragments used as a pool of templates (Lee et al., J. Molec.
Catalysis 26:119-129 (2003)); Degenerate Oligonucleotide Gene
Shuffling (DOGS), in which degenerate primers are used to control
recombination between molecules; (Bergquist and Gibbs, Methods Mol.
Biol 352:191-204 (2007); Bergquist et al., Biomol. Eng 22:63-72
(2005): Gibbs et al., Gene 271:13-20 (2001)); Incremental
Truncation for the Creation of Hybrid Enzymes (ITCHY), which
creates a combinatorial library with 1 base pair deletions of a
gene or gene fragment of interest (Ostermeier et al., Proc. Natl.
Acad. Sci. USA 96:3562-3567 (1999); and Ostermeier et al., Nat.
Biotechnol. 17:1205-1209 (1999)); Thio-Incremental Truncation for
the Creation of Hybrid Enzymes (THIO-ITCHY), which is similar to
ITCHY except that phosphothioate dNTPs are used to generate
truncations (Lutz et al., Nucleic Acids Res 29:E16 (2001));
SCRATCHY, which combines two methods for recombining genes, ITCHY
and DNA shuffling (Lutz et al., Proc. Natl. Acad. Sci. USA
98:11248-11253 (2001)); Random Drift Mutagenesis (RNDM), in which
mutations made via epPCR are followed by screening/selection for
those retaining usable activity (Bergquist et al., Biomol. Eng.
22:63-72 (2005)); Sequence Saturation Mutagenesis (SeSaM), a random
mutagenesis method that generates a pool of random length fragments
using random incorporation of a phosphothioate nucleotide and
cleavage, which is used as a template to extend in the presence of
"universal" bases such as inosine, and replication of an
inosine-containing complement gives random base incorporation and,
consequently, mutagenesis (Wong et al., Biotechnol. J. 3:74-82
(2008); Wong et al., Nucleic Acids Res. 32:e26 (2004); and Wong et
al., Anal. Biochem. 341:187-189 (2005)); Synthetic Shuffling, which
uses overlapping oligonucleotides designed to encode "all genetic
diversity in targets" and allows a very high diversity for the
shuffled progeny (Ness et al., Nat. Biotechnol. 20:1251-1255
(2002)); Nucleotide Exchange and Excision Technology NexT, which
exploits a combination of dUTP incorporation followed by treatment
with uracil DNA glycosylase and then piperidine to perform endpoint
DNA fragmentation (Muller et al., Nucleic Acids Res. 33:e117
(2005)).
[0120] Further methods include Sequence Homology-Independent
Protein Recombination (SHIPREC), in which a linker is used to
facilitate fusion between two distantly related or unrelated genes,
and a range of chimeras is generated between the two genes,
resulting in libraries of single-crossover hybrids (Sieber et al.,
Nat. Biotechnol. 19:456-460 (2001)): Gene Site Saturation
Mutagenesis.TM. (GSSM.TM.), in which the starting materials include
a supercoiled double stranded DNA (dsDNA) plasmid containing an
insert and two primers which are degenerate at the desired site of
mutations (Kretz et al., Methods Enzymol. 388:3-11 (2004));
Combinatorial Cassette Mutagenesis (CCM), which involves the use of
short oligonucleotide cassettes to replace limited regions with a
large number of possible amino acid sequence alterations
(Reidhaar-Olson et al. Methods Enzymol. 208:564-586 (1991); and
Reidhaar-Olson et al. Science 241:53-57 (1988)); Combinatorial
Multiple Cassette Mutagenesis (CMCM), which is essentially similar
to CCM and uses epPCR at high mutation rate to identify hot spots
and hot regions and then extension by CMCM to cover a defined
region of protein sequence space (Reetz et al., Angew. Chem. Int.
Ed Engl. 40:3589-3591 (2001)); the Mutator Strains technique, in
which conditional ts mutator plasmids, utilizing the mutD5 gene,
which encodes a mutant subunit of DNA polymerase III, to allow
increases of 20 to 4000-X in random and natural mutation frequency
during selection and block accumulation of deleterious mutations
when selection is not required (Selifonova et al., Appl. Environ.
Microbiol. 67:3645-3649 (2001)): Low et al., J. Mol. Biol.
260:359-3680 (1996)).
[0121] Additional exemplary methods include Look-Through
Mutagenesis (LTM), which is a multidimensional mutagenesis method
that assesses and optimizes combinatorial mutations of selected
amino acids (Rajpal et al., Proc. Natl. Acad. Sci. USA
102:8466-8471 (2005)); Gene Reassembly, which is a DNA shuffling
method that can be applied to multiple genes at one time or to
create a large library of chimeras (multiple mutations) of a single
gene (Tunable GeneReassembly.TM. (TGR.TM.) Technology supplied by
Verenium Corporation), in Silico Protein Design Automation (PDA),
which is an optimization algorithm that anchors the structurally
defined protein backbone possessing a particular fold, and searches
sequence space for amino acid substitutions that can stabilize the
fold and overall protein energetics, and generally works most
effectively on proteins with known three-dimensional structures
(Hayes et al., Proc. Natl. Acad. Sci. USA 99:15926-15931 (2002));
and Iterative Saturation Mutagenesis (ISM), which involves using
knowledge of structure/function to choose a likely site for enzyme
improvement, performing saturation mutagenesis at chosen site using
a mutagenesis method such as Stratagene QuikChange (Stratagene; San
Diego Calif.), screening/selecting for desired properties, and,
using improved clone(s), starting over at another site and continue
repeating until a desired activity is achieved (Reetz et al., Nat.
Protoc. 2:891-903 (2007); and Reetz et al., Angew. Chem. Int. Ed
Engl. 45:7745-7751 (2006)).
[0122] Any of the aforementioned methods for mutagenesis can be
used alone or in any combination. Additionally, any one or
combination of the directed evolution methods can be used in
conjunction with adaptive evolution techniques, as described
herein.
Example I
Aniline Biosynthesis Using p-Aminobenzoate as the Precursor
[0123] This Example describes the generation of a microbial
organism capable of producing aniline using chorismate as the
precursor.
[0124] Escherichia coli is used as a target organism to engineer
the pathway using the enzymes aminodeoxychorismate synthase,
aminodeoxychorismate lyase, and 4-aminobenzoate carboxylyase as
shown in FIG. 2. E. coli provides a good host for generating a
non-naturally occurring microorganism capable of producing aniline.
E. coli is amenable to genetic manipulation and is known to be
capable of producing various products, like ethanol, acetic acid,
formic acid, lactic acid, and succinic acid, effectively under
anaerobic or microaerobic conditions.
[0125] To generate an E. coli strain engineered to produce aniline,
nucleic acids encoding the enzymes utilized in the disclosed
pathway, as described previously, are expressed in E. coli to the
desired extent using well known molecular biology techniques (see,
for example, Sambrook, supra, 2001; Ausubel supra, 1999: Roberts et
al., supra, 1989).
[0126] The native enzymes in E. coli can be modified or
heterologous enzymes can be introduced to produce significant
quantities of p-aminobenzoate. Further, 4-aminobenzoate
carboxylyase activity can be incorporated into the strain by
introducing the appropriate genes, such as shdB. C and D from
Sedimentibacter hydroxybenzoicus. The genes are cloned into the
pZE13 vector (Expressys, Ruelzheim, Germany) under the PA1/lacO
promoter. The plasmid is transformed into the recombinant E. coli
strain producing p-aminobenzoate to express the proteins and
enzymes required for aniline synthesis from this metabolite.
[0127] The resulting genetically engineered organism is cultured in
glucose containing medium following procedures well known in the
art (see, for example, Sambrook et al., supra. 2001). The
expression of the pathway genes is corroborated using methods well
known in the art for determining polypeptide expression or
enzymatic activity, including, for example, Northern blots, PCR
amplification of mRNA, immunoblotting. Enzymatic activities of the
expressed enzymes are confirmed using assays specific for the
individually activities. The ability of the engineered E. coli
strain to produce aniline is confirmed using HPLC, gas
chromatography-mass spectrometry (GCMS) or liquid
chromatography-mass spectrometry (LCMS).
[0128] Microbial strains engineered to have a functional aniline
synthesis pathway are further augmented by optimization for
efficient utilization of the pathway. Briefly, the engineered
strain is assessed to determine whether any of the exogenous genes
are expressed at a rate limiting level. Expression is increased for
any enzymes expressed at low levels that can limit the flux through
the pathway by, for example, introduction of additional gene copy
numbers.
[0129] To generate better producers, metabolic modeling is utilized
to optimize growth conditions. Modeling is also used to design gene
knockouts that additionally optimize utilization of the pathway
(see, 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).
Modeling analysis allows reliable predictions of the effects on
cell growth of shifting the metabolism towards more efficient
production of aniline. One modeling method is the bilevel
optimization approach, OptKnock (Burgard et al., Biotechnol.
Bioengineer. 84:647-657 (2003)), which is applied to select gene
knockouts that collectively result in better production of aniline.
Adaptive evolution also can be used to generate better producers
of, for example, the intermediate, chorismate or the product,
aniline. Adaptive evolution is performed to improve both growth and
production characteristics (Fong and Palsson, Nat. Genet.
36:1056-1058 (2004); Alper et al., Science 314:1565-1568 (2006)).
Based on the results, subsequent rounds of modeling, genetic
engineering and adaptive evolution can be applied to the aniline
producer to further increase production.
[0130] For large-scale production of aniline, the recombinant
organism is cultured in a fermenter using a medium known in the art
to support growth of the organism under anaerobic conditions.
Fermentations are performed in either a batch, fed-batch or
continuous manner. Anaerobic conditions are maintained by first
sparging the medium with nitrogen and then sealing culture vessel
(e.g., flasks can be sealed with a septum and crimp-cap).
Microaerobic conditions also can be utilized by providing a small
hole for limited aeration. The pH of the medium is maintained at a
pH of 7 by addition of an acid, such as H.sub.2SO.sub.4. The growth
rate is determined by measuring optical density using a
spectrophotometer (600 nm), and the glucose uptake rate by
monitoring carbon source depletion over time. Byproducts such as
undesirable alcohols, organic acids, and residual glucose can be
quantified by HPLC (Shimadzu) with an HPX-087 column (BioRad),
using a refractive index detector for glucose and alcohols, and a
UV detector for organic acids (Lin et al., Biotechnol Bioeng.
90:775-779 (2005)).
[0131] Throughout this application various publications have been
referenced. The disclosures of these publications in their
entireties, including GenBank and GI number publications, 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 examples provided above, it should be understood that
various modifications can be made without departing from the spirit
of the invention.
[0132] 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.
* * * * *