U.S. patent application number 14/609420 was filed with the patent office on 2015-07-30 for recombinant microorganisms and methods of use thereof.
The applicant listed for this patent is LANZATECH NEW ZEALAND LIMITED. Invention is credited to Bakir Al-Sinawi, Sashini De Tissera, Michael Koepke, Shilpa Nagaraju.
Application Number | 20150210987 14/609420 |
Document ID | / |
Family ID | 53678453 |
Filed Date | 2015-07-30 |
United States Patent
Application |
20150210987 |
Kind Code |
A1 |
Nagaraju; Shilpa ; et
al. |
July 30, 2015 |
RECOMBINANT MICROORGANISMS AND METHODS OF USE THEREOF
Abstract
The invention provides a carboxydotrophic acetogenic bacterium
comprising a disrupting mutation in a lactate biosynthesis pathway
enzyme and a method of producing a product by culturing the
bacterium in the presence of a substrate comprising carbon
monoxide. Preferably, the lactate biosynthesis pathway enzyme is
lactate dehydrogenase (LDH) or another enzyme that converts
pyruvate to lactate, wherein the disrupting mutation reduces or
eliminates the expression or activity of the enzyme such that the
bacterium produces a reduced amount of lactate or no lactate.
Inventors: |
Nagaraju; Shilpa; (Skokie,
IL) ; Al-Sinawi; Bakir; (Sydney, AU) ; De
Tissera; Sashini; (Auckland, NZ) ; Koepke;
Michael; (Skokie, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LANZATECH NEW ZEALAND LIMITED |
AUCKLAND |
|
NZ |
|
|
Family ID: |
53678453 |
Appl. No.: |
14/609420 |
Filed: |
January 29, 2015 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61933815 |
Jan 30, 2014 |
|
|
|
61944541 |
Feb 25, 2014 |
|
|
|
Current U.S.
Class: |
435/115 ;
435/116; 435/136; 435/139; 435/143; 435/144; 435/145; 435/146;
435/158; 435/161; 435/252.3 |
Current CPC
Class: |
C12P 7/18 20130101; Y02E
50/10 20130101; C12P 7/54 20130101; C12N 15/52 20130101; C12P 7/065
20130101; C12N 9/0006 20130101 |
International
Class: |
C12N 9/04 20060101
C12N009/04; C12N 15/52 20060101 C12N015/52; C12N 1/20 20060101
C12N001/20 |
Claims
1. A carboxydotrophic acetogenic bacterium comprising a disrupting
mutation in a lactate biosynthesis pathway enzyme.
2. The bacterium of claim 1, wherein the mutation reduces or
eliminates the expression or activity of the enzyme.
3. The bacterium of claim 1, wherein the bacterium produces a
reduced amount of lactate compared to a parental bacterium.
4. The bacterium of claim 1, wherein the bacterium produces
substantially no lactate.
5. The bacterium of claim 1, wherein the bacterium produces one or
more of ethanol, 2,3-butanediol, formate, pyruvate, succinate,
valine, leucine, isoleucine, malate, fumarate, 2-oxogluterate,
citrate, and citramalate.
6. The bacterium of claim 1, wherein the bacterium produces an
increased amount of one or more of ethanol, 2,3-butanediol,
formate, pyruvate, succinate, valine, leucine, isoleucine, malate,
fumarate, 2-oxogluterate, citrate, and citramalate compared to a
parental bacterium.
7. The bacterium of claim 1, wherein the enzyme natively converts
pyruvate to lactate.
8. The bacterium of claim 1, wherein the enzyme is lactate
dehydrogenase (LDH).
9. The bacterium of claim 1, wherein the bacterium is derived from
a parental bacterium selected from the group consisting of
Clostridium autoethanogenum, Clostridium ljungdahlii, and
Clostridium ragsdalei.
10. The bacterium of claim 9, wherein the parental bacterium is
Clostridium autoethanogenum deposited under DSMZ accession number
DSM23693.
11. A method of producing a product comprising culturing the
bacterium of claim 1 in the presence of a substrate comprising CO
whereby the bacterium produces a product.
12. The method of claim 11, wherein the mutation reduces or
eliminates the expression or activity of the enzyme.
13. The method of claim 11, wherein the bacterium produces a
reduced amount of lactate compared to a parental bacterium.
14. The method of claim 11, wherein the bacterium produces
substantially no lactate.
15. The method of claim 11, wherein the product comprises one or
more of ethanol, 2,3-butanediol, formate, pyruvate, succinate,
valine, leucine, isoleucine, malate, fumarate, 2-oxogluterate,
citrate, and citramalate.
16. The method of claim 11, wherein the bacterium produces an
increased amount of the product compared to a parental
bacterium.
17. The method of claim 11, wherein the enzyme natively converts
pyruvate to lactate.
18. The method of claim 11, wherein the enzyme is lactate
dehydrogenase (LDH).
19. The method of claim 11, wherein the bacterium is derived from a
parental bacterium selected from the group consisting of
Clostridium autoethanogenum, Clostridium ljungdahlii, and
Clostridium ragsdalei.
20. The method of claim 19, wherein the parental bacterium is
Clostridium autoethanogenum deposited under DSMZ accession number
DSM23693.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Patent Application 61/933,815 filed Jan. 30, 2014 and U.S.
Provisional Patent Application 61/944,541 filed Feb. 25, 2014, the
entirety of which are incorporated herein by reference.
SEQUENCE LISTING
[0002] This application includes a nucleotide/amino acid sequence
listing submitted concurrently herewith and identified as follows:
23,322 byte ASCII (text) file named "LT102US1_ST25.txt" created on
Jan. 29, 2015, the entirety of which is incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0003] An acetogen is a microorganism that generates or is capable
of generating acetate as a product of anaerobic respiration.
Typically, acetogens are obligately anaerobic bacteria that use the
Wood-Ljungdahl pathway as their main mechanism for energy
conservation and for synthesis of acetyl-CoA and acetyl-CoA-derived
products, such as acetate and ethanol (Ragsdale, Biochim Biophys
Acta, 1784: 1873-1898, 2008).
[0004] Many acetogens naturally produce at least two or more
products. However, this is not necessarily desirable on a
commercial scale, since the production of multiple products is
detrimental to the efficiency and yield of each individual product.
In particular, byproducts may divert carbon away from the
biosynthetic pathways of a target product, introduce toxicity
concerns, impede the recovery and separation of a target product,
complicate the control of fermentation conditions favoring a target
product, and serve as a substrate for contaminating
microorganisms.
[0005] For instance, acetogens such as Clostridium autoethanogenum,
Clostridium ljungdahlii, Clostridium ragsdalei (Kopke, Appl Environ
Microbiol, 77: 5467-5475, 2011), and Butyribacterium
methylotrophicum (Heiskanen, Enzyme Microb Technol, 41 362-367,
2007) may produce lactate as a byproduct. This production of
lactate reduces the efficiency and yield of target products, such
as ethanol, butanol, or 2,3-butanediol. Additionally, lactate may
be toxic to acetogens such as Clostridium autoethanogenum even at
low concentrations (Kopke, Appl Environ Microbiol, 77: 5467-5475,
2011) and may serve as a substrate for other bacteria, increasingly
the likelihood of bacterial contamination when lactate is produced.
Furthermore, separating lactate from other products, such as
ethanol, may require cumbersome processing steps.
[0006] Accordingly, there is a strong need for microorganisms and
methods that reduce or eliminate the production of byproducts, such
as lactate.
SUMMARY OF THE INVENTION
[0007] The invention provides a carboxydotrophic acetogenic
bacterium comprising a disrupting mutation in a lactate
biosynthesis pathway enzyme. In one embodiment, the disrupting
mutation reduces or eliminates the expression or activity of the
lactate biosynthesis pathway enzyme.
[0008] The disrupting mutation affects the ability of the bacterium
to produce lactate. In one embodiment, the bacterium of the
invention produces a reduced amount of lactate compared to a
parental bacterium. In one embodiment, the bacterium of the
invention produces substantially no lactate.
[0009] The bacterium of the invention may produce products, such as
one or more of ethanol, 2,3-butanediol, formate, pyruvate,
succinate, valine, leucine, isoleucine, malate, fumarate,
2-oxogluterate, citrate, and citramalate. In one embodiment, the
bacterium of the invention produces an increased amount of one or
more of ethanol, 2,3-butanediol, formate, pyruvate, succinate,
valine, leucine, isoleucine, malate, fumarate, 2-oxogluterate,
citrate, and citramalate compared to a parental bacterium.
[0010] In one embodiment, the lactate biosynthesis pathway enzyme
is an enzyme that natively converts pyruvate to lactate. In a
preferred embodiment, the lactate biosynthesis pathway enzyme is
lactate dehydrogenase (LDH).
[0011] The bacterium of the invention may be derived from a
parental bacterium, such as Clostridium autoethanogenum,
Clostridium ljungdahlii, and Clostridium ragsdalei. In a preferred
embodiment, the parental bacterium is Clostridium autoethanogenum
deposited under DSMZ accession number DSM23693.
[0012] The invention further provides a method of producing a
product comprising culturing the bacterium of the invention in the
presence of a substrate comprising CO whereby the bacterium
produces a product.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a diagram showing a LDH knockout strategy and the
primers used for screening.
[0014] FIG. 2 is a set of gel images. The first gel image shows
screening for single crossover integration of knockout plasmid
using primers Og24r/Og35f for 5' crossover and Og21f/Og36r for 3'
crossover in wild type (w) and transconjugant clone 6 (6). The
second gel image shows screening for double crossover using outer
flanking primers Og35f/Og36r and Og21f/Og24r.
[0015] FIG. 3 is a gel image showing colony PCR for Gene ID: 126803
Target 129S using primers LdhAF/R. A PCR product of 100 bp
indicated a wild-type genotype, while a product size of
approximately 1.9 kb confirmed the insertion of the group II intron
in the target site.
[0016] FIG. 4 is a gel image showing plasmid loss with primers
CatPR/RepHF. The plasmid loss was checked by amplification of the
resistance marker (catP) and the gram positive origin of
replication (pCB102).
[0017] FIG. 5A is a graph showing HPLC analysis of C.
autoethanogenum after 6 days of growth in serum bottles with 30 psi
steel mill off-gas (44% CO, 22% CO.sub.2, 2% H.sub.2, 32% N.sub.2)
as substrate. FIG. 5B is a graph showing HPLC analysis of C.
autoethanogenum with inactivated lactate dehydrogenase after 6 days
of growth in serum bottles with 30 psi steel mill off-gas (44% CO,
22% CO.sub.2, 2% H.sub.2, 32% N.sub.2) as substrate.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The inventors have discovered that disruption of the lactate
biosynthesis pathway in an acetogenic bacterium results in
increased or more efficient production of products, such as
ethanol, 2,3-butanediol, formate, succinate, 2-oxogluterate,
valine, leucine, and isoleucine, compared to a parental
microorganism, and may also result in increased or more efficient
production of pyruvate, malate, fumarate, and citrate, which are
precursors of succinate, 2-oxogluterate, valine, leucine, and
isoleucine. The production of valine, leucine, formate, and
pyruvate also obviates the need to supplement culture media with
these compounds, which may result in further cost savings.
Furthermore, reduction or elimination of lactate production by a
bacterium reduces or eliminates the toxic effects of lactate on the
bacterium.
[0019] The invention provides a carboxydotrophic acetogenic
bacterium comprising a disrupting mutation in a lactate
biosynthesis pathway enzyme.
[0020] "Mutation" refers to a modification in a nucleic acid or
protein in the bacterium of the invention compared to the wild-type
or parental microorganism from which the bacterium of the invention
is derived. The term "genetic modification" encompasses the term
"mutation." In one embodiment, the mutation may be a deletion,
insertion, or substitution of one or more nucleotides in a gene
encoding an enzyme. In another embodiment, the mutation may be a
deletion, insertion, or substitution of one or more amino acids in
an enzyme.
[0021] Typically, the mutation is a "disrupting mutation" that
reduces or eliminates (i.e., "disrupts") the expression or activity
of a lactate biosynthesis pathway enzyme. The disrupting mutation
may partially inactivate, fully inactivate, or delete a lactate
biosynthesis pathway enzyme or a gene encoding the enzyme. The
disrupting mutation may be a knockout (KO) mutation. The disrupting
mutation may be any mutation that reduces, prevents, or blocks the
biosynthesis of lactate. The disrupting mutation may include, for
example, a mutation in a gene encoding a lactate biosynthesis
pathway enzyme, a mutation in a genetic regulatory element involved
in the expression of a gene encoding a lactate biosynthesis pathway
enzyme, the introduction of a nucleic acid which produces a protein
that reduces or inhibits the activity of a lactate biosynthesis
pathway enzyme, or the introduction of a nucleic acid (e.g.,
antisense RNA, siRNA, CRISPR) or protein which inhibits the
expression of a lactate biosynthesis pathway enzyme.
[0022] The disrupting mutation results in a bacterium of the
invention that produces no lactate or substantially no lactate or a
reduced amount of lactate compared to the parental bacterium from
which the bacterium is derived. For example, the bacterium of the
invention may produce no lactate or at least about 1%, 3%, 5%, 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% less lactate than
the parental bacterium. For example, the bacterium of the invention
may produce less than about 0.001, 0.01, 0.10, 0.30, 0.50, or 1.0
g/L lactate. In contrast, depending on fermentation conditions,
unmodified C. autoethanogenum LZ1561 may produce up to about 2 g/L
lactate. Other unmodified bacterial strains may produce even more
lactate.
[0023] The disrupting mutation may be introduced using any method
known in the art. Exemplary methods include heterologous gene
expression, gene or promoter insertion or deletion, altered gene
expression or inactivation, enzyme engineering, directed evolution,
knowledge-based design, random mutagenesis methods, gene shuffling,
and codon optimization. Such methods are described, for example, in
Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Pleiss,
Curr Opin Biotechnol, 22: 611-617, 2011; and Park, Protein
Engineering and Design, CRC Press, 2010. The disrupting mutation
may be introduced using nucleic acids, such as single-stranded or
double-stranded DNA, RNA, cDNA, or combinations thereof, as is
appropriate. The nucleic acids may be referred to as constructs or
vectors, and may include one or more regulatory elements, origins
of replication, multicloning sites, and/or selectable markers. In
one embodiment, the nucleic acid may be adapted to disrupt a gene
encoding a lactate biosynthesis pathway enzyme in a parental
bacterium. In one embodiment, the nucleic acid may be adapted to
allow expression of one or more genes encoded by the nucleic acid.
Constructs or vectors may include plasmids (e.g., pMTL, pIMP,
pJIR), viruses (including bacteriophages), cosmids, and artificial
chromosomes. The constructs may remain extra-chromosomal upon
transformation of a parental bacterium or may be adapted for
integration into the genome of the bacterium. Accordingly,
constructs may include nucleic acid sequences adapted to assist
integration (e.g., a region which allows for homologous
recombination and targeted integration into the host genome) or
expression and replication of an extrachromosomal construct (e.g.,
origin of replication, promoter, and other regulatory
sequences).
[0024] The nucleic acids may be introduced using homologous
recombination. Such nucleic acids may include arms homologous to a
region within or flanking the gene to be disrupted ("homology
arms"). These homology arms allow homologous recombination and the
introduction, deletion, or substitution of one or more nucleotides
within the gene to be disrupted. While it is preferred that the
homology arms have 100% complementarity to the target region in the
genome, 100% complementarity is not required so long that the
sequence is sufficiently complementary to allow for targeted
recombination with the target region in the genome. Typically, the
homology arms will have a level of homology which would allow for
hybridization to a target region under stringent conditions
(Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). Knowledge
of the target nucleic acid sequences in a parental bacterium (i.e.,
the sequence of a target gene or target region in a parental
bacterium) is generally sufficient to design appropriate homology
arms. For example, to disrupt LDH, the flanking homology arms
described herein may be used (e.g., SEQ ID NOs: 1-2). In C.
ljungdahlii, homology arms may be designed based on GenBank
CP001666.1. For other strains, homology arms may be designed based
on other publically available nucleic acid sequence
information.
[0025] The "lactate biosynthesis pathway" is a pathway of reactions
resulting in the production of lactate. In one embodiment, the
lactate biosynthesis pathway comprises one or more enzymes that
convert pyruvate to lactate. In one embodiment, the lactate
biosynthesis pathway comprises a lactate dehydrogenase enzyme.
Depending on the bacterium, a number of different enzymes may be
involved in the lactate biosynthesis pathway. When a bacterium
comprises two or more enzymes in the lactate biosynthesis pathway,
e.g., two or more enzymes capable of converting pyruvate to
lactate, disrupting more than one such enzyme may have the effect
of increasing the production of a product above the level that may
be achieved by disrupting a single enzyme. In one embodiment, the
bacterium comprises disrupting mutations in two, three, four, five,
or more enzymes capable of converting pyruvate to lactate. While
disrupting expression and/or activity of all such enzymes may
provide some advantage in terms of product production, it is not
generally necessary to disrupt expression and/or activity of all
such enzymes to gain the benefits of the invention, namely
increased production of one or more main or target products.
[0026] In one embodiment, the lactate biosynthesis pathway enzyme
natively (i.e., endogenously or naturally) converts pyruvate to
lactate, such that the enzyme has lactate dehydrogenase activity.
The enzyme may have additional catalytic functions so long as it
also converts pyruvate to lactate. For example, the enzyme may be
any dehydrogenase having lactate dehydrogenase activity. The
introduction of a disrupting mutation to the enzyme that converts
pyruvate to lactate reduces or eliminates (i.e., "disrupts") the
expression or activity of that enzyme.
[0027] In a preferred embodiment, the lactate biosynthesis pathway
enzyme is lactate dehydrogenase (LDH). The introduction of a
disrupting mutation to LDH reduces or eliminates (i.e., "disrupts")
the expression or activity of LDH.
[0028] The bacterium of the invention may comprise one or more
other genetic modifications in addition to a disrupting mutation in
a lactate biosynthesis pathway enzyme, including genetic
modifications of one or more genes or proteins not associated with
the lactate biosynthesis pathway.
[0029] In one particular embodiment, the bacterium of the invention
may express an inhibitor of a lactate biosynthesis pathway enzyme
in addition to or instead of comprising a disrupting mutation in a
lactate biosynthesis pathway enzyme.
[0030] "Enzyme activity" refers broadly to enzymatic activity,
including, but not limited, to the activity of an enzyme, the
amount of an enzyme, or the availability of an enzyme to catalyze a
reaction. Accordingly, "decreasing" or "reducing" enzyme activity
includes decreasing or reducing the activity of an enzyme, the
amount of an enzyme, or the availability of an enzyme to catalyze a
reaction. An enzyme is "capable of converting" a first compound or
substrate into a second compound or product, if it can catalyze a
reaction in which at least a portion of the first compound is
converted into the second compound.
[0031] The term "variants" includes nucleic acids and proteins
whose sequence varies from the sequence of a reference nucleic acid
and protein, such as a sequence of a reference nucleic acid and
protein disclosed in the prior art or exemplified herein. The
invention may be practiced using variant nucleic acids or proteins
that perform substantially the same function as the reference
nucleic acid or protein. For example, a variant protein may perform
substantially the same function or catalyze substantially the same
reaction as a reference protein. A variant gene may encode the same
or substantially the same protein as a reference gene. A variant
promoter may have substantially the same ability to promote the
expression of one or more genes as a reference promoter.
[0032] Variant nucleic acids or proteins with substantially the
same level of activity as a reference nucleic acid or protein may
be referred to herein as "functionally equivalent variants." By way
of example, functionally equivalent variants of a nucleic acid may
include allelic variants, fragments of a gene, mutated genes,
polymorphisms, and the like. Homologous genes from other
microorganisms are also examples of functionally equivalent
variants. These include homologous genes in species such as
Clostridium acetobutylicum, Clostridium beijerinckii, or
Clostridium ljungdahlii, the details of which are publicly
available on websites such as Genbank or NCBI. Functionally
equivalent variants also includes nucleic acids whose sequence
varies as a result of codon optimization for a particular organism.
A functionally equivalent variant of a nucleic acid will preferably
have at least approximately 70%, approximately 80%, approximately
85%, approximately 90%, approximately 95%, approximately 98%, or
greater nucleic acid sequence identity (percent homology) with the
referenced nucleic acid. A functionally equivalent variant of a
protein will preferably have at least approximately 70%,
approximately 80%, approximately 85%, approximately 90%,
approximately 95%, approximately 98%, or greater amino acid
identity (percent homology) with the referenced protein. The
functional equivalence of a variant nucleic acid or protein may be
evaluated using any method known in the art.
[0033] However, variant nucleic acids or proteins may also have a
reduced level of activity compared to a reference nucleic acid or
protein. For example, a variant nucleic acid may have a reduced
level of expression or a variant enzyme may have a reduced ability
to catalyze a particular reaction compared to a reference nucleic
acid or enzyme, respectively. Enzyme assays and kits for assessing
the activity of enzymes in the lactate biosynthesis pathway are
known in the art (Wang, J Bacteriol, 195: 4373-4386, 2013;
Sigma-Aldrich (MAK066), Thermo (88953); Worthington Biochemical
Corporation (LS002755)).
[0034] Nucleic acids may be delivered to a bacterium of the
invention using any method known in the art. For example, nucleic
acids may be delivered as naked nucleic acids or may be formulated
with one or more agents (e.g., liposomes). Restriction inhibitors
may be used in certain embodiments (Murray, Microbiol Molec Biol
Rev, 64: 412-434, 2000). By way of example, transformation
(including transduction or transfection) may be achieved by
electroporation, ultrasonication, polyethylene glycol-mediated
transformation, chemical or natural competence, protoplast
transformation, prophage induction, or conjugation (see, e.g.,
Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). The use
of electroporation has been reported for several carboxydotrophic
acetogens, including Clostridium ljungdahlii (Koepke, PNAS,
107:13087-13092, 2010; WO/2012/053905), Clostridium autoethanogenum
(WO/2012/053905), Clostridium aceticum (Schiel-Bengelsdorf,
Synthetic Biol, 15: 2191-2198, 2012), and Acetobacterium woodii
(Stratz, Appl Environ Microbiol, 60: 1033-1037, 1994). The use of
electroporation has also been reported in Clostridia, including
Clostridium acetobutylicum (Mermelstein, Biotechnol, 10: 190-195,
1992), and Clostridium cellulolyticum (Jennert, Microbiol, 146:
3071-3080, 2000). Prophage induction has been demonstrated for
carboxydotrophic acetogens, including Clostridium scatologenes
(Parthasarathy, Development of a Genetic Modification System in
Clostridium scatologenes ATCC 25775 for Generation of Mutants,
Masters Project, Western Kentucky University, 2010), and
conjugation been described for many Clostridia, including
Clostridium difficile (Herbert, FEMS Microbiol Lett, 229: 103-110,
2003) and Clostridium acetobuylicum (Williams, J Gen Microbiol,
136: 819-826, 1990). In certain embodiments having active
restriction enzyme systems, it may be necessary to methylate a
nucleic acid before introduction of the nucleic acid into the
bacterium of the invention (WO 2012/105853).
[0035] The term "recombinant" indicates that a nucleic acid,
protein, or microorganism is the product of genetic modification,
mutation, or recombination. Generally, the term "recombinant"
refers to a nucleic acid, protein, or microorganism that contains
or is encoded by genetic material derived from multiple sources,
such as two or more different strains or species of microorganisms.
As used herein, the term "recombinant" may also be used to describe
a microorganism that comprises a mutated nucleic acid or protein,
including a mutated form of an endogenous nucleic acid or
protein.
[0036] A "parental bacterium" is a bacterium used to generate a
bacterium of the invention. The parental bacterium may be a
naturally-occurring bacterium (i.e., a wild-type bacterium) or a
bacterium that has been previously modified (i.e., a mutant or
recombinant bacterium). The bacterium of the invention may be
modified to express a lower amount of an enzyme compared to the
parental bacterium, or the bacterium of the invention may be
modified to not express an enzyme that is expressed by the parental
bacterium. In one embodiment, the parental bacterium is Clostridium
autoethanogenum, Clostridium ljungdahlii, or Clostridium ragsdalei.
In a preferred embodiment, the parental bacterium is Clostridium
autoethanogenum deposited under DSMZ accession DSM23693 (i.e.,
Clostridium autoethanogenum LZ1561).
[0037] The term "derived from" indicates that a nucleic acid,
protein, or microorganism is modified or adapted from a different
(e.g., a parental or wild-type) nucleic acid, protein, or
microorganism, so as to produce a new nucleic acid, protein, or
microorganism. Such modifications or adaptations typically include
insertion, deletion, mutation, or substitution of nucleic acids or
genes. Generally, the bacterium of the invention is derived from a
parental bacterium. In one embodiment, the bacterium of the
invention is derived from Clostridium autoethanogenum, Clostridium
ljungdahlii, or Clostridium ragsdalei. In a preferred embodiment,
the bacterium of the invention is derived from Clostridium
autoethanogenum LZ1561, which is deposited under DSMZ accession
DSM23693.
[0038] In one embodiment, the parental bacterium is selected from
the group of carboxydotrophic acetogenic bacteria comprising the
species Clostridium autoethanogenum, Clostridium ljungdahlii,
Clostridium ragsdalei, Clostridium coskatii, Clostridium
carboxidivorans, Clostridium drakei, Clostridium scatologenes,
Clostridium aceticum, Clostridium formicoaceticum, Clostridium
magnum, Acetobacterium woodii, Alkalibaculum bacchii, Moorella
thermoacetica, Sporomusa ovate, Butyribacterium methylotrophicum,
Blautia producta, Eubacterium limosum, and Thermoanaerobacter
kiuvi. These carboxydotrophic acetogenic bacteria are defined by
their ability to grow chemoautotrophically on gaseous one-carbon
sources such as carbon monoxide (CO) and carbon dioxide (CO.sub.2),
use carbon monoxide (CO) and/or hydrogen (H.sub.2) as energy
sources under anaerobic conditions, and produce acetyl-CoA,
acetate, and other products. They share the same mode of
fermentation, the Wood-Ljungdahl or reductive acetyl-CoA pathway,
and are defined by the presence of the enzyme set consisting of
carbon monoxide dehydrogenase (CODH), hydrogenase, formate
dehydrogenase, formyl-tetrahydrofolate synthetase,
methylene-tetrahydrofolate dehydrogenase, formyl-tetrahydrofolate
cyclohydrolase, methylene-tetrahydrofolate reductase, and carbon
monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS), which
combination is characteristic and unique to this type of bacteria
(Drake, The Prokaryotes, 354-420, Springer, New York, N.Y., 2006).
In contrast to chemoheterotrophic growth of sugar-fermenting
bacteria that convert a substrate into biomass, secondary
metabolites, and pyruvate, from which products are formed directly
or via acetyl-CoA, acetogens channel a substrate directly into
acetyl-CoA, from which products, biomass, and secondary metabolites
are formed.
[0039] In a preferred embodiment, the bacterium of the invention is
derived from a parental microorganism comprising a lactate
dehydrogenase, wherein the bacterium of the invention comprises a
disrupting mutation in the lactate dehydrogenase. For example, the
parental microorganism may be C. autoethanogenum comprising a
nucleic acid sequence comprising GenBank AEI90736.1 or an amino
acid sequence comprising GenBank CP006763.1, KEGG
CAETHG.sub.--1147, or GenBank HQ876025.1. The parental
microorganism may be C. ljungdahlii comprising a nucleic acid
sequence comprising GenBank YP.sub.--003781368.1 or an amino acid
sequence comprising GenBank CP001666.1 or KEGG CLJU_c32190. The
parental microorganism may be C. ragsdalei comprising a nucleic
acid sequence comprising GenBank AEI90737.1 or an amino acid
sequence comprising GenBank HQ876026.1. Other parental bacteria may
have other nucleic acid and amino acid sequences.
[0040] A "carboxydotroph" is a microorganism capable of tolerating
a high concentration of carbon monoxide (CO). Typically, the
bacterium of the invention is a carboxydotroph.
[0041] The bacterium of the invention may be derived from the
cluster of carboxydotrophic Clostridia comprising the species
Clostridium autoethanogenum, Clostridium ljungdahlii, Clostridium
ragsdalei, and related isolates, including, but not limited to,
strains Clostridium autoethanogenum JAI-1T (DSM10061) (Abrini, Arch
Microbiol, 161: 345-351, 1994), Clostridium autoethanogenum LBS1560
(DSM19630) (WO 2009/064200), Clostridium autoethanogenum LZ1561
(DSM23693), Clostridium ljungdahlii PETCT (DSM13528=ATCC 55383)
(Tanner, Int J Syst Bacteriol, 43: 232-236, 1993), Clostridium
ljungdahlii ERI-2 (ATCC 55380) (U.S. Pat. No. 5,593,886),
Clostridium ljungdahlii C-01 (ATCC 55988) (U.S. Pat. No.
6,368,819), Clostridium ljungdahlii O-52 (ATCC 55989) (U.S. Pat.
No. 6,368,819), Clostridium ragsdalei P11T (ATCC BAA-622) (WO
2008/028055), related isolates such as "Clostridium coskatii" (U.S.
Publication 2011/0229947), or mutated strains such as Clostridium
ljungdahlii OTA-1 (Tirado-Acevedo, Production of Bioethanol from
Synthesis Gas Using Clostridium ljungdahlii, PhD thesis, North
Carolina State University, 2010).
[0042] These strains form a subcluster within the Clostridial rRNA
cluster I and their 16S rRNA gene is more than 99% identical with a
similar low GC content of around 30%. However, DNA-DNA
reassociation and DNA fingerprinting experiments showed that these
strains belong to distinct species (WO 2008/028055). The strains of
this cluster are defined by common characteristics, having both a
similar genotype and phenotype, and they all share the same mode of
energy conservation and fermentative metabolism. Furthermore, the
strains of this cluster lack cytochromes and conserve energy via an
Rnf complex. All species of this cluster have a similar morphology
and size (logarithmic growing cells are between 0.5-0.7.times.3-5
.mu.m), are mesophilic (optimal growth temperature between
30-37.degree. C.), and are strictly anaerobic (Abrini, Arch
Microbiol, 161: 345-351, 1994; Tanner, Int J Syst Bacteriol, 43:
232-236, 1993; and WO 2008/028055). Moreover, they all share the
same major phylogenetic traits, such as same pH range (pH 4-7.5,
with an optimal initial pH of 5.5-6), strong autotrophic growth on
CO-containing gases with similar growth rates, and a similar
metabolic profile with ethanol and acetic acid as main fermentation
end products, and small amounts of 2,3-butanediol and lactic acid
formed under certain conditions (Abrini, Arch Microbiol, 161:
345-351, 1994; Kopke, Curr Opin Biotechnol, 22: 320-325, 2011;
Tanner, Int J Syst Bacteriol, 43: 232-236, 1993; and WO
2008/028055). Indole production was observed with all three species
as well.
[0043] However, the species differentiate in substrate utilization
of various sugars (e.g., rhamnose, arabinose), acids (e.g.,
gluconate, citrate), amino acids (e.g., arginine, histidine), or
other substrates (e.g., betaine, butanol). Moreover, some of the
species were found to be auxotrophic to certain vitamins (e.g.,
thiamine, biotin) while others were not. The organization and
number of Wood-Ljungdahl pathway genes, responsible for gas uptake,
has been found to be the same in all species, despite differences
in nucleic and amino acid sequences (Kopke, Curr Opin Biotechnol,
22: 320-325, 2011). Also, reduction of carboxylic acids into their
corresponding alcohols has been shown in a range of these
microorganisms (Perez, Biotechnol Bioeng, 110:1066-1077, 2012).
These traits are therefore not specific to one microorganism, like
Clostridium autoethanogenum or Clostridium ljungdahlii, but rather
general traits for carboxydotrophic, ethanol-synthesizing
Clostridia and it can be anticipated that mechanisms work similarly
across these strains, although there may be differences in
performance.
[0044] An "acetogen" is a microorganism that generates or is
capable of generating acetate as a product of anaerobic
respiration. Typically, acetogens are obligately anaerobic bacteria
that use the Wood-Ljungdahl pathway as their main mechanism for
energy conservation and for synthesis of acetyl-CoA and
acetyl-CoA-derived products, such as acetate (Ragsdale, Biochim
Biophys Acta, 1784: 1873-1898, 2008). In a preferred embodiment,
the bacterium of the invention is an acetogen.
[0045] The invention further provides a method of producing a
product comprising culturing the bacterium of the invention in the
presence of a substrate comprising CO whereby the bacterium of the
invention produces a product.
[0046] The term "substrate" refers to a carbon and/or energy source
for the bacterium of the invention. Typically, the substrate is a
gaseous substrate that comprises carbon monoxide (CO). The
substrate may comprise a major proportion of CO, such as about 20%
to 100%, 20% to 70%, 30% to 60%, or 40% to 55% CO by volume. In
particular embodiments, the substrate comprises about 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, or 60% CO by volume. The bacterium of
the invention generally converts at least a portion of the CO in
the substrate to a product.
[0047] While it is not necessary for the substrate to contain any
hydrogen (H.sub.2), the presence of H.sub.2 should not be
detrimental to product formation and may result improved overall
efficiency. For example, in particular embodiments, the substrate
may comprise an approximate ratio of H.sub.2:CO of 2:1, 1:1, or
1:2. In one embodiment, the substrate comprises less than about
30%, 20%, 15%, or 10% H.sub.2 by volume. In other embodiments, the
substrate comprises low concentrations of H.sub.2, for example,
less than 5%, less than 4%, less than 3%, less than 2%, or less
than 1% H.sub.2. In further embodiments, the substrate contains
substantially no H.sub.2.
[0048] The substrate may also contain carbon dioxide (CO.sub.2),
for example, about 1% to 80% or 1% to 30% CO.sub.2 by volume. In
one embodiment, the substrate comprises less than about 20%
CO.sub.2 by volume. In further embodiments, the substrate comprises
less than about 15%, 10%, or 5% CO.sub.2 by volume. In another
embodiment, the substrate contains substantially no CO.sub.2.
[0049] Although the substrate is typically gaseous, the substrate
may also be provided in alternative forms. For example, the
substrate may be dissolved in a liquid saturated with a
CO-containing gas using a microbubble dispersion generator
(Hensirisak, Appl Biochem Biotechnol, 101: 211-227, 2002). By way
of further example, the substrate may be adsorbed onto a solid
support.
[0050] The substrate may be a waste gas obtained as a by-product of
an industrial process or from some other source, such as from
automobile exhaust fumes or biomass gasification. In certain
embodiments, the industrial process is selected from the group
consisting of ferrous metal products manufacturing, such as a steel
mill manufacturing, non-ferrous products manufacturing, petroleum
refining processes, coal gasification, electric power production,
carbon black production, ammonia production, methanol production,
and coke manufacturing. In these embodiments, the CO-containing gas
may be captured from the industrial process before it is emitted
into the atmosphere, using any convenient method. The CO may be a
component of syngas, i.e., a gas comprising carbon monoxide and
hydrogen. The CO produced from industrial processes is normally
flared off to produce CO.sub.2 and therefore the invention has
particular utility in reducing CO.sub.2 greenhouse gas emissions.
The composition of the substrate may have a significant impact on
the efficiency and/or cost of the reaction. For example, the
presence of oxygen (O.sub.2) may reduce the efficiency of an
anaerobic fermentation process. Depending on the composition of the
substrate, it may be desirable to treat, scrub, or filter the
substrate to remove any undesired impurities, such as toxins,
undesired components, or dust particles, and/or increase the
concentration of desirable components.
[0051] The bacterium of the invention may be cultured to produce
one or more products. Generally, the bacterium of the invention
produces one or more products selected from the group consisting of
ethanol, 2,3-butanediol, formate, pyruvate, succinate, valine,
leucine, isoleucine, malate, fumarate, 2-oxogluterate, citrate, and
citramalate. The bacterium of the invention may also produce other
products, such as acetolactate or acetoin malate.
[0052] In a preferred embodiment, the bacterium of the invention
produces an increased amount of one or more of ethanol,
2,3-butanediol, formate, pyruvate, succinate, valine, leucine,
isoleucine, malate, fumarate, 2-oxogluterate, citrate, and
citramalate compared to a parental bacterium. For example, the
bacterium of the invention may produce about 1%, 3%, 5%, 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%,
200%, 300%, 400%, or 500% more of one or more products compared to
the parental bacterium from which the bacterium of the invention is
derived. This increase in product production may be due, at least
in part, to the disrupting mutation in the lactate biosynthesis
pathway enzyme, which diverts carbon and energy away from the
production of lactate and towards the production of other
products.
[0053] The term "main product" refers to the single product
produced in the highest concentration and/or yield. In one
embodiment, the main product is ethanol or 2,3-butanediol.
[0054] Additionally, it is possible to engineer the bacterium of
the invention to favor the production of one or more products over
one or more other products. For example, disrupting the conversion
of pyruvate to lactate may favor the production of 2,3-butanediol,
formate, malate, fumarate, citrate, succinate and 2-oxogluterate
over the production of valine, leucine and isoleucine.
[0055] Herein, recitation of a product (e.g., citrate) includes
both salt (e.g., citrate) and acid (e.g., citric acid) forms of the
product. Oftentimes, a mixture of the salt and acid forms of the
product will be present in a fermentation broth, in a ratio that
varies depending on the pH of the broth. As further examples, the
term "acetate" encompasses acetate and acetic acid, the term
"formate" encompasses formate and formic acid, the term "malate"
encompasses malate and malic acid, and the term "lactate"
encompasses lactate and lactic acid.
[0056] Unless the context requires otherwise, reference to any
compound herein which may exist in one or more isomeric forms (for
example, D, L, meso, S, R, cis, or trans forms) should be taken
generally to encompass any one or more such isomers of the
compound. For example, reference to "lactate" generally encompasses
both the D and L isomers of lactate.
[0057] Typically, the culture is performed in a bioreactor. The
term "bioreactor" includes a culture/fermentation device consisting
of one or more vessels, towers, or piping arrangements, such as a
continuous stirred tank reactor (CSTR), immobilized cell reactor
(ICR), trickle bed reactor (TBR), bubble column, gas lift
fermenter, static mixer, or other vessel or other device suitable
for gas-liquid contact. In some embodiments, the bioreactor may
comprise a first growth reactor and a second culture/fermentation
reactor. The substrate may be provided to one or both of these
reactors. As used herein, the terms "culture" and "fermentation"
are used interchangeably. These terms encompass both the growth
phase and product biosynthesis phase of the culture/fermentation
process.
[0058] The culture is generally maintained in an aqueous culture
medium that contains nutrients, vitamins, and/or minerals
sufficient to permit growth of the bacterium. Preferably the
aqueous culture medium is a minimal anaerobic microbial growth
medium. Suitable media are known in the art and described, for
example, in U.S. Pat. No. 5,173,429, U.S. Pat. No. 5,593,886, and
WO 2002/008438.
[0059] The culture/fermentation should desirably be carried out
under appropriate conditions for production of the target product.
Reaction conditions to consider include pressure (or partial
pressure of CO), temperature, gas flow rate, liquid flow rate,
media pH, media redox potential, agitation rate (if using a
continuous stirred tank reactor), inoculum level, maximum gas
substrate concentrations to ensure that CO in the liquid phase does
not become limiting, and maximum product concentrations to avoid
product inhibition. In particular, the rate of introduction of the
CO-containing substrate may be controlled to ensure that the
concentration of CO in the liquid phase does not become limiting,
since products may be consumed by the culture under CO-limited
conditions.
[0060] The terms "increasing the efficiency," "increased
efficiency," and the like, when used in relation to a fermentation
process, include, but are not limited to, increasing one or more of
the rate of growth of microorganisms catalyzing the fermentation,
the growth and/or product production rate, the volume of desired
product (such as alcohols) produced per volume of substrate
consumed, the rate of production or level of production of the
desired product, and the relative proportion of the desired product
produced compared with other by-products of the fermentation.
[0061] Operating a bioreactor at elevated pressures allows for an
increased rate of CO mass transfer from the gas phase to the liquid
phase. Accordingly, it is generally preferable to perform the
culture/fermentation at pressures higher than atmospheric pressure.
Also, since a given CO conversion rate is, in part, a function of
the substrate retention time and retention time dictates the
required volume of a bioreactor, the use of pressurized systems can
greatly reduce the volume of the bioreactor required and,
consequently, the capital cost of the culture/fermentation
equipment. According to examples in U.S. Pat. No. 5,593,886,
reactor volume can be reduced in linear proportion to increases in
reactor operating pressure. In other words, a bioreactor operated
at 10 atmospheres of pressure need only be one tenth the volume of
a bioreactor operated at 1 atmosphere of pressure. Additionally, WO
2002/008438 describes gas-to-ethanol fermentations performed under
pressures of 30 psig and 75 psig, giving ethanol productivities of
150 g/L/day and 369 g/L/day, respectively. In contrast,
fermentations performed using similar media and input gas
compositions at atmospheric pressure were found to produce between
10 and 20 times less ethanol per litre per day.
[0062] The method of the invention may further comprise recovering
or purifying one or more products. For example, ethanol or a mixed
alcohol stream containing ethanol and/or other products may be
recovered from a fermentation broth by any method known in the art,
including fractional distillation, evaporation, pervaporation, or
extractive fermentation (e.g., liquid-liquid extraction).
Byproducts, such as acetate or acids, may also be recovered from a
fermentation broth using any method known in the art, including
activated charcoal adsorption systems, electrodialysis, or
continuous gas stripping. In one embodiment, a product may be
recovered from a fermentation broth by continuously removing a
portion of the broth from the bioreactor, separating microbial
cells from the broth (conveniently by filtration), and recovering
the product from the broth. The separated microbial cells may be
returned to the bioreactor. Additionally, cell-free permeate may
also be returned to the bioreactor after the product has been
removed, optionally with supplementation of nutrients, such as B
vitamins.
[0063] Succinate can be recovered from a fermentation broth using,
for example, acidification, electrodialysis coupled with
ion-exchange chromatography (Song, Enzyme Microb Technol, 39:
352-361, 2006), precipitation with Ca(OH) coupled with filtration
and addition of sulfuric acid (Lee, Appl Microbiol Biotechnol, 79:
11-22, 2008), or reactive extraction with amine-based extractants
such as tri-n-octylamine (Huhet, Proc Biochem 41: 1461-1465, 2006).
For all methods, it is crucial to have the free acid form, not the
salt. Most biotechnological production processes for succinic acid,
however, operate at a neutral or slightly acidic pH of 6-7. Given
the pKa of succinic acid (pKa=4.16 and 5.61), the majority of
succinic acid is present as salt and not as free acid under these
conditions. C. autoethanogenum and other carboxydotrophic
acetogens, however, are known to tolerate and grow at a desirably
low pH of 4-6.
[0064] Branched-chain amino acids, such as valine, leucine, and
isoleucine, may be recovered from a fermentation broth using
concentration (e.g., via reverse osmosis), crystallization or
removal of the biomass (e.g., via ultrafiltration or
centrifugation), or ion exchange chromatography (Ikeda, Microbial
Production of L-Amino Acids, 1-35, 2003).
[0065] 2,3-butanediol, formate, 2-oxogluterate, and other products
may be recovered from a fermentation broth using any method known
in the art. For example, low concentrations of 2,3-butanediol may
be recovered using membrane techniques, such as electrodialysis,
involving the application of a suitable potential across a
selective ion permeable membrane. Other suitable techniques include
nanofiltration, wherein monovalent ions selectively pass through a
membrane under pressure.
EXAMPLES
[0066] The following examples further illustrate the invention but,
of course, should not be construed to limit its scope in any
way.
Example 1
[0067] This example describes general materials and methods.
[0068] C. autoethanogenum DSM10061 and DSM23693 (a derivate of
DSM10061) and C. ljungdahlii DSM13528 were sourced from DSMZ (The
German Collection of Microorganisms and Cell Cultures,
Inhoffenstra.beta.e 7 B, 38124 Braunschweig, Germany). C. ragsdalei
ATCC BAA-622 was sourced from ATCC (American Type Culture
Collection, Manassas, Va. 20108, USA). E. coli DH5.alpha. was
sourced from Invitrogen (Carlsbad, Calif. 92008, USA).
[0069] E. coli was grown aerobic at 37.degree. C. in LB
(Luria-Bertani) medium. Solid media contained 1.5% agar.
TABLE-US-00001 Amount per 1.0 L LB medium component of LB medium
Tryptone 10 g Yeast extract 5 g NaCl 10 g
[0070] Clostridium strains were grown at 37.degree. C. in PETC
medium at pH 5.6 using standard anaerobic techniques (Hungate,
Methods Microbiol, 3B: 117-132, 1969; Wolfe, Adv Microbiol Physiol,
6: 107-146, 1971). Fructose (heterotrophic growth) or 30 psi
CO-containing steel mill gas (collected from New Zealand Steel site
in Glenbrook, NZ; composition: 44% CO, 32% N.sub.2, 22% CO.sub.2,
2% H.sub.2) in the headspace (autotrophic growth) was used as
substrate. For solid media, 1.2% bacto agar (BD, Franklin Lakes,
N.J. 07417, USA) was added.
TABLE-US-00002 Amount per 1.0 L of PETC medium component PETC
medium NH.sub.4Cl 1 g KCl 0.1 g MgSO.sub.4.cndot.7H.sub.2O 0.2 g
NaCl 0.8 g KH.sub.2PO.sub.4 0.1 g CaCl.sub.2 0.02 g Trace metal
solution (see below) 10 ml Wolfe's vitamin solution (see below) 10
ml Yeast extract (optional) 1 g Resazurin (2 g/L stock) 0.5 ml
NaHCO.sub.3 2 g Reducing agent solution (see below) 0.006-0.008%
(v/v) Fructose (for heterotrophic growth) 5 g
TABLE-US-00003 Amount per 1.0 L of Trace metal solution component
trace metal solution Nitrilotriacetic acid 2 g
MnSO.sub.4.cndot.H.sub.2O 1 g
Fe(SO.sub.4).sub.2(NH.sub.4).sub.2.cndot.6H.sub.2O 0.8 g
CoCl.sub.2.cndot.6H.sub.2O 0.2 g ZnSO.sub.4.cndot.7H.sub.2O 0.2 mg
CuCl.sub.2.cndot.2H.sub.2O 0.02 g NaMoO.sub.4.cndot.2H.sub.2O 0.02
g Na.sub.2SeO.sub.3 0.02 g NiCl.sub.2.cndot.6H.sub.2O 0.02 g
Na.sub.2WO.sub.4.cndot.2H.sub.2O 0.02 g
TABLE-US-00004 Amount per 1.0 L of Wolfe's vitamin solution
component Wolfe's vitamin solution Biotin 2 mg Folic acid 2 mg
Pyridoxine hydrochloride 10 mg Thiamine HCl 5 mg Riboflavin 5 mg
Nicotinic acid 5 mg Calcium D-(+)-pantothenate 5 mg Vitamin B12 0.1
mg P-aminobenzoic acid 5 mg Thioctic acid 5 mg
TABLE-US-00005 Reducing agent Amount per 100 mL of solution
component reducing agent solution NaOH 0.9 g Cysteine-HCl 4 g
Na.sub.2S 4 g
[0071] Fermentations with C. autoethanogenum DSM23693 were carried
out in 1.5 L bioreactors at 37.degree. C. using CO-containing steel
mill gas as sole energy and carbon source. A defined medium was
prepared, containing: MgCl, CaCl.sub.2 (0.5 mM), KCl (2 mM),
H.sub.3PO.sub.4 (5 mM), Fe (100 .mu.M), Ni, Zn (5 .mu.M), Mn, B, W,
Mo, Se (2 .mu.M). The medium was transferred into the bioreactor
and autoclaved at 121.degree. C. for 45 minutes. After autoclaving,
the medium was supplemented with thiamine, pantothenate (0.05
mg/l), and biotin (0.02 mg/l) and reduced with 3 mM cysteine-HCl.
To achieve anaerobic conditions, the reactor vessel was sparged
with nitrogen through a 0.2 .mu.m filter. Prior to inoculation, the
gas was switched to CO-containing steel mill gas, feeding
continuously to the reactor. The gas flow was initially set at 80
ml/min and increased to 200 ml/min during mid-exponential phase,
while the agitation was increased from 200 rpm to 350 rmp.
Na.sub.2S was dosed into the bioreactor at 0.25 ml/hr. Once the
OD600 reached 0.5, the bioreactor was switched to continuous mode
at a rate of 1.0 ml/min (dilution rate 0.96 d.sup.-1). Samples were
taken to measure the biomass and metabolites. Additionally,
headspace analysis of the in- and out-flowing gas was performed on
regular basis.
[0072] Gas composition of the headspace was measured on a Varian
CP-4900 micro GC with two installed channels. Channel 1 was a 10 m
Mol-sieve column running at 70.degree. C., 200 kPa argon and a
backflush time of 4.2 s, while channel 2 was a 10 m PPQ column
running at 90.degree. C., 150 kPa helium and no backflush. The
injector temperature for both channels was 70.degree. C. Runtimes
were set to 120 s, but all peaks of interest would usually elute
before 100 s.
[0073] HPLC analysis of metabolic end products was performed using
an Agilent 1100 Series HPLC system equipped with a RID (Refractive
Index Detector) operated at 35.degree. C. and an Alltech IOA-2000
organic acid column (150.times.6.5 mm, particle size 5 .mu.m) kept
at 60.degree. C. Slightly acidified water was used (0.005 M
H.sub.2SO.sub.4) as mobile phase with a flow rate of 0.7 ml/min. To
remove proteins and other cell residues, 400 .mu.l samples were
mixed with 100 .mu.l of a 2% (w/v) 5-sulfosalicylic acid and
centrifuged at 14,000.times.g for 3 min to separate precipitated
residues. 10 .mu.l of the supernatant were then injected into the
HPLC for analyses.
[0074] GC analysis of metabolic end products was performed using an
Agilent 6890N headspace GC equipped with a Supelco PDMS 100 1 cm
fiber, an Alltech EC-1000 (30 m.times.0.25 mm.times.0.25 .mu.m)
column, and a flame ionization detector (FID). 5 ml samples were
transferred into a Hungate tube, heated to 40.degree. C. in a water
bath and exposed to the fiber for exactly 5 min. The injector was
kept at 250.degree. C. and helium with a constant flow of 1 ml/min
was used as carrier gas. The oven program was 40.degree. C. for 5
min, followed by an increase of 10.degree. C./min up to 200.degree.
C. The temperature was then further increased to 220.degree. C.
with a rate of 50.degree. C./min followed by a 5 min hold at this
temperature, before the temperature was decreased to 40.degree. C.
with a rate of 50.degree. C./min and a final 1 min hold. The FID
was kept at 250.degree. C. with 40 ml/min hydrogen, 450 ml/min air
and 15 ml/min nitrogen as make up gas.
[0075] During the complete transformation experiment, C.
autoethanogenum DSM23693 was grown in YTF medium in the presence of
reducing agents and with 30 psi steel mill waste gas (collected
from New Zealand Steel site in Glenbrook, NZ; composition: 44% CO,
32% N.sub.2, 22% CO.sub.2, 2% H.sub.2) at 37.degree. C. using
standard anaerobic techniques (Hungate, Methods Microbiol, 3B:
117-132, 1969; Wolfe, Adv Microbiol Physiol, 6: 107-146, 1971).
TABLE-US-00006 YTF medium component Amount per 1.0 L of YTF medium
Yeast extract 10 g Tryptone 16 g Sodium chloride 0.2 g Fructose 10
g Distilled water to 1.0 L
TABLE-US-00007 Reducing agent Amount per 100 mL of solution
component reducing agent solution NaOH 0.9 g Cysteine-HCl 4 g
Na.sub.2S 4 g Distilled water to 100 ml
[0076] To make competent cells, a 50 ml culture of C.
autoethanogenum DSM23693 was subcultured to fresh YTF media for 5
consecutive days. These cells were used to inoculate 50 ml YTF
media containing 40 mM DL-threonine at an OD.sub.600 nm of 0.05.
When the culture reached an OD.sub.600 nm of 0.5, the cells were
incubated on ice for 30 minutes and then transferred into an
anaerobic chamber and harvested at 4,700.times.g and 4.degree. C.
The culture was twice washed with ice-cold electroporation buffer
(270 mM sucrose, 1 mM MgCl.sub.2, 7 mM sodium phosphate, pH 7.4)
and finally suspended in a volume of 600 .mu.l fresh
electroporation buffer. This mixture was transferred into a
pre-cooled electroporation cuvette with a 0.4 cm electrode gap
containing 2 .mu.g of the methylated plasmid mix and 1 .mu.l type 1
restriction inhibitor (Epicentre Biotechnologies) and immediately
pulsed using the Gene pulser Xcell electroporation system (Bio-Rad)
with the following settings: 2.5 kV, 600.OMEGA., and 25 .mu.F. Time
constants of 3.7-4.0 ms were achieved. The culture was transferred
into 5 ml fresh YTF medium. Regeneration of the cells was monitored
at a wavelength of 600 nm using a Spectronic Helios Epsilon
Spectrophotometer (Thermo) equipped with a tube holder. After an
initial drop in biomass, the cells started growing again. Once the
biomass doubled from that point, about 200 .mu.l of culture was
spread on YTF-agar plates and PETC agar plates containing 5 g/1
fructose (both containing 1.2% bacto agar and 15 .mu.g/ml
thiamphenicol). After 3-4 days of incubation with 30 psi steel mill
gas at 37.degree. C., 500 colonies per plate were clearly
visible.
[0077] C. autoethanogenum: To verify the identity of the six clones
and the DNA transfer, genomic DNA was isolated from all 6
colonies/clones in PETC liquid media using PURELINK.TM. Genomic DNA
mini kit (Invitrogen) according to manufacturer's instruction.
These genomic DNA along with that of wild-type C. autoethanogenum
DSM23693 were used as a template in PCR. The PCR was performed with
iproof High Fidelity DNA Polymerase (Bio-Rad Labratories), specific
primers as described in examples below and the following program:
initial denaturation at 98.degree. C. for 2 min, followed by 25
cycles of denaturation (98.degree. C. for 10 s), annealing
(61.degree. C. for 15 s) and elongation (72.degree. C. for 90 s),
before a final extension step (72.degree. C. for 7 min). The
genomic DNA from wild-type C. autoethanogenum DSM23693 was used as
template in control PCR.
[0078] To confirm the identity of the clones, PCR was also
performed against the 16s rRNA gene using primers fD1 (SEQ ID NO:
10) and rP2 (SEQ ID NO: 11) and using PCR conditions as described
above. The PCR products were purified using Zymo CLEAN AND
CONCENTRATOR.TM. kit and sequenced using primer rP2.
Example 2
[0079] This example demonstrates the genetic modification of C.
autoethanogenum to eliminate lactate dehydrogenase activity.
[0080] Demonstration of inactivation of the identified (Kopke, Appl
Environ Microbiol, 77: 5467-5475, 2011) lactate dehydrogenase
(AEI90736.1) gene ldh (HQ876025.1) of C. autoethanogenum was
demonstrated by using two methodologies: homologous recombination
and ClosTron.
[0081] Homologous Recombination:
[0082] To create a C. autoethanogenum strain that can no longer
produce lactate, a knock out construct was designed to disrupt ldh
by double homologous recombination. Approximately 1 kb homology
arms (SEQ ID NOs: 1-2) flanking the ldh gene were cloned into
pMTL85151 plasmid (FIG. 1) and the resulting plasmid
pMTL85151-ldh-ko (SEQ ID NO: 3). Standard recombinant DNA and
molecular cloning techniques are known in the art (Sambrook,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 2001; Ausubel, Current
Protocols in Molecular Biology. Wiley, 1987). Genomic DNA from C.
autoethanogenum DSM23693 was isolated using Purelink Genomic DNA
mini kit from Invitrogen, according to the manufacturer's
instruction.
[0083] Transformation to introduce DNA was carried out as described
above or in WO 2012/053905.
[0084] Following selection, colonies was screened for single
crossover integration (FIG. 2A) and then for double-crossover
mutants (FIG. 2B). A 3' crossover event was seen in clone 6 (FIG.
2A) and knockout of ldh gene was observed when screened with outer
flanking primers (FIG. 2B). Oligonucleotides Og21f (SEQ ID NO: 4),
Og24r (SEQ ID NO: 5), Og35f (SEQ ID NO: 6, and Og36r (SEQ ID NO: 7)
were used for identification of the double-crossover lactate
dehydrogenase deletion.
[0085] The same strategy and plasmid can also be used, for example,
in C. ljungdahlii or C. ragsdalei. Transformation protocols have
been described in the art (WO 2012/053905 Leang, Applied Environ
Microbiol, 79: 1102-1109, 2013).
[0086] ClosTron:
[0087] ClosTron (Heap, J Microbiol Methods, 70: 452-464, 2007), an
intron design tool hosted on the ClosTron website, was used to
design a 344 bp targeting region 129s (SEQ ID NO: 8) and identify a
target site (SEQ ID NO: 9). The targeting region was chemically
synthesized in the vector pMTL007C-E2 containing a
retro-transposition activated ermB marker (RAM) by DNA2.0 (Menlo
Park) (SEQ ID NO: 12).
[0088] The vectors were introduced into C. autoethanogenum as
described in WO 2012/053905. Single colonies grown on PETC MES with
15 .mu.g/ml thiamphenicol were streaked on PETC MES with 5 .mu.g/ml
clarothromycin. Colonies from each target were randomly picked and
screened for the insertion using flanking primers 155F (SEQ ID NO:
4), and 939R (SEQ ID NO: 5). Amplification was performed using the
iNtron Maxime PCR premix. A PCR product of 100 bp indicated a
wild-type genotype, while a product size of approximately 1.9 kb
suggests the insertion of the group II intron in the target site
(FIG. 3). The loss of the plasmid was checked by amplification of
the resistance marker (catP) and the gram positive origin of
replication (pCB102) (FIG. 4).
TABLE-US-00008 SEQ ID NO Description 1 left homology arm for
disruption of the lactate dehydrogenase gene 2 right homology arm
for disruption of the lactate dehydrogenase gene 3 plasmid
pMTL85151-ldh-ko 4 oligonucleotide Og21f 5 oligonucleotide Og24r 6
oligonucleotide Og35f 7 oligonucleotide Og35f 8 ClosTron targeting
region 9 ClosTron target site 10 oligonucleotide fD1 11
oligonucleotide rP2 12 ClosTron plasmid pMTL007C-E2-ldh::129s
[0089] The same strategy and plasmid can also be used in C.
ljungdahlii or C. ragsdalei. Transformation protocols have been
described in the art (WO 2012/053905; Leang, Appl Environ
Microbiol, 79: 1102-1109, 2013).
Example 3
[0090] This example describes growth experiments comparing the
product profile of C. autoethanogenum strains with inactivated
lactate dehydrogenase to unmodified C. autoethanogenum.
[0091] Cultures of C. autoethanogenum and a C. autoethanogenum
strain with an inactivated lactate dehydrogenase were grown in PETC
media with 10 g/L MES buffer in serum bottles. The inoculum was 10%
of the media volume and the volume of the media was 10 ml. The
cultures were gassed with steel mill off-gas (44% CO, 22% CO.sub.2,
2% H.sub.2, 32% N.sub.2) 30 psi and incubated at 37.degree. C. The
pH of the media was 5.7.
[0092] During the growth period, samples were taken for the
measurement of OD600 and for analysis by HPLC. The bottles were
gassed every day with 30 psi mill gas. The experiment was performed
in triplicate.
[0093] While the unmodified C. autoethanogenum strain produced
0.263.+-.0.041 g/L lactate after 6 days of growth (FIG. 5A), the C.
autoethanogenum strain with inactivated lactate dehydrogenase
produced no lactate after 6 days of growth (FIG. 5B). Additionally,
the C. autoethanogenum strain with inactivated lactate
dehydrogenase produced increased amounts of acetate, ethanol, and
2,3-butanediol. The two strains otherwise had a similar growth
profile and reached a similar OD600 nm of 2.69 and 2.365,
respectively.
[0094] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein. The reference to any prior art in
this specification is not, and should not be taken as, an
acknowledgement that that prior art forms part of the common
general knowledge in the field of endeavour in any country.
[0095] The use of the terms "a" and "an" and "the" and similar
referents in the context of describing the invention (especially in
the context of the following claims) are to be construed to cover
both the singular and the plural, unless otherwise indicated herein
or clearly contradicted by context. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0096] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
1211000DNAArtificial sequenceSynthetic 1tggtattcta gcttgaaatt
tgaacttata catgaaatat aatgacaaaa ttgaaatcac 60atttaaaaac ttttttaaat
aagtcttagc ttggtatttt agaaaacctt aggaacttag 120atatgtttga
ggtacgagtt gatctaagtt cctttaggtt ttcaaaatac caagctttag
180acttattaaa agtttttata gtgatgaaat ttgtcattat atgatattgt
ataagtgaac 240tttcaaccta gaaatatcta aatgtctaaa attatatctt
ctaaacttct ttttggtaca 300tgatggactt tattctcgtc tctccagtac
ttcacatcat tatttgaatc catagcttct 360ataaccactt gttcctttgg
ctttccaaga gctactacca atataatttc atatttttca 420tccaaattaa
gatttgattt taattctttt ttattcacat ttcccagcat acatccgcca
480aatccttttt ctacagctcc tagtaaaata gtttgagctg ctattccagg
atcaaatgat 540gggcttttac taatagatgt gtcattaagt ataacaacat
aaccacttgg tttttcacct 600tcttcaggtc catcccagtc ctttaaatat
cctgcccaac ccaaagtctt aaatattttt 660tcattattat cttctgtatt
tgatataaca tattttagtg gctgcaaatt tgaacctgat 720gctgataatc
tggcaaggtt caccaaatat tttaaagttt ccacagttat tttttcattc
780tgatagaatc ttctacagga tctatttttt aaaactagtt cttttatcat
attcatccct 840ccaagctaat aacttaatat cattacaatt atcatatctc
taaacaaatt gaatatcaat 900caaatatagt aaatataaag gtaatattga
attatatata atgttactat aaaattatat 960atagtaaata ttattcataa
aaagggggag ctagaataca 100021000DNAArtificial sequenceSynthetic
2acaaaagttc ttatgcactc ttcttttttt gataaaagaa gagtgcattg attttaatgt
60ttttttatct tatttataga atatagaaat cttagcaact agacttaaac cataatcaag
120ttaaaatctt ggtaaatcct ttaaattttc aacatccata acatcagggt
cagctactaa 180atattctcct ccatctttac ctttaaatac gccaactcct
tcaattatgt gctcccttgc 240catcattata gcatgattta aaggaagcac
ctcaccattt tcaagtaata catctgtaat 300cttaccctca tgatctttcc
ttactttaat tatttttgaa tttttcatta tatacctcct 360gattagatat
atcattaata tatattttga gagaaaactc aactacttat acaaaatatt
420tttacattat atttacacaa ttataacttt tgtggttgca attaaagtta
taattgaacc 480aaatcttgtt attgacagta caaaatgcaa gtcatatatt
tatccgatga ctacccgctc 540taatactttc cctcttttcc aagtgggagt
aaagagcgga tacgtccctg gataacgatt 600tttcctaaag gataacgcct
tctaagtgct gaagcactaa gaatcctgtt aataagcatc 660aggtggagtt
aaacctccat ctgatactaa gaactctgtt tataacatga tttgcaaata
720tatcactttg gaggaatttt atgaaaaaaa atattattaa aagttccata
gtatttatgg 780tgatttttgc tttatttttt atagcatcag ataaatcaac
tgttcatgca ttaaattgct 840atacagtaag cttgtcaaat tttaaagagg
tatcccaaaa catttatgta cagccaaata 900cttctgataa agatataaat
aatatattga gtaccatttc caaatctaaa aacattgtgg 960ccaatttata
tggaagtttc aatgctaaac ctgtctttat 100034746DNAArtificial
sequenceSynthetic 3cctgcaggtt aagagcaaca ctgtacgtgg tattctagct
tgaaatttga acttatacat 60gaaatataat gacaaaattg aaatcacatt taaaaacttt
tttaaataag tcttagcttg 120gtattttaga aaaccttagg aacttagata
tgtttgaggt acgagttgat ctaagttcct 180ttaggttttc aaaataccaa
gctttagact tattaaaagt ttttatagtg atgaaatttg 240tcattatatg
atattgtata agtgaacttt caacctagaa atatctaaat gtctaaaatt
300atatcttcta aacttctttt tggtacatga tggactttat tctcgtctct
ccagtacttc 360acatcattat ttgaatccat agcttctata accacttgtt
cctttggctt tccaagagct 420actaccaata taatttcata tttttcatcc
aaattaagat ttgattttaa ttctttttta 480ttcacatttc ccagcataca
tccgccaaat cctttttcta cagctcctag taaaatagtt 540tgagctgcta
ttccaggatc aaatgatggg cttttactaa tagatgtgtc attaagtata
600acaacataac cacttggttt ttcaccttct tcaggtccat cccagtcctt
taaatatcct 660gcccaaccca aagtcttaaa tattttttca ttattatctt
ctgtatttga tataacatat 720tttagtggct gcaaatttga acctgatgct
gataatctgg caaggttcac caaatatttt 780aaagtttcca cagttatttt
ttcattctga tagaatcttc tacaggatct attttttaaa 840actagttctt
ttatcatatt catccctcca agctaataac ttaatatcat tacaattatc
900atatctctaa acaaattgaa tatcaatcaa atatagtaaa tataaaggta
atattgaatt 960atatataatg ttactataaa attatatata gtaaatatta
ttcataaaaa gggggagcta 1020gaatacaatg aaagtgctag cgaaggcaaa
gaaaatccat ggcaaattga ataaacaaaa 1080gttcttatgc actcttcttt
ttttgataaa agaagagtgc attgatttta atgttttttt 1140atcttattta
tagaatatag aaatcttagc aactagactt aaaccataat caagttaaaa
1200tcttggtaaa tcctttaaat tttcaacatc cataacatca gggtcagcta
ctaaatattc 1260tcctccatct ttacctttaa atacgccaac tccttcaatt
atgtgctccc ttgccatcat 1320tatagcatga tttaaaggaa gcacctcacc
attttcaagt aatacatctg taatcttacc 1380ctcatgatct ttccttactt
taattatttt tgaatttttc attatatacc tcctgattag 1440atatatcatt
aatatatatt ttgagagaaa actcaactac ttatacaaaa tatttttaca
1500ttatatttac acaattataa cttttgtggt tgcaattaaa gttataattg
aaccaaatct 1560tgttattgac agtacaaaat gcaagtcata tatttatccg
atgactaccc gctctaatac 1620tttccctctt ttccaagtgg gagtaaagag
cggatacgtc cctggataac gatttttcct 1680aaaggataac gccttctaag
tgctgaagca ctaagaatcc tgttaataag catcaggtgg 1740agttaaacct
ccatctgata ctaagaactc tgtttataac atgatttgca aatatatcac
1800tttggaggaa ttttatgaaa aaaaatatta ttaaaagttc catagtattt
atggtgattt 1860ttgctttatt ttttatagca tcagataaat caactgttca
tgcattaaat tgctatacag 1920taagcttgtc aaattttaaa gaggtatccc
aaaacattta tgtacagcca aatacttctg 1980ataaagatat aaataatata
ttgagtacca tttccaaatc taaaaacatt gtggccaatt 2040tatatggaag
tttcaatgct aaacctgtct ttataattag caaagattca acagccttaa
2100aaaaatttgg tgttgaaaat aaaacaggag ctacacaaaa aactatacta
ggtagctaca 2160tagttctggg accagaggga ttaaatcggc gcgccgcatt
cacttctttt ctatataaat 2220atgagcgaag cgaataagcg tcggaaaagc
agcaaaaagt ttcctttttg ctgttggagc 2280atgggggttc agggggtgca
gtatctgacg tcaatgccga gcgaaagcga gccgaagggt 2340agcatttacg
ttagataacc ccctgatatg ctccgacgct ttatatagaa aagaagattc
2400aactaggtaa aatcttaata taggttgaga tgataaggtt tataaggaat
ttgtttgttc 2460taatttttca ctcattttgt tctaatttct tttaacaaat
gttctttttt ttttagaaca 2520gttatgatat agttagaata gtttaaaata
aggagtgaga aaaagatgaa agaaagatat 2580ggaacagtct ataaaggctc
tcagaggctc atagacgaag aaagtggaga agtcatagag 2640gtagacaagt
tataccgtaa acaaacgtct ggtaacttcg taaaggcata tatagtgcaa
2700ttaataagta tgttagatat gattggcgga aaaaaactta aaatcgttaa
ctatatccta 2760gataatgtcc acttaagtaa caatacaatg atagctacaa
caagagaaat agcaaaagct 2820acaggaacaa gtctacaaac agtaataaca
acacttaaaa tcttagaaga aggaaatatt 2880ataaaaagaa aaactggagt
attaatgtta aaccctgaac tactaatgag aggcgacgac 2940caaaaacaaa
aatacctctt actcgaattt gggaactttg agcaagaggc aaatgaaata
3000gattgacctc ccaataacac cacgtagtta ttgggaggtc aatctatgaa
atgcgattaa 3060gggccggcca gtgggcaagt tgaaaaattc acaaaaatgt
ggtataatat ctttgttcat 3120tagagcgata aacttgaatt tgagagggaa
cttagatggt atttgaaaaa attgataaaa 3180atagttggaa cagaaaagag
tattttgacc actactttgc aagtgtacct tgtacctaca 3240gcatgaccgt
taaagtggat atcacacaaa taaaggaaaa gggaatgaaa ctatatcctg
3300caatgcttta ttatattgca atgattgtaa accgccattc agagtttagg
acggcaatca 3360atcaagatgg tgaattgggg atatatgatg agatgatacc
aagctataca atatttcaca 3420atgatactga aacattttcc agcctttgga
ctgagtgtaa gtctgacttt aaatcatttt 3480tagcagatta tgaaagtgat
acgcaacggt atggaaacaa tcatagaatg gaaggaaagc 3540caaatgctcc
ggaaaacatt tttaatgtat ctatgatacc gtggtcaacc ttcgatggct
3600ttaatctgaa tttgcagaaa ggatatgatt atttgattcc tatttttact
atggggaaat 3660attataaaga agataacaaa attatacttc ctttggcaat
tcaagttcat cacgcagtat 3720gtgacggatt tcacatttgc cgttttgtaa
acgaattgca ggaattgata aatagttaac 3780ttcaggtttg tctgtaacta
aaaacaagta tttaagcaaa aacatcgtag aaatacggtg 3840ttttttgtta
ccctaagttt aaactccttt ttgataatct catgaccaaa atcccttaac
3900gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
tcttcttgag 3960atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
aaaaccaccg ctaccagcgg 4020tggtttgttt gccggatcaa gagctaccaa
ctctttttcc gaaggtaact ggcttcagca 4080gagcgcagat accaaatact
gttcttctag tgtagccgta gttaggccac cacttcaaga 4140actctgtagc
accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
4200gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
gataaggcgc 4260agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
cttggagcga acgacctaca 4320ccgaactgag atacctacag cgtgagctat
gagaaagcgc cacgcttccc gaagggagaa 4380aggcggacag gtatccggta
agcggcaggg tcggaacagg agagcgcacg agggagcttc 4440cagggggaaa
cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
4500gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
agcaacgcgg 4560cctttttacg gttcctggcc ttttgctggc cttttgctca
catgttcttt cctgcgttat 4620cccctgattc tgtggataac cgtattaccg
cctttgagtg agctgatacc gctcgccgca 4680gccgaacgac cgagcgcagc
gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca 4740gggccc
4746439DNAArtificial sequenceSynthetic 4attcatcctg caggttaaga
gcaacactgt acgtggtat 39537DNAArtificial sequenceSynthetic
5gactggcgcg ccatttaatc cctctggtcc cagaact 37625DNAArtificial
sequenceSynthetic 6tgaactaaaa agtttcgtac gatgt 25725DNAArtificial
sequenceSynthetic 7tcctttatcc attgtccctc agagt 258309DNAArtificial
sequenceSynthetic 8ttatccttag acttcgccaa ggtgcgccca gatagggtgt
taagtcaagt agtttaaggt 60actactctgt aagataacac agaaaacagc caacctaacc
gaaaagcgaa agctgatacg 120ggaacagagc acggttggaa agcgatgagt
tacctaaaga caatcgggta cgactgagtc 180gcaatgttaa tcagatataa
ggtataagtt gtgtttactg aacgcaagtt tctaatttcg 240attaagtctc
gatagaggaa agtgtctgaa acctctagta caaagaaagg taagttaccc 300ttggcgact
309945DNAArtificial sequenceSynthetic 9ccaactatgg aaaatgcaga
cttggccaag ggatttgact gcatc 451037DNAArtificial sequenceSynthetic
10ccgaattcgt cgacaacaga gtttgatcct ggctcag 371137DNAArtificial
sequenceSynthetic 11cccgggatcc aagcttacgg ctaccttgtt acgactt
37129034DNAArtificial sequenceSynthetic 12agaaggccat cctgacggat
ggcctttttg cgtttctaca aactcttcct gtcgtcatat 60ctacaagcca tccccccaca
gatacgggcg cgccgccatt atttttttga acaattgaca 120attcatttct
tattttttat taagtgatag tcaaaaggca taacagtgct gaatagaaag
180aaatttacag aaaagaaaat tatagaattt agtatgatta attatactca
tttatgaatg 240tttaattgaa tacaaaaaaa aatacttgtt atgtattcaa
ttacgggtta aaatatagac 300aagttgaaaa atttaataaa aaaataagtc
ctcagctctt atatattaag ctaccaactt 360agtatataag ccaaaactta
aatgtgctac caacacatca agccgttaga gaactctatc 420tatagcaata
tttcaaatgt accgacatac aagagaaaca ttaactatat atattcaatt
480tatgagatta tcttaacaga tataaatgta aattgcaata agtaagattt
agaagtttat 540agcctttgtg tattggaagc agtacgcaaa ggctttttta
tttgataaaa attagaagta 600tatttatttt ttcataatta atttatgaaa
atgaaagggg gtgagcaaag tgacagagga 660aagcagtatc ttatcaaata
acaaggtatt agcaatatca ttattgactt tagcagtaaa 720cattatgact
tttatagtgc ttgtagctaa gtagtacgaa agggggagct ttaaaaagct
780ccttggaata catagaattc ataaattaat ttatgaaaag aagggcgtat
atgaaaactt 840gtaaaaattg caaagagttt attaaagata ctgaaatatg
caaaatacat tcgttgatga 900ttcatgataa aacagtagca acctattgca
gtaaatacaa tgagtcaaga tgtttacata 960aagggaaagt ccaatgtatt
aattgttcaa agatgaaccg atatggatgg tgtgccataa 1020aaatgagatg
ttttacagag gaagaacaga aaaaagaacg tacatgcatt aaatattatg
1080caaggagctt taaaaaagct catgtaaaga agagtaaaaa gaaaaaataa
tttatttatt 1140aatttaatat tgagagtgcc gacacagtat gcactaaaaa
atatatctgt ggtgtagtga 1200gccgatacaa aaggatagtc actcgcattt
tcataataca tcttatgtta tgattatgtg 1260tcggtgggac ttcacgacga
aaacccacaa taaaaaaaga gttcggggta gggttaagca 1320tagttgaggc
aactaaacaa tcaagctagg atatgcagta gcagaccgta aggtcgttgt
1380ttaggtgtgt tgtaatacat acgctattaa gatgtaaaaa tacggatacc
aatgaaggga 1440aaagtataat ttttggatgt agtttgtttg ttcatctatg
ggcaaactac gtccaaagcc 1500gtttccaaat ctgctaaaaa gtatatcctt
tctaaaatca aagtcaagta tgaaatcata 1560aataaagttt aattttgaag
ttattatgat attatgtttt tctattaaaa taaattaagt 1620atatagaata
gtttaataat agtatatact taatgtgata agtgtctgac agtgtcacag
1680aaaggatgat tgttatggat tataagcggc cggccagtgg gcaagttgaa
aaattcacaa 1740aaatgtggta taatatcttt gttcattaga gcgataaact
tgaatttgag agggaactta 1800gatggtattt gaaaaaattg ataaaaatag
ttggaacaga aaagagtatt ttgaccacta 1860ctttgcaagt gtaccttgta
cctacagcat gaccgttaaa gtggatatca cacaaataaa 1920ggaaaaggga
atgaaactat atcctgcaat gctttattat attgcaatga ttgtaaaccg
1980ccattcagag tttaggacgg caatcaatca agatggtgaa ttggggatat
atgatgagat 2040gataccaagc tatacaatat ttcacaatga tactgaaaca
ttttccagcc tttggactga 2100gtgtaagtct gactttaaat catttttagc
agattatgaa agtgatacgc aacggtatgg 2160aaacaatcat agaatggaag
gaaagccaaa tgctccggaa aacattttta atgtatctat 2220gataccgtgg
tcaaccttcg atggctttaa tctgaatttg cagaaaggat atgattattt
2280gattcctatt tttactatgg ggaaatatta taaagaagat aacaaaatta
tacttccttt 2340ggcaattcaa gttcatcacg cagtatgtga cggatttcac
atttgccgtt ttgtaaacga 2400attgcaggaa ttgataaata gttaacttca
ggtttgtctg taactaaaaa caagtattta 2460agcaaaaaca tcgtagaaat
acggtgtttt ttgttaccct aagtttaaac tcctttttga 2520taatctcatg
accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
2580agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct
gctgcttgca 2640aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
gatcaagagc taccaactct 2700ttttccgaag gtaactggct tcagcagagc
gcagatacca aatactgtcc ttctagtgta 2760gccgtagtta ggccaccact
tcaagaactc tgtagcaccg cctacatacc tcgctctgct 2820aatcctgtta
ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
2880aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt
cgtgcacaca 2940gcccagcttg gagcgaacga cctacaccga actgagatac
ctacagcgtg agcattgaga 3000aagcgccacg cttcccgaag ggagaaaggc
ggacaggtat ccggtaagcg gcagggtcgg 3060aacaggagag cgcacgaggg
agcttccagg gggaaacgcc tggtatcttt atagtcctgt 3120cgggtttcgc
cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
3180cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt
gctggccttt 3240tgctcacatg ttctttcctg cgttatcccc tgattctgtg
gataaccgta ttaccgcctt 3300tgagtgagct gataccgctc gccgcagccg
aacgaccgag cgcagcgagt cagtgagcga 3360ggaagcggaa gagcgcccaa
tacgcagggc cccctgcttc ggggtcatta tagcgatttt 3420ttcggtatat
ccatcctttt tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga
3480ctttccttgg tgtatccaac ggcgtcagcc gggcaggata ggtgaagtag
gcccacccgc 3540gagcgggtgt tccttcttca ctgtccctta ttcgcacctg
gcggtgctca acgggaatcc 3600tgctctgcga ggctggccgg ctaccgccgg
cgtaacagat gagggcaagc ggatggctga 3660tgaaaccaag ccaaccagga
agggcagccc acctatcaag gtgtactgcc ttccagacga 3720acgaagagcg
attgaggaaa aggcggcggc ggccggcatg agcctgtcgg cctacctgct
3780ggccgtcggc cagggctaca aaatcacggg cgtcgtggac tatgagcacg
tccgcgagct 3840ggcccgcatc aatggcgacc tgggccgcct gggcggcctg
ctgaaactct ggctcaccga 3900cgacccgcgc acggcgcggt tcggtgatgc
cacgatcctc gccctgctgg cgaagatcga 3960agagaagcag gacgagcttg
gcaaggtcat gatgggcgtg gtccgcccga gggcagagcc 4020atgacttttt
tagccgctaa aacggccggg gggtgcgcgt gattgccaag cacgtcccca
4080tgcgctccat caagaagagc gacttcgcgg agctggtgaa gtacatcacc
gacgagcaag 4140gcaagaccga tcgggccccc tgcagggtgt agtagcctgt
gaaataagta aggaaaaaaa 4200agaagtaagt gttatatatg atgattattt
tgtagatgta gataggataa tagaatccat 4260agaaaatata ggttatacag
ttatataaaa attactttaa aaattaataa aaacatggta 4320aaatataaat
cgtataaagt tgtgtaattt ttaagcttta taattatcct tagacttcgc
4380caaggtgcgc ccagataggg tgttaagtca agtagtttaa ggtactactc
tgtaagataa 4440cacagaaaac agccaaccta accgaaaagc gaaagctgat
acgggaacag agcacggttg 4500gaaagcgatg agttacctaa agacaatcgg
gtacgactga gtcgcaatgt taatcagata 4560taaggtataa gttgtgttta
ctgaacgcaa gtttctaatt tcgattaagt ctcgatagag 4620gaaagtgtct
gaaacctcta gtacaaagaa aggtaagtta cccttggcga cttatctgtt
4680atcaccacat ttgtacaatc tgtaggagaa cctatgggaa cgaaacgaaa
gcgatgccga 4740gaatctgaat ttaccaagac ttaacactaa ctggggatac
cctaaacaag aatgcctaat 4800agaaaggagg aaaaaggcta tagcactaga
gcttgaaaat cttgcaaggg tacggagtac 4860tcgtagtagt ctgagaaggg
taacgccctt tacatggcaa aggggtacag ttattgtgta 4920ctaaaattaa
aaattgatta gggaggaaaa cctcaaaatg aaaccaacaa tggcaatttt
4980agaaagaatc agtaaaaatt cacaagaaaa tatagacgaa gtttttacaa
gactttatcg 5040ttatctttta cgtccagata tttattacgt ggcgacgcgt
gaagttccta tactttctag 5100agaataggaa cttcgcgact catagaatta
tttcctcccg ttaaataata gataactatt 5160aaaaatagac aatacttgct
cataagtaac ggtacttaaa ttgtttactt tggcgtgttt 5220cattgcttga
tgaaactgat ttttagtaaa cagttgacga tattctcgat tgacccattt
5280tgaaacaaag tacgtatata gcttccaata tttatctgga acatctgtgg
tatggcgggt 5340aagttttatt aagacactgt ttacttttgg tttaggatga
aagcattccg ctggcagctt 5400aagcaattgc tgaatcgaga cttgagtgtg
caagagcaac cctagtgttc ggtgaatatc 5460caaggtacgc ttgtagaatc
cttcttcaac aatcagatag atgtcagacg catggctttc 5520aaaaaccact
tttttaataa tttgtgtgct taaatggtaa ggaatactcc caacaatttt
5580atacctctgt ttgttaggga attgaaactg tagaatatct tggtgaatta
aagtgacacg 5640agtattcagt tttaattttt ctgacgataa gttgaataga
tgactgtcta attcaataga 5700cgttacctgt ttacttattt tagccagttt
cgtcgttaaa tgccctttac ctgttccaat 5760ttcgtaaacg gtatcggttt
cttttaaatt caattgtttt attatttggt tgagtacttt 5820ttcactcgtt
aaaaagtttt gagaatattt tatatttttg ttcataccag caccagaagc
5880accagcatct cttgggttaa ttgaggcctg agtataaggt gacttatact
tgtaatctat 5940ctaaacgggg aacctctcta gtagacaatc ccgtgctaaa
ttgtaggact gccctttaat 6000aaatacttct atatttaaag aggtatttat
gaaaagcgga atttatcaga ttaaaaatac 6060tttctctaga gaaaatttcg
tctggattag ttacttatcg tgtaaaatct gataaatgga 6120attggttcta
cataaatgcc taacgactat ccctttgggg agtagggtca agtgactcga
6180aacgatagac aacttgcttt aacaagttgg agatatagtc tgctctgcat
ggtgacatgc 6240agctggatat aattccgggg taagattaac gaccttatct
gaacataatg ccatatgaat 6300ccctcctaat ttatacgttt tctctaacaa
cttaattata cccactatta ttatttttat 6360caatatagaa gttcctatac
tttctagaga ataggaactt cacgcgttgg gaaatggcaa 6420tgatagcgaa
acaacgtaaa actcttgttg tatgctttca ttgtcatcgt cacgtgattc
6480ataaacacaa gtgaatgtcg acagtgaatt tttacgaacg aacaataaca
gagccgtata 6540ctccgagagg ggtacgtacg gttcccgaag agggtggtgc
aaaccagtca cagtaatgtg 6600aacaaggcgg tacctcccta cttcaccata
tcattttctg cagcccccta gaaataattt 6660tgtttaactt taagaaggag
atatacatat atggctagat cgtccattcc gacagcatcg 6720ccagtcacta
tggcgtgctg ctagcgctat atgcgttgat gcaatttcta tgcactcgta
6780gtagtctgag aagggtaacg ccctttacat ggcaaagggg tacagttatt
gtgtactaaa 6840attaaaaatt gattagggag gaaaacctca aaatgaaacc
aacaatggca attttagaaa 6900gaatcagtaa aaattcacaa gaaaatatag
acgaagtttt tacaagactt tatcgttatc 6960ttttacgtcc agatatttat
tacgtggcgt atcaaaattt atattccaat aaaggagctt 7020ccacaaaagg
aatattagat gatacagcgg atggctttag tgaagaaaaa ataaaaaaga
7080ttattcaatc tttaaaagac ggaacttact atcctcaacc tgtacgaaga
atgtatattg 7140caaaaaagaa ttctaaaaag atgagacctt taggaattcc
aactttcaca gataaattga 7200tccaagaagc tgtgagaata attcttgaat
ctatctatga accggtattc gaagatgtgt 7260ctcacggttt tagacctcaa
cgaagctgtc acacagcttt gaaaacaatc aaaagagagt 7320ttggcggcgc
aagatggttt gtggagggag atataaaagg ctgcttcgat aatatagacc
7380acgttacact cattggactc atcaatctta aaatcaaaga tatgaaaatg
agccaattga 7440tttataaatt tctaaaagca ggttatctgg aaaactggca
gtatcacaaa acttacagcg 7500gaacacctca aggtggaatt ctatctcctc
ttttggccaa catctatctt catgaattgg 7560ataagtttgt tttacaactc
aaaatgaagt ttgaccgaga aagtccagaa agaataacac 7620ctgaatatcg
ggagctccac aatgagataa aaagaatttc tcaccgtctc aagaagttgg
7680agggtgaaga aaaagctaaa gttcttttag aatatcaaga aaaacgtaaa
agattaccca 7740cactcccctg tacctcacag acaaataaag tattgaaata
cgtccggtat gcggacgact 7800tcattatctc tgttaaagga agcaaagagg
actgtcaatg gataaaagaa caattaaaac 7860tttttattca taacaagcta
aaaatggaat tgagtgaaga aaaaacactc atcacacata 7920gcagtcaacc
cgctcgtttt ctgggatatg atatacgagt aaggagatct ggaacgataa
7980aacgatctgg taaagtcaaa aagagaacac tcaatgggag tgtagaactc
cttattcctc 8040ttcaagacaa aattcgtcaa tttatttttg acaagaaaat
agctatccaa aagaaagata 8100gctcatggtt tccagttcac aggaaatatc
ttattcgttc aacagactta gaaatcatca 8160caatttataa ttctgaactc
cgcgggattt gtaattacta cggtctagca agtaatttta 8220accagctcaa
ttattttgct tatcttatgg aatacagctg tctaaaaacg atagcctcca
8280aacataaggg aacactttca aaaaccattt ccatgtttaa agatggaagt
ggttcgtggg 8340ggatcccgta tgagataaag caaggtaagc agcgccgtta
ttttgcaaat tttagtgaat 8400gtaaatcccc ttatcaattt acggatgaga
taagtcaagc tcctgtattg tatggctatg 8460cccggaatac tcttgaaaac
aggttaaaag ctaaatgttg tgaattatgt gggacgtctg 8520atgaaaatac
ttcctatgaa attcaccatg tcaataaggt caaaaatctt aaaggcaaag
8580aaaaatggga aatggcaatg atagcgaaac aacgtaaaac tcttgttgta
tgctttcatt 8640gtcatcgtca cgtgattcat aaacacaagt gaatgtcgag
cacccgttct cggagcactg 8700tccgaccgct ttggccgccg cccagtcctg
ctcgcttcgc tacttggagc cactatcgac 8760tacgcgatca tggcgaccac
acccgtcctg tggatcgcca agccgccgat ggtagtgtgg 8820ggtctcccca
tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg
8880aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
gagtaggaca 8940aatccgccgg gagcggattt gaacgttgcg aagcaacggc
ccggagggtg gcgggcagga 9000cgcccgccat aaactgccag gcatcaaatt aagc
9034
* * * * *