U.S. patent application number 10/997308 was filed with the patent office on 2006-03-16 for method for the positive selection of chromosomal mutations in c1 metabolizing bacteria via homologous recombination.
Invention is credited to Pamela L. Sharpe.
Application Number | 20060057726 10/997308 |
Document ID | / |
Family ID | 34681523 |
Filed Date | 2006-03-16 |
United States Patent
Application |
20060057726 |
Kind Code |
A1 |
Sharpe; Pamela L. |
March 16, 2006 |
Method for the positive selection of chromosomal mutations in C1
metabolizing bacteria via homologous recombination
Abstract
A method for the positive selection of allelic exchange mutants
is provided for C1 metabolizing bacteria. The chromosomal
integration vectors, based on the pGP704 suicide vector, comprise
at least one genetic selectable marker and the sacB gene, encoding
levansucrase. A one- and two-step selection strategy is provided
for the facile identification of double-crossover mutations in C1
metabolizing bacteria. This methodology enables production of
"markerless" transformants, such that multiple rounds of mutation
can be performed. Optimized conditions for conjugal transfer,
homologous recombination, transformant purification, and screening
are also presented.
Inventors: |
Sharpe; Pamela L.; (Newark,
DE) |
Correspondence
Address: |
E I DU PONT DE NEMOURS AND COMPANY;LEGAL PATENT RECORDS CENTER
BARLEY MILL PLAZA 25/1128
4417 LANCASTER PIKE
WILMINGTON
DE
19805
US
|
Family ID: |
34681523 |
Appl. No.: |
10/997308 |
Filed: |
November 24, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60527877 |
Dec 8, 2003 |
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60527083 |
Dec 3, 2003 |
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Current U.S.
Class: |
435/471 |
Current CPC
Class: |
C12N 9/0083 20130101;
C12R 2001/26 20210501; C12N 15/52 20130101; C12N 9/00 20130101;
C12N 9/0004 20130101; C12N 9/1051 20130101; C12N 15/74 20130101;
C12Y 205/01029 20130101; C12N 9/1085 20130101; C12N 9/0071
20130101; C12N 9/001 20130101; C12P 23/00 20130101; C12N 1/205
20210501; C12N 9/90 20130101 |
Class at
Publication: |
435/471 |
International
Class: |
C12N 15/74 20060101
C12N015/74 |
Claims
1. A method for the positive selection of double-crossover events
in a C1 metabolizing bacterial host cell comprising: a) providing a
C1 metabolizing bacteria selected from the group consisting of
methanotrophs and methylotrophs; b) providing a positive selection
vector comprising: (i) a first genetic selectable marker; (ii) an
origin of transfer for a C1 metabolizing bacteria; (iii) plasmid
mobilization genes; (iv) a sacB coding region encoding a
levansucrase enzyme, under the control of a suitable promoter; (v)
a replacement nucleotide sequence of interest having at least two
regions homology to a chromosomal nucleotide sequence of interest
in the C1 metabolizing bacteria, said replacement nucleotide
sequence of interest being disrupted by a second genetic selectable
marker; wherein the first and second genetic selectable markers are
different and the vector is unable to replicate in the C1
metabolizing bacteria; c) transforming the C1 metabolizing bacteria
of (a) with the vector of (b) d) selecting the transformants of (c)
on the basis of the genetic selectable markers and sacB gene
expression wherein, the transformants which are positive for the
second selectable marker and grow in the presence of sucrose, but
are negative for the first selectable marker have undergone
double-crossover events; and e) recovering the selected
transformants of (d) which have undergone double-crossover
events.
2. A method for the positive selection of double-crossover events
in C1 metabolizing bacterial host cells comprising: a) providing a
C1 metabolizing bacteria selected from the group consisting of
methanotrophs and methylotrophs; b) providing a positive selection
vector comprising: (i) a genetic selectable marker; (ii) an origin
of transfer for a C1 metabolizing bacteria; (iii) plasmid
mobilization genes; (iv) a sacB coding region encoding a
levansucrase enzyme under the control of a suitable promoter; (v) a
mutant replacement nucleotide sequence of interest having at least
two regions of homology to a chromosomal nucleotide sequence of
interest in the C1 metabolizing bacteria; the vector being unable
to replicate in the C1 metabolizing bacteria chromosome; c)
transforming the C1 metabolizing bacteria of (a) with the vector of
(b); d) isolating the transformants of (c) on the basis of the
genetic selectable marker; and e) isolating the transformants of
(d) which grow in the presence of sucrose, wherein said
transformants contain double-crossover events.
3. A method according to either of claims 1 or 2 wherein the
regions of homology are at least about 50 bp in length.
4. A method according to claim 2 wherein the mutation in the mutant
nucleotide of interest is at least about 50 bp downstream of the 5'
end of the sequence.
5. A method according to either of claims 1 or 2, wherein the C1
metabolizing bacteria is selected from the group consisting of
Methylomonas, Methylobacter, Methylococcus, Methylosinus,
Methylocyctis, Methylomicrobium, Methanomonas, Methylophilus,
Methylobacillus, Methylobacterium, Hyphomicrobium, Xanthobacter,
Bacillus, Paracoccus, Nocardia, Arthrobacter, Rhodopseudomonas, and
Pseudomonas.
6. A method according to either of claims 1 or 2, wherein the
suitable promoter is a npr promoter.
7. A method according to either of claims 1 or 2, wherein said sacB
coding region is derived from the group consisting of Bacillus
subtilis, Bacillus pumilus, Bacillus loceniformis, Bacillus
amyloliquifaciens, Bacillus thurgiensis, Bacillus
stearothermophilus, and Bacillus sphaericus.
8. A method according to either of claims 1 or 2, wherein the
positive selection vector is pGP704::sacB.
9. A method according to either of claims 1 or 2 wherein any of the
genetic selectable markers are independently selected from the
group consisting of antibiotic resistance markers, metal resistance
markers, substrate-utilization markers, genes encoding fluorescent
and bioluminescent proteins, lacZ, gfp, cat, galK, inaZ, luc,
luxAB, bgaB, nptll, phoA, uidA, and xylE.
10. A C1 metabolizing bacteria transformant selected by the process
of either claim 1 or claim 2 employing a positive selection
vector.
11. A recombinant C1 metabolizing bacteria transformant of claim 10
wherein the genome of said transformant lacks any sequence of the
positive selection vector, other than the replacement nucleotide
sequence of interest.
12. A recombinant C1 metabolizing bacteria transformant of claim 10
wherein the genome of said transformant lacks any genetic
selectable marker sequence.
13. A positive selection vector for the positive selection of
double-crossover events in a C1 metabolizing bacterial host cell
comprising: (i) at least one genetic selectable marker; (ii) an
origin of transfer for a C1 metabolizing bacteria; (iii) plasmid
mobilization genes; (iv) a sacB coding region encoding a
levansucrase enzyme under the control of a suitable promoter; (v) a
replacement nucleotide sequence of interest having at least two
regions of homology to a chromosomal nucleotide sequence of
interest in the C1 metabolizing bacteria; the vector being unable
to replicate in the C1 metabolizing bacteria chromosome.
14. A vector according to claim 13 wherein said sacB coding region
is derived from the group consisting of Bacillus subtilis, Bacillus
pumilus, Bacillus loceniformis, Bacillus amyloliquifaciens,
Bacillus thurgiensis, Bacillus stearothermophilus, and Bacillus
sphearicus.
15. A vector according to claim 13 wherein the suitable promoter is
the npr promoter.
16. A vector according to claim 13 wherein the replacement
nucleotide of interest is disrupted by at least one second genetic
selectable marker.
17. A vector according to claim 13 or 16 wherein the genetic
selectable marker is selected independently from the group
consisting of antibiotic resistance markers, metal resistance
markers, substrate-utilization markers, genes encoding fluorescent
and bioluminescent proteins, lacZ, gfp, cat, galK, inaZ, luc,
luxAB, bgaB, nptll, phoA, uidA, and xylE.
18. A vector according to claim 13 wherein the regions of homology
are at least about 50 bp in length.
19. A vector according to claim 13 wherein the replacement
nucleotide sequence of interest is a mutant replacement nucleotide
sequence of interest.
20. A vector according to claim 13 wherein the vector is
pGP704::sacB.
21. A transformed C1 metabolizing bacteria comprising the vector of
claim 13.
22. A transformed C1 metabolizing bacteria of claim 21 selected
from the group consisting of methanotrophs and methylotrophs.
23. A methylotroph of claim 22 selected from the group consisting
of Methylomonas, Methylobacter, Methylococcus, Methylosinus,
Methylocyctis, Methylomicrobium, Methanomonas, Methylophilus,
Methylobacillus, Methylobacterium, Hyphomicrobium, Xanthobacter,
Bacillus, Paracoccus, Nocardia, Arthrobacter, Rhodopseudomonas, and
Pseudomonas.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/527,877 filed Dec. 8, 2003 and U.S. Provisional
Application No. 60/527,083 filed Dec. 3, 2003.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of molecular
biology and the use of selection vectors for the identification of
recombinant bacteria containing heterologous DNA. More
specifically, vectors have been constructed for the positive
selection of double-crossover mutants in C1 metabolizing
bacteria.
BACKGROUND INFORMATION
[0003] There are a number of microorganisms that utilize single
carbon substrates as their sole energy source. Such microorganisms
are referred to herein as "C1 metabolizers". These organisms are
characterized by the ability to use carbon substrates lacking
carbon to carbon bonds as a sole source of energy and biomass. All
C1 metabolizing microorganisms are generally classified as
methylotrophs. Methylotrophs may be defined as any organism capable
of oxidizing organic compounds that do not contain carbon-carbon
bonds. However, facultative methylotrophs, obligate methylotrophs,
and obligate methanotrophs are all various subsets of
methylotrophs. Specifically: [0004] Facultative methylotrophs have
the ability to oxidize organic compounds which do not contain
carbon-carbon bonds, but may also use other carbon substrates such
as sugars and complex carbohydrates for energy and biomass; [0005]
Obligate methylotrophs are those organisms which are limited to the
use of organic compounds that do not contain carbon-carbon bonds
for the generation of energy; and [0006] Obligate methanotrophs are
those obligate methylotrophs that have the distinct ability to
oxidize methane. Additionally, the ability to utilize single carbon
substrates is not limited to bacteria but extends also to yeasts
and fungi. A number of yeast genera are able to use single carbon
substrates as energy sources in addition to more complex materials
(i.e., the methylotrophic yeasts).
[0007] The ability of obligate methanotrophic bacteria to use
methane as their sole source of carbon and energy under ambient
conditions, in conjunction with the abundance of methane, makes the
biotransformation of methane a potentially unique and valuable
process. As such, several have attempted to harness the unique
natural abilities of these organisms for commercial
applications.
[0008] Historically, the commercial applications for the
biotransformation of methane have fallen broadly into three
categories: 1.) production of single cell protein; 2.) epoxidation
of alkenes for production of chemicals; and 3.) biodegradation of
chlorinated pollutants. Only epoxidation of alkenes has experienced
slight commercial success due to low product yields, toxicity of
products, and the large amount of cell mass required to generate
products. Large-scale protein production from methane, termed
single cell protein (or "SCP"), has been technically feasible and
commercialized at large scale (Villadsen, J., Recent Trends Chem.
React. Eng., [Proc. Int. Chem. React. Eng. Conf.], 2.sup.nd ed.;
Kulkarni, B. D., Mashelkar, R. A., and Sharma, M. M., Eds.; Wiley
East: New Delhi, India (1987); Vol 2, pp 320-33). However, SCP has
not been economically successful thus far due to the relatively
high cost of producing microbial protein, as compared to
agriculturally derived protein (i.e., soy protein). This makes SCP
a relatively low value product whose economic production cannot
tolerate heavy bioprocessing costs. Thus, the yield of the
methanotrophic strain in the process may be critical to the overall
economic viability of the process. Microbial biomass produced by
methanotrophic bacteria is typically very high in protein content
(.about.70-80% by weight), which can restrict the direct use of
this protein to certain types of animal feed.
[0009] In addition to the synthesis of SCP, methanotrophic cells
can further build the oxidation products of methane (i.e., methanol
and formaldehyde) into more complex molecules such as carbohydrate,
lipids, and other proteins. For example, under certain conditions
methanotrophs are known to produce exopolysaccharides (U.S. Pat.
No. 6,537,786; U.S. Pat. No. 6,689,601; Ivanova et al.,
Mikrobiologiya, 57(4):600-5 (1988); Kilbane, John J., II, Gas, Oil,
Coal, Environ. Biotechnol. 3, [Pap. IGT's Int. Symp.], 3.sup.rd
ed., Meeting Date 1990; Akin, C. and J. Smith, Eds; IGT: Chicago,
Ill. (1991); pp 207-26). Similarly, methanotrophs are known to
accumulate both isoprenoid compounds and carotenoid pigments of
various carbon lengths (U.S. Pat. No. 6,660,507; U.S. Pat. No.
6,689,601; Urakami et al., J. Gen. Appl. Microbiol., 32(4):317-41
(1986)).
[0010] Most recently, the natural abilities of methanotrophic
organisms have been stretched by the advances of genetic
engineering. Odom et al. has investigated Methylomonas sp. 16a as a
microbial platform of choice for production of a variety of
materials beyond single cell protein, including carbohydrates,
pigments, terpenoid compounds and aromatic compounds (U.S. Pat. No.
6,689,601 and U.S. Pat. No. 09/941947, herein incorporated entirely
by reference). This particular methanotrophic bacterial strain is
capable of efficiently using either methanol or methane as a carbon
substrate, is metabolically versatile in that it contains multiple
pathways for the incorporation of carbon from formaldehyde into
3-carbon units, and is capable of genetic exchange with donor
species such as Escherichia coli via bacterial conjugation. Thus,
Methylomonas sp. 16a can be engineered to produce new classes of
products other than those naturally produced from methane and/or
methanol. Further advancement in the metabolic engineering of C1
metabolizing microorganisms such as Methylomonas sp. 16a, however,
is currently limited by the lack of efficient tools for generating
defined mutants by homologous recombination.
[0011] The ability to produce specific defined mutations in a
microorganism frequently relies on exploitation of the native
homologous recombination properties of the cell to replace a
nucleotide sequence of interest with a modified copy. Most
frequently, the nucleotide sequence of interest is a particular
functional gene of interest, which is then inactivated by the
insertion of an antibiotic-resistance marker. In theory, this type
of recombination event is easily detected on a selective medium;
however, performing allelic exchange in C1 metabolizing
microorganisms has been relatively cumbersome due to the organisms'
slow growth rates and the rarity of double-crossover events (which
require extensive screening to isolate an allelic-exchange mutant).
Despite these difficulties, there are examples of successful
identification of allelic exchange in slow-growing C1 metabolizing
microorganisms using suicide vectors (Naumov, G. N. et al., Mol.
Gen Mikrobiol. Virusol., 11:44-48 (1986) (abstract); Toyama and
Lidstrom, Microbiol., 144:183-191 (1998); Stolyar, S. et al.,
Microbiology, 145(5):1235-1244 (1999)). None of these previous
studies, however, have utilized positive selection as a method for
the rapid identification of allelic exchange mutants. Clearly, the
identification of allelic exchange mutants obtained by targeted
homologous recombination would greatly benefit from a system
allowing for the positive selection of mutants.
[0012] The positive selection (or direct genetic selection) of
mutant bacteria is possible whenever survival of the recombinant
bacteria depends upon the presence or absence of a particular
function encoded by the DNA that is introduced into the organism.
The advantage of a selection method over a screening method is that
growth of bacteria with the specific desired mutation is greatly
favored over bacteria lacking that specific mutation, thus
facilitating the identification of the preferred mutants.
[0013] Direct or positive selection vectors containing genes that
convey lethality to the host are well known. For example,
expression of the Bacillus subtilis or the B. amyloliquefaciens
sacB gene in the presence of sucrose is lethal to E. coli and a
variety of other Gram-negative and Gram-positive bacteria. The sacB
gene encodes levansucrase, which catalyzes both the hydrolysis of
sucrose and the polymerization of sucrose to form the lethal
product levan. Although the basis for the lethality of levansucrase
in the presence of sucrose is not fully understood, the inability
of E. coli and many other gram negative bacteria to grow when sacB
is expressed can be exploited to directly select for cells that
have lost the sacB gene via homologous recombination. Numerous
methods have been developed for the selection of various bacterial
mutants, based on sacB. See for example: U.S. Pat. No. 6,048,694
(Bramucci et al.) concerning Bacillus; U.S. Pat. No. 5,843,664
(Pelicic et al.) concerning mycobacterium; U.S. Pat. No. 5,380,657
(Schaefer et al.) concerning Coryneform bacteria; Hoang et al.
(Gene, 212(1):77-86 (1998)) concerning Pseudomonas aeruginosa;
Copass et al. (Infection and Immun., 65(5):1949-1952 (1997))
concerning Helicobacter pylon; and Kamoun et al. (Mol. Microbiol.,
6(6):809-816 (1992)) concerning Xanthomonas. However, no one has
previously attempted to use sacB in a positive selection method for
C1 metabolizing bacteria.
[0014] The problem to be solved, therefore, is to develop an
efficient means of generating defined mutants by homologous
recombination in C1 metabolizing bacteria. This method must permit
transformants that have undergone allelic exchange to be selected
for growth.
[0015] The present problem has been solved through the development
of positive selection vectors for C1 metabolizing bacteria. These
vectors, comprise at least one genetic selectable marker and the
sacB gene, encoding levansucrase. Thus, the selectable marker can
be used for the primary selection of transformants, while the sacB
gene has a conditionally dominant lethal effect, to enable
counter-selection against those clones which have chromosomally
integrated the vector DNA. This eliminates the need for extensive
screening. A one- and two-step selection strategy is provided for
the facile identification of allelic exchange mutations in C1
metabolizing bacteria. Further, this methodology enables production
of "markerless" transformants, such that multiple rounds of
mutation can be performed.
SUMMARY OF THE INVENTION
[0016] The present invention provides methods for the integration
of nucleotide sequences into the chromosome of a C1 metabolizing
host microbe. The method employs a positive selection vector
comprising the necessary genetic elements for replication of the
plasmid and transfer of the plasmid into the host cell.
Additionally, the vector comprises a sacB coding region and an
variety or genetic selectable markers. The vector may be used in a
number of conformations providing for targeted gene replacement as
well as transformants free of any vector or marker sequences.
[0017] Accordingly the invention provides a method for the positive
selection of double-crossover events in a C1 metabolizing bacterial
host cell comprising: [0018] a) providing a C1 metabolizing
bacteria selected from the group consisting of methanotrophs and
methylotrophs; [0019] b) providing a positive selection vector
comprising: [0020] (i) a first genetic selectable marker; [0021]
(ii) an origin of transfer for a Cl metabolizing bacteria; [0022]
(iii) plasmid mobilization genes; [0023] (iv) a sacB coding region
encoding a levansucrase enzyme, under the control of a suitable
promoter; [0024] (v) a replacement nucleotide sequence of interest
having at least two regions homology to a chromosomal nucleotide
sequence of interest in the C1 metabolizing bacteria, said
replacement nucleotide sequence of interest being disrupted by a
second genetic selectable marker; [0025] wherein the first and
second genetic selectable markers are different and the vector is
unable to replicate in the Cl metabolizing bacteria; [0026] c)
transforming the C1 metabolizing bacteria of (a) with the vector of
(b) [0027] d) selecting the transformants of (c) on the basis of
the genetic selectable markers and sacB gene expression wherein,
those transformants which are positive for the second selectable
marker and grow in the presence of sucrose, but are negative for
the first selectable marker have undergone double-crossover events;
and [0028] e) recovering the selected transformants of (d) which
have undergone double-crossover events.
[0029] In similar fashion, the invention provides a method for the
positive selection of double-crossover events in C1 metabolizing
bacterial host cells comprising: [0030] a) providing a C1
metabolizing bacteria selected from the group consisting of
methanotrophs and methylotrophs; [0031] b) providing a positive
selection vector comprising: [0032] (i) a genetic selectable
marker; [0033] (ii) an origin of transfer for a C1 metabolizing
bacteria; [0034] (iii) plasmid mobilization genes; [0035] (iv) a
sacB coding region encoding a levansucrase enzyme under the control
of a suitable promoter; [0036] (v) a mutant replacement nucleotide
sequence of interest having at least two regions of homology to a
chromosomal nucleotide sequence of interest in the C1 metabolizing
bacteria; [0037] the vector being unable to replicate in the C1
metabolizing bacteria chromosome; [0038] c) transforming the C1
metabolizing bacteria of (a) with the vector of (b); [0039] d)
isolating the transformants of (c) on the basis of the genetic
selectable marker; and [0040] e) isolating the transformants of (d)
which grow in the presence of sucrose, wherein said transformants
contain double-crossover events.
[0041] C1 metabolizing bacteria transformants selected by the
methods of the invention are also provided.
[0042] Additionally, the invention provides a positive selection
vector for the positive selection of double-crossover events in a
C1 metabolizing bacterial host cell comprising: [0043] (i) at least
one genetic selectable marker; [0044] (ii) an origin of transfer
for a C1 metabolizing bacteria; [0045] (iii) plasmid mobilization
genes; [0046] (iv) a sacB coding region encoding a levansucrase
enzyme under the control of a suitable promoter; [0047] (v) a
replacement nucleotide sequence of interest having at least two
regions of homology to a chromosomal nucleotide sequence of
interest in the C1 metabolizing bacteria; the vector being unable
to replicate in the C1 metabolizing bacteria chromosome, wherein
the vector of the invention may optionally comprise an additional
genetic selectable marker disrupting the replacement nucleotide
sequence of interest.
BRIEF DESCRIPTION OF THE FIGURES. SEQUENCE DESCRIPTIONS, AND
BIOLOGICAL DEPOSITS
[0048] FIG. 1 is a schematic diagram illustrating the single-step
selection protocol, useful for identifying double-crossover mutants
containing a marker in C1 metabolizing bacteria.
[0049] FIG. 2 is a schematic diagram illustrating the two-step
selection protocol, useful for identifying "markerless"
double-crossover mutants in C1 metabolizing bacteria.
[0050] FIG. 3 illustrates the organization of the crtN1 gene
cluster in Methylomonas sp. 16a.
[0051] FIG. 4 is a plasmid map of pGP704.
[0052] The invention can be more fully understood from the
following detailed description and the accompanying sequence
descriptions, which form a part of this application.
[0053] The following sequences conform with 37 C.F.R. 1.821-1.825
("Requirements for Patent Applications Containing Nucleotide
Sequences and/or Amino Acid Sequence Disclosures--the Sequence
Rules") and are consistent with World Intellectual Property
Organization (WIPO) Standard ST.25 (1998) and the sequence listing
requirements of the EPO and PCT (Rules 5.2 and 49.5(a-bis), and
Section 208 and Annex C of the Administrative Instructions). The
symbols and format used for nucleotide and amino acid sequence data
comply with the rules set forth in 37 C.F.R. .sctn.1.822.
[0054] SEQ ID NOs:1-3 are the full-length Methylomonas sp. 16a
genes known as crtN1, ald, and crtN2, respectively.
[0055] SEQ ID NOs:4 and 5 encode primers Drdl/npr-sacB and
Tthlll/npr-sacB, respectively, used for amplification of the
npr-sacB cassette from plasmid pBE83.
[0056] SEQ ID NOs:6 and 7 encode primers crtN#2-ctg288+1 kB/Bg/ll
and crtN#2-ctg 288+1kB/Xbal, respectively, used for amplification
of the crtN2 gene from Methylomonas sp. 16a.
[0057] SEQ ID NOs:8 and 9, respectively, encode the
transposon-specific primers KAN-2FP-1 and KAN-2RP-1.
[0058] SEQ ID NOs:10 and 11, respectively, encode primers
OP#1crtN#2 and OP#2crtN#2, used for confirming the double-crossover
crtN2 mutant.
[0059] SEQ ID NOs:12 and 13 encode primers Bg/ll/ald (deletion)#1
and Sphl-Notl/ald (deletion)#1, respectively, used for the
amplification of aldehyde deletion fragment #1 when constructing a
deletion of the ald gene in Methylomonas sp. 16a chromosomal
DNA.
[0060] SEQ ID NOs:14 and 15 encode primers Notl/ald (deletion)#2
and Sphl/ald (deletion)#2, respectively, used for the amplification
of aldehyde deletion fragment #2 when constructing a deletion of
the ald gene in Methylomonas sp. 16a chromosomal DNA.
[0061] Applicants made the following biological deposit under the
terms of the Budapest Treaty on the International Recognition of
the Deposit of Micro-organisms for the Purposes of Patent
Procedure: TABLE-US-00001 Depositor International Identification
Depository Reference Designation Date of Deposit Methylomonas 16a
ATCC PTA 2402 Aug. 22, 2000
[0062] As used herein, "ATCC" refers to the American Type Culture
Collection International Depository Authority located at ATCC,
10801 University Blvd., Manassas, Va. 20110-2209, U.S.A. The
"International Depository Designation" is the accession number to
the culture on deposit with ATCC.
[0063] The listed deposit will be maintained in the indicated
international depository for at least thirty (30) years and will be
made available to the public upon the grant of a patent disclosing
it. The availability of a deposit does not constitute a license to
practice the subject invention in derogation of patent rights
granted by government action.
DETAILED DESCRIPTION OF THE INVENTION
[0064] The present invention describes a process for the positive
selection of allelic exchange mutants in a C1 metabolizing
bacteria. Thus, using the native homologous recombination machinery
of the C1 metabolizing host cell it is possible to replace a
specific chromosomal nucleotide sequence of interest in a host
organism's genome with a modified sequence (replacement nucleotide
sequence of interest) via a double-crossover event.
[0065] In a single-step protocol, a suicide vector containing at
least two genetic selectable markers (one marker positioned within
the replacement nucleotide sequence of interest), a replacement
nucleotide sequence of interest having homology to the chromosomal
nucleotide sequence of interest, and the sacB gene are used to
produce allelic exchange mutants that can be directly selected on
sucrose and the selectable marker located within the disrupted
nucleotide sequence.
[0066] In a two-step selection procedure, the suicide vector
comprises at least one genetic selectable marker, a replacement
nucleotide sequence of interest having homology to the chromosomal
nucleotide sequence of interest, and a sacB coding sequence under
the control of a suitable promoter. Following transformation of the
host bacteria, transformants that underwent a single-crossover
event are first selected based on the genetic selectable marker.
Then, by plating on sucrose, the mutants that have lost the sacB
gene during a second crossover event can be positively
selected.
[0067] Thus, depending on the specific vector construct used and
the choice of selection strategy, it is possible to produce both
allelic exchange transformants containing a marker at the site of
DNA replacement and markerless transformants.
DEFINITIONS
[0068] In this disclosure, a number of terms and abbreviations are
used. The following definitions are provided.
[0069] As used herein, the term "comprising" means the presence of
the stated features, integers, steps, or components as referred to
in the claims, but that it does not preclude the presence or
addition of one or more other features, integers, steps, components
or groups thereof.
[0070] "Polymerase chain reaction" is abbreviated PCR.
[0071] "Kanamycin" is abbreviated Kan.
[0072] "Ampicillin" is abbreviated Amp.
[0073] The term "positive selection" means a selection method that
enables only those cells that carry a DNA insert integrated at a
specific chromosomal location to grow under particular conditions.
In contrast, negative selection is based on selection methods
whereby only those individuals that do not possess a certain
character (e.g., cells that do not carry a DNA insert integrated at
a specific chromosomal location) are selected.
[0074] The term "homologous recombination" refers to the exchange
of DNA fragments between two DNA molecules (during cross over). The
fragments that are exchanged are flanked by sites of identical
nucleotide sequences between the two DNA molecules (i.e., "regions
of homology"). Homologous recombination is the most common means
for generated genetic diversity in microbes.
[0075] The term "regions of homology" refer to stretches of
nucleotide sequence on nucleic acid fragments that participate in
homologous recombination that have homology to each other.
Effective homologous recombination will take place where these
regions of homology are at least about 10 bp in length where at
least about 50 bp in length is preferred. Typically, fragments that
are intended for recombination contain at least two regions of
homology where targeted gene disruption or replacement is
desired.
[0076] The term "chromosomal integration" means that a chromosomal
integration vector becomes congruent with the chromosome of a
microorganism through recombination between homologous DNA regions
on the chromosomal integration vector and within the
chromosome.
[0077] The term "chromosomal integration vector" means an
extra-chromosomal vector that is capable of integrating into the
host's genome through homologous recombination.
[0078] The term "suicide vector" or "positive selection vector"
refers to a type of chromosomal integration vector that is capable
of replicating in one host but not in another. Thus, the vector is
conditional for its replication.
[0079] The terms "single-crossover event" and "plasmid integration"
are used interchangeably and mean the incorporation of a
chromosomal integration vector into the genome of a host via
homologous recombination between regions of homology between DNA
present within the chromosomal integration vector and the host's
chromosomal DNA. A "5single-crossover mutant" refers to a cell that
has undergone a single-crossover event.
[0080] The terms "double-crossover event", "allelic exchange" and
"gene replacement" are used interchangeably and mean the homologous
recombination between a DNA region within the chromosomal
integration vector and a region within the chromosome that results
in the replacement of the functional chromosomal nucleotide
sequence of interest (i.e., chr-NSI) with a homologous plasmid
region (i.e., the replacement nucleotide sequence of interest, or
re-NSI). A "double-crossover mutant" or "allelic exchange mutant"
is the result of a double-crossover event. This mutant can be
generated by two simultaneous reciprocal breakage and reunion
events between the same two DNA fragments; alternatively, a
double-crossover mutant can be the result of two single-crossovers
that occur non-simultaneously.
[0081] The term "chromosomal nucleotide sequence of interest" is
abbreviated "chr-NSI" and refers to a specific chromosomal sequence
that is targeted for homologous recombination. In a preferred
embodiment, the chr-NSI encodes a native gene of the C1
metabolizing bacteria.
[0082] The term "replacement nucleotide sequence of interest" is
abbreviated "re-NSI" and refers to a nucleotide sequence of
interest that is cloned into a chromosomal integration vector for
the purpose of inducing homologous recombination with a chromosomal
sequence. The re-NSI is modified with respect to chr-NSI by the
addition, deletion, or substitution of at least one nucleotide.
Sufficient homology must exist, however, between the two nucleotide
sequences of interest to enable homologous recombination to occur.
Similarly the term "mutant replacement nucleotide sequence of
interest" refers to a nucleic acid sequence having homology to a
sequence in a microbial genome, that has nucleotide additions,
deletions or substitutions in the sequence that differ from the
chromosomal sequence.
[0083] The terms "genetic selectable marker" or "selectable marker"
will be used interchangeably and means a phenotypic trait, encoded
by a genetic element that can be visualized under special
conditions. For example, an antibiotic resistance marker serves as
a useful selectable marker, since it enables detection of cells
which are resistant to the antibiotic, when cells are grown on
media containing that particular antibiotic.
[0084] The term "markerless mutants" or "markerless transformants"
refers to an allelic exchange mutant, wherein the mutant allele
does not carry a genetic marker.
[0085] The term "SacB" means a Bacillus encoded protein that
catalyzes the conversion of sucrose into levan, a product that is
toxic to most Gram negative microorganisms. The term "sacB" means a
gene that encodes the "SacB" protein.
[0086] The terms "transposon" and "transposable element" are used
interchangeably and refer to a relatively small DNA transposable or
movable genetic element that has the ability to move from one
chromosomal position to another within a DNA molecule, using a
process known as transposition. Transposition in bacteria does not
require extensive DNA homology between the transposon and the
target DNA. A well-known example of a bacterial transposon is Tn5,
which carries the genes for resistance to the antibiotics neomycin
and kanamycin and a transposase. The term "transposase" means a
protein that catalyses the chemical steps, i.e., breakage and
joining, of a transposition reaction.
[0087] The term "in vitro transposition" means a biochemical
reaction that is initiated outside the cell that catalyzes the
movement of a transposable element from one site into a different
site within a DNA molecule.
[0088] As used herein, the term "C.sub.1 carbon substrate" refers
to any carbon-containing molecule that lacks a carbon-carbon bond.
Non-limiting examples are methane, methanol, formaldehyde, formic
acid, formate, methylated amines (e.g., mono-, di-, and tri-methyl
amine), methylated thiols, and carbon dioxide. In one embodiment,
the C.sub.1 carbon substrate is selected from the group consisting
of methanol and/or methane.
[0089] The term "C.sub.1 metabolizer" refers to a microorganism
that has the ability to use a single carbon substrate as its sole
source of energy and biomass. C.sub.1 metabolizers will typically
be methylotrophs and/or methanotrophs.
[0090] The term "C.sub.1 metabolizing bacteria" refers to bacteria
that have the ability to use a single carbon substrate as their
sole source of energy and biomass. C.sub.1 metabolizing bacteria, a
subset of C.sub.1 metabolizers, will typically be methylotrophs
and/or methanotrophs.
[0091] The term "methylotroph" means an organism capable of
oxidizing organic compounds that do not contain carbon-carbon
bonds. Where the methylotroph is able to oxidize CH.sub.4, the
methylotroph is also a methanotroph. In one embodiment, the
methylotroph organism is a methylotrophic bacteria capable of using
methanol and/or methane as single carbon substrate.
[0092] The term "methanotroph" or "methanotrophic bacteria" means a
prokaryote capable of utilizing methane and/or methanol as a source
of carbon and energy. Complete oxidation of methane to carbon
dioxide occurs by aerobic degradation pathways. Typical examples of
methanotrophs useful in the present invention include (but are not
limited to) the genera Methylomonas, Methylobacter, Methylococcus,
and Methylosinus.
[0093] The term "high growth methanotrophic bacterial strain"
refers to a bacterium capable of growth with methane or methanol as
the sole carbon and energy source and which possesses a functional
Embden-Meyerof carbon flux pathway, resulting in a high rate of
growth and yield of cell mass per gram of C.sub.1 substrate
metabolized (U.S. Pat. No. 6,689,601). The specific "high growth
methanotrophic bacterial strain" described herein is referred to as
"Methylomonas 16a", "16a" or "Methylomonas sp. 16a", which terms
are used interchangeably and which refer to the Methylomonas strain
used in the present invention.
[0094] The term "carotenoid" refers to a class of hydrocarbons
having a conjugated polyene carbon skeleton formally derived from
isoprene. This class of molecules is composed of C.sub.30
diapocarotenoids and C.sub.40 carotenoids and their oxygenated
derivatives; and, these molecules typically have strong light
absorbing properties.
[0095] "C.sub.30 diapocarotenoids" consist of six isoprenoid units
joined in such a manner that the arrangement of isoprenoid units is
reversed at the center of the molecule so that the two central
methyl groups are in a 1,6-positional relationship and the
remaining nonterminal methyl groups are in a 1,5-positional
relationship. All C.sub.30 carotenoids may be formally derived from
the acyclic C.sub.30H.sub.42 structure (Formula I below,
hereinafter referred to as "diapophytoene"), having a long central
chain of conjugated double bonds, by: (i) hydrogenation (ii)
dehydrogenation, (iii) cyclization, (iv) oxidation, (v)
esterification/glycosylation, or any combination of these
processes. ##STR1##
[0096] The term "CrtN1" refers to an enzyme encoded by the crtN1
gene, active in the native carotenoid biosynthetic pathway of
Methylomonas sp. 16a. This gene is located within an operon
comprising crtN2 and ald.
[0097] The term "CrtN2" refers to an enzyme encoded by the crtN2
gene, active in the native carotenoid biosynthetic pathway of
Methylomonas sp. 16a. This gene is located within an operon
comprising CrtN1 and ald.
[0098] The term "ALD" refers to an enzyme (an aldehyde
dehydrogenase) encoded by the ald gene, active in the native
carotenoid biosynthetic pathway of Methylomonas sp. 16a. This gene
is located within an operon comprising CrtN1 and CrtN2.
[0099] A "nucleic acid" is a polymeric compound comprised of
covalently linked subunits called nucleotides. Nucleic acids
include polyribonucleic acid (RNA) and polydeoxyribonucleic acid
(DNA), both of which may be single-stranded or double-stranded. DNA
includes cDNA, genomic DNA, synthetic DNA, and semi-synthetic
DNA.
[0100] As used herein, an "isolated nucleic acid fragment" is a
polymer of RNA or DNA that is single- or double-stranded,
optionally containing synthetic, non-natural or altered nucleotide
bases. An isolated nucleic acid fragment in the form of a polymer
of DNA may be comprised of one or more segments of cDNA, genomic
DNA or synthetic DNA.
[0101] A nucleic acid fragment is "hybridizable" to another nucleic
acid fragment, such as a cDNA, genomic DNA, or RNA, when a single
stranded form of the nucleic acid fragment can anneal to the other
nucleic acid fragment under the appropriate conditions of
temperature and solution ionic strength. Hybridization and washing
conditions are well known and exemplified in Sambrook, J., Fritsch,
E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual,
2.sup.nd ed., Cold Spring Harbor Laboratory: Cold Spring Harbor
(1989), particularly Chapter 1 1 and Table 11.1 therein (entirely
incorporated herein by reference). The conditions of temperature
and ionic strength determine the "stringency" of the hybridization.
Stringency conditions can be adjusted to screen for moderately
similar fragments (such as homologous sequences from distantly
related organisms) to highly similar fragments (such as genes that
duplicate functional enzymes from closely related organisms).
Post-hybridization washes determine stringency conditions. One set
of preferred conditions uses a series of washes starting with
6.times.SSC, 0.5% SDS at room temperature for 15 min, then repeated
with 2.times.SSC, 0.5% SDS at 45.degree. C. for 30 min, and then
repeated twice with 0.2.times.SSC, 0.5% SDS at 50.degree. C. for 30
min. A more preferred set of stringent conditions uses higher
temperatures in which the washes are identical to those above
except for the temperature of the final two 30 min washes in
0.2.times.SSC, 0.5% SDS was increased to 60.degree. C. Another
preferred set of highly stringent conditions uses two final washes
in 0.1.times.SSC, 0.1% SDS at 65.degree. C. An additional set of
stringent conditions include hybridization at 0.1.times.SSC, 0.1%
SDS, 65.degree. C. and washed with 2.times.SSC, 0.1% SDS followed
by 0.1.times.SSC, 0.1% SDS, for example.
[0102] Hybridization requires that the two nucleic acids contain
complementary sequences, although depending on the stringency of
the hybridization, mismatches between bases are possible. The
appropriate stringency for hybridizing nucleic acids depends on the
length of the nucleic acids and the degree of complementation,
variables well known in the art. The greater the degree of
similarity or homology between two nucleotide sequences, the
greater the value of Tm for hybrids of nucleic acids having those
sequences. The relative stability (corresponding to higher Tm) of
nucleic acid hybridizations decreases in the following order:
RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100
nucleotides in length, equations for calculating Tm have been
derived (see Sambrook et al., supra, 9.50-9.51). For hybridizations
with shorter nucleic acids, i.e., oligonucleotides, the position of
mismatches becomes more important, and the length of the
oligonucleotide determines its specificity (see Sambrook et al.,
supra, 11.7-11.8). In one embodiment the length for a hybridizable
nucleic acid is at least about 10 nucleotides. Preferable a minimum
length for a hybridizable nucleic acid is at least about 15
nucleotides; more preferably at least about 20 nucleotides; and
most preferably the length is at least 30 nucleotides. Furthermore,
the skilled artisan will recognize that the temperature and wash
solution salt concentration may be adjusted as necessary according
to factors such as the length of the probe.
[0103] The term "oligonucleotide" refers to a nucleic acid,
generally of at least about 18 nucleotides, that is hybridizable to
a genomic DNA molecule, a cDNA molecule, or an mRNA molecule.
[0104] "Gene" refers to a nucleic acid fragment that expresses a
specific protein, including regulatory sequences preceding (5'
non-coding sequences) and following (3' non-coding sequences) the
coding sequence. "Native gene" refers to a gene as found in nature
with its own regulatory sequences. "Chimeric gene" refers to any
gene that is not a native gene, comprising regulatory and coding
sequences that are not found together in nature. Accordingly, a
chimeric gene may comprise regulatory sequences and coding
sequences that are derived from different sources, or regulatory
sequences and coding sequences derived from the same source, but
arranged in a manner different than that found in nature.
"Endogenous gene" refers to a native gene in its natural location
in the genome of an organism. A "foreign" gene refers to a gene not
normally found in the host organism, but that is introduced into
the host organism by gene transfer. Foreign genes can comprise
native genes inserted into a non-native organism, or chimeric
genes. A "transgene" is a gene that has been introduced into the
genome by a transformation procedure.
[0105] "Chemically synthesized", as related to a sequence of DNA,
means that the component nucleotides were assembled in vitro.
Manual chemical synthesis of DNA may be accomplished using
well-established procedures, or automated chemical synthesis can be
performed using one of a number of commercially available
machines.
[0106] "Synthetic genes" can be assembled from oligonucleotide
building blocks that are chemically synthesized using procedures
known to those skilled in the art. These building blocks are
ligated and annealed to form gene segments that are then
enzymatically assembled to construct the entire gene. Accordingly,
the genes can be tailored for optimal gene expression based on
optimization of nucleotide sequence to reflect the codon bias of
the host cell. The skilled artisan appreciates the likelihood of
successful gene expression if codon usage is biased towards those
codons favored by the host. Determination of preferred codons can
be based on a survey of genes derived from the host cell where
sequence information is available.
[0107] As used herein, the term "homolog", as applied to a gene,
means any gene derived from the same or a different microbe having
the same function. A homologous gene may have significant sequence
similarity.
[0108] "Coding sequence" or "coding region of interest" refers to a
DNA sequence that codes for a specific amino acid sequence.
[0109] "Suitable regulatory sequences" refer to nucleotide
sequences located upstream (5' non-coding sequences), within, or
downstream (3' non-coding sequences) of a coding sequence, and
which influence the transcription, RNA processing or stability, or
translation of the associated coding sequence. Regulatory sequences
may include promoters, translation leader sequences, RNA processing
sites, effector binding sites and stem-loop structures.
[0110] "Transcriptional and translational control sequences" are
DNA regulatory sequences, such as promoters, enhancers,
terminators, and the like, that provide for the expression of a
coding sequence in a host cell. "Promoter" refers to a DNA sequence
capable of controlling the expression of a coding sequence or
functional RNA. In general, a coding sequence is located 3' to a
promoter sequence. Promoters may be derived in their entirety from
a native gene, or be composed of different elements derived from
different promoters found in nature, or even comprise synthetic DNA
segments. It is understood by those skilled in the art that
different promoters may direct the expression of a gene at
different stages of development, or in response to different
environmental or physiological conditions. Promoters that cause a
gene to be expressed in most cell types at most times are commonly
referred to as "constitutive promoters". It is further recognized
that since in most cases the exact boundaries of regulatory
sequences have not been completely defined, DNA fragments of
different lengths may have identical promoter activity.
[0111] The "3' non-coding sequences" refer to DNA sequences located
downstream of a coding sequence and include sequences encoding
regulatory signals capable of affecting mRNA processing or gene
expression.
[0112] The term "operably linked" refers to the association of
nucleic acid sequences on a single nucleic acid fragment so that
the function of one is affected by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
the coding sequence is under the transcriptional control of the
promoter). Coding sequences can be operably linked to regulatory
sequences in sense or antisense orientation.
[0113] "Transformation" refers to the transfer of a nucleic acid
fragment into the genome of a host organism, resulting in
genetically stable inheritance. As used herein, the host cell's
genome is comprised of chromosomal and extrachromosomal (e.g.
plasmid) genes. Host organisms containing the transformed nucleic
acid fragments are referred to as "transgenic" or "recombinant" or
"transformed" organisms.
[0114] "Conjugation" refers to a particular type of transformation
in which a unidirectional transfer of DNA (e.g., from a bacterial
plasmid) occurs from one bacterium cell (i.e., the "donor") to
another (i.e., the "recipient"). The process involves direct
cell-to-cell contact.
[0115] The terms "plasmid", "vector" and "cassette" refer to an
extra chromosomal element often carrying genes which are not part
of the central metabolism of the cell, and usually in the form of
circular double-stranded DNA fragments. Such elements may be
autonomously replicating sequences, genome integrating sequences,
phage or nucleotide sequences, linear or circular, of a single- or
double-stranded DNA or RNA, derived from any source, in which a
number of nucleotide sequences have been joined or recombined into
a unique construction. "Transformation cassette" refers to a
specific vector containing a foreign gene and having elements (in
addition to the foreign gene) that facilitate transformation of a
particular host cell. "Expression cassette" refers to a specific
vector containing a foreign gene and having elements in addition to
the foreign gene that allow for enhanced expression of that gene in
a foreign host.
[0116] The term "altered biological activity" or "altered activity"
will refer to an activity, associated with a protein encoded by a
nucleotide sequence which can be measured by an assay method, where
that activity is either greater than or less than the activity
associated with the native sequence. "Enhanced biological activity"
refers to an altered activity that is greater than that associated
with the native sequence. "Diminished biological activity" is an
altered activity that is less than that associated with the native
sequence.
[0117] The term "sequence analysis software" refers to any computer
algorithm or software program that is useful for the analysis of
nucleotide or amino acid sequences. "Sequence analysis software"
may be commercially available or independently developed. Typical
sequence analysis software will include, but is not limited to: the
GCG suite of programs (Wisconsin Package Version 9.0, Genetics
Computer Group (GCG), Madison, Wis.); BLASTP, BLASTN, BLASTX
(Altschul et al., J. Mol. Biol. 215:403410 (1990)); DNASTAR
(DNASTAR, Inc., Madison, Wis.); and the FASTA program incorporating
the Smith-Waterman algorithm (W. R. Pearson, Comput. Methods Genome
Res., [Proc. Int. Symp.], Meeting Date 1992,111-20. Suhai, Sandor,
Ed.; Plenum: New York, N.Y. (1994)). Within the context of this
application it will be understood that where sequence analysis
software is used for analysis, the results of the analysis are
based on the "default values" of the program referenced, unless
otherwise specified. As used herein "default values" will mean any
set of values or parameters which originally load with the software
when first initialized.
[0118] Standard recombinant DNA and molecular cloning techniques
used here are well known in the art and are described by Sambrook,
J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A
Laboratory Manual, 2.sup.nd ed., Cold Spring Harbor Laboratory:
Cold Spring Harbor, N.Y. (1989) (hereinafter "Maniatis"); and by
Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with
Gene Fusions, Cold Spring Harbor Laboratory: Cold Spring Harbor,
N.Y. (1984); and by Ausubel, F. M. et al., Current Protocols in
Molecular Biology, published by Greene Publishing Assoc. and
Wiley-Interscience (1987).
Overview of the One-Step and Two-Step Selection Strategy
[0119] The principles of the one-step and two-step selection
strategies of the present invention are illustrated in FIGS. 1 and
2, respectively. Each of these methods relies on the application of
a positive selection vector, in a preferred embodiment derived from
the suicide delivery vector pGP704, which is able to integrate into
the chromosome of C1 metabolizing bacteria to produce mutations
that are the result of both single- or double-crossover events. The
positive selection vector must contain an origin of transfer
suitable for a C1 metabolizing bacteria, as well as plasmid
mobilization genes.
[0120] The one-step selection strategy for the positive selection
of the double-crossover events so produced in C1 metabolizing
bacterial host cells is illustrated in FIG. 1. Specifically, the
positive selection vector comprises: [0121] (i) at least one gene
encoding resistance to a first genetic selectable marker (e.g.,
Amp); [0122] (ii) a sacB coding region encoding a levansucrase
enzyme under the control of a suitable promoter; and [0123] (iii) a
replacement nucleotide sequence of interest (i.e., re-NSI) that is
disrupted by a gene encoding resistance to a second genetic
selectable marker, wherein the second genetic selectable marker is
different than the first genetic selectable marker (e.g., Kan). The
re-NSI in the positive selection vector must have at least two
regions homology to a nucleotide sequence of interest in the
chromosome of the C1 metabolizing bacteria (i.e., chr-NSI), in
order to undergo allelic exchange.
[0124] Upon transformation of C1 metabolizing bacteria with the
positive selection vector described above, a double-crossover event
by homologous recombination may occur between chr-NSI and re-NSI,
such that the chr-NSI is replaced with re-NSI from the vector.
Direct selection of these allelic exchange transformants is
possible by growing the transformants in the presence of the
genetic selectable markers and sucrose. Specifically, Table 1
illustrates the specific type of transformants that would be
identified using various combinations of selection, according to
the illustration shown in FIG. 1 (although the selection markers
illustrated therein are by no means to be construed as a limitation
to the present invention). TABLE-US-00002 TABLE 1 Identification of
Single and Double Crossover Mutants using Various Strategies of
Selection Selection Bacteria Identified by Selection sucrose only
wildtype and double-crossover mutants Kan only single- and
double-crossover mutants Amp only single-crossover mutants Amp +
Kan single-crossover mutants Kan + sucrose double-crossover
mutants
[0125] Clearly, facile identification of transformants which have
undergone double-crossover events is possible by growing
transformants in the presence of the second genetic selectable
marker (i.e., that disrupted re-NSI) and sucrose.
[0126] In FIG. 2, the two-step selection strategy for the positive
selection of the double-crossover events produced in C1
metabolizing bacterial host cells is illustrated. For this
strategy, the positive selection vector comprises: [0127] (i) at
least one gene encoding resistance to a first genetic selectable
marker (e.g., Amp); [0128] (ii) a sacB coding region encoding a
levansucrase enzyme under the control of a suitable promoter; and
[0129] (iii) a replacement nucleotide sequence of interest (i.e.,
re-NSI), which one desires to insert into the chromosome of the C1
metabolizing bacteria as a replacement to an existing nucleotide
sequence of interest in the bacterial chromosome (i.e., chr-NSI).
Thus, re-NSI is a mutant re-NSI modified with respect to chr-NSI by
the addition, substitution, or deletion of at least one
nucleotide.
[0130] Upon transformation of C1 metabolizing bacteria with the
positive selection vector described above, a single-crossover event
by homologous recombination occurs between chr-NSI and re-NSI, such
that the entire positive selection vector is integrated into the
bacterial chromosome at the site of crossover. These events can be
selected by growth on the first genetic selectable marker (e.g.,
Amp), whereby a complete copy of chr-NSI and a complete copy of
re-NSI are present in the chromosome. Upon removal of selection by
the first genetic selectable marker, a second crossover event may
occur, resulting in the "looping out" of the positive selection
vector, to yield transformants containing either the chr-NSI or the
re-NSI in the chromosome. Direct selection of these allelic
exchange transformants is possible by growing the transformants in
the presence of sucrose, since single-crossover mutants will be
killed under these conditions.
[0131] Although both the one-step versus two-step selection
strategy will result in the facile identification allelic exchange
mutants, use of the one-step selection strategy will produce
allelic exchange transformants containing a marker at the site of
DNA replacement while use of the two-step selection strategy will
produce allelic exchange transformants that are markerless. Each
methodology will therefore have advantages that must be considered
with respect to subsequent applications.
Chromosomal Integration Vectors for Generating and Isolating
Single- and Double-Crossover Mutants
[0132] Suicide plasmids can be used to create defined modifications
within a targeted chromosome of a host organism's genome.
Frequently, these defined modifications result in disruptions,
insertions, and replacements upstream, downstream, and within
native genes via homologous recombination. A single-or
double-crossover event can occur via targeted homologous
recombination using the following methodology. First, a specific
nucleotide sequence of interest (i.e., re-NSI) is cloned into a
chromosomal integration vector and the vector is subsequently
introduced into the specific host organism (the "recipient"). The
transformed host is then placed under conditions where the plasmid
cannot replicate.
[0133] Single-crossover mutants can be identified by selection for
a selectable marker on the plasmid. These transformants have
integrated the plasmid into the host chromosome via homology
between re-NSI and chr-NSI. In one embodiment, it is possible to
produce a single-crossover mutant whereby two non-functional copies
of the nucleotide sequence of interest are present in the recipient
chromosome. This requires one to clone an internal portion of the
chr-NSI (i.e., wherein the 5' and 3' portion of chr-NSI are not
present) into the chromosomal integration vector as re-NSI. Upon
homologous recombination, the functional 5' portion of chr-NSI is
joined with the non-functional 3' portion of re-NSI; in like
manner, the non-functional 5' portion of re-NSI is joined with the
functional 3' portion of chr-NSI. Thus, both chromosomal copies of
the nucleotide sequence of interest are non-functional.
[0134] In contrast to single-crossover mutants, double-crossover
mutants occur with less frequency and are typically much more
difficult to isolate. Identification of double-crossover events,
whereby two crossovers between re-NSI and chr-NSI must occur,
requires either the one-step or two-step selection procedure of the
present invention. In one embodiment, re-NSI must possess an easily
selectable phenotype (e.g., when disrupted with a Kan.sup.R
insertion), thus enabling a one-step procedure for selection (FIG.
1). Alternatively, when direct selection of the mutation is not
possible (e.g., when re-NSI is marked with only a point mutation) a
two-step procedure is used instead (FIG. 2). First, the entire
plasmid is integrated into the chromosome by a single-crossover
between the homologous nucleotide sequences of interest, producing
a chromosomal duplication. Wild-type cells are selected against
while those transformants possessing a single-crossover are
selected for according to the genetic selectable marker present on
the suicide vector. Then, the chromosomal duplication must undergo
a second homologous recombination event between the two copies of
the nucleotide sequences of interest. Depending on the specific
site of this second recombination event, a single copy of the
nucleotide sequence of interest remains on the chromosome--either
the wild-type copy (i.e., chr-NSI) or the mutant replacement copy
(i.e., re-NSI). In all cases where a second recombination event
occurs, however, the integrated plasmid is excised from the
recient's chromosome. Based on the rare occurrence of this second
homologous recombination event, a second selection strategy must
exist to differentiate between single- and double-crossover
mutants. One way of selecting against the integrated plasmid is to
use a plasmid that also carries the sacB gene. This enables a
direct selection for loss of the plasmid.
[0135] Based on the description above for the generation and
selection of allelic exchange mutants, chromosomal integration
vectors utilized for the generation of double-crossover mutations
in plasmids will generally possess the following characteristics:
1.) The plasmid will be conditional for replication to permit
integration into the chromosome. This can be achieved by using a
plasmid that is able to replicate autonomously only in permissive
hosts or by using conditional replicons (e.g., a plasmid that is
temperature-sensitive for replication; 2.) the plasmid will
typically carry at least one genetic selectable marker; 3.) it is
desirable if the plasmid is transferable to a variety of other
bacteria; and 4.) preferably, the plasmid will possess a variety of
unique cloning sites.
[0136] In addition to the above characteristics of a chromosomal
integration vector, the vector in the present invention must be
suitable for integration into C1 metabolizing bacterial host cells.
Thus, the chromosomal integration vector suitable for positive
selection in the present invention will generally possess the
following characteristics: [0137] 1. The plasmid will typically be
conditional for replication to permit integration into the
chromosome; [0138] 2. The plasmid will generally possess at least
one genetic selectable marker; [0139] 3. The plasmid will have an
origin of transfer for a C1 metabolizing bacteria; [0140] 4. The
plasmid will generally possess mobilization genes, such that it is
transferable to other bacteria (e.g., Escherchia coli); [0141] 5.
The plasmid will possess a sacB coding region (encoding
levansucrase) under the control of a suitable promoter; and, [0142]
6. The plasmid will typically contain a nucleotide sequence of
interest having homology to a nucleotide sequence of interest in
the chromosome of the C1 metabolizing bacteria. [0143] 7.
Preferably, the plasmid will contain a variety of unique cloning
sites. Each of these components of the positive selection vector
will be discussed in detail below.
[0144] 1. Conditional Replication and Chromosomal Integration
[0145] In general, the act of plasmid replication requires two
components: a DNA segment on the vector DNA known as an origin of
replication (i.e., the site on the plasmid where DNA replication
initiates) and vector-encoded proteins that control replication
through interactions with the DNA sequences. Conditional
replication occurs when a plasmid replicates only under a specific
set of conditions. There are several types of conditional
replication systems known in the art. For example, some vectors
have the replication origin of R6K, which requires the trans-acting
factor (.PI. protein) encoded by the pir gene to be present for
replication to occur. By selecting the particular host background
in which the vector is located (i.e., by selecting hosts that do or
do not possess the .PI. protein), it is therefore possible to
control replication of the vector. In other conditional replication
vectors, replication is controlled by temperature. Specifically,
under one set of growth conditions, the temperature-sensitive
vector is capable of replication; however, under a second set of
growth conditions, replication is impaired.
[0146] The conditional replication of some vectors has been
exploited to allow for the targeted alteration of the host's
genome. When conditions prevent vector replication, vector
integration is stimulated. Thus, if DNA sequences are present on
the vector (i.e., re-NSI sequences) that share sequence homology
with a region within the chromosome (i.e., chr-NSI sequences), the
vector sequences can be integrated into the host genome via
homologous recombination.
[0147] 2. A Selectable Or Screenable Genetic Marker
[0148] The chromosomal integration vector will contain at least one
genetic selectable or screenable genetic marker that is suitable
for use in the C1 metabolizing bacterial host cell. Genetic
selectable markers of the invention are those that are encoded by a
genetic element and result in a phenotypic change in the
transformant such that a genetic event may be identified and
tracked. Genetic selectable markers may encoded polypeptides that
serve as markers in and of them selves (i.e. lac), or may encode
polypeptides that result in a specific a phenotype under specific
conditions (antibiotic resistance). A person of skill in the art
will be able to quickly determine suitable markers. In a preferred
embodiment, antibiotic markers are used, such as those markers for
kanamycin, tetracycline, ampicillin, chloramphenicol, and
streptinomycin resistance. Additionally, metal resistance markers,
substrate-utilization markers, genes encoding fluorescent and
bioluminescent proteins, lacZ, gfp, cat, galK, inaZ, luc, luxAB,
bgaB, nptll, phoA, uidA and xylE are also considered suitable
selectable or screenable genetic marker(s), although this list is
not intended to be limiting.
[0149] 3. An Origin Of Transfer ("oriT") For A C1 Metabolizing
Bacteria
[0150] The mobilization of vector DNA into a C1 metabolizing
bacteria typically occurs via conjugation. Conjugation is a process
by which DNA is transferred from a donor cell to a recipient cell,
mediated by physical contact between the cells. Once the cells are
brought into close contact, the donor vector is nicked at a
specific site within the origin of transfer (oriT) by a
plasmid-encoded protein (i.e., a DNA relaxase). The vector DNA is
passed from the donor cell into the recipient cell as a single
strand via a process known as rolling circle replication. Once the
single strand of vector has been transferred into the recipient
cell, replication of the single strand occurs to re-generate a
double-stranded molecule.
[0151] The oriT itself is not influenced by the host background;
thus, any oriT that is recognized by the DNA relaxase encoded by
the chromosomal integration vector of the present invention should
be functional in C1 metabolizing bacteria.
[0152] 4. Plasmid Mobilization Genes
[0153] Most plasmids are classified on the basis of their intrinsic
properties, according to their "incompatibility groups" or "Inc"
groups. This classification reflects the similarities in sequence,
function, and the nature of the replicon (as replicons of the same
type are unable to co-exist in a cell, while replicons from
different incompatibility groups may exist simultaneously in a
single cell). Natural plasmid isolates of gram-negative bacteria
that belong to incompatibility groups C, N, P, Q and W display
replication and maintenance proficiency in a diversity of bacterial
species.
[0154] The chromosomal integration vector must encode mobilization
proteins, known as "Mob" proteins such that it is transferable from
donor to recipient bacteria cells via conjugation; however, the
specific Inc group of the vector affects the mob genes that are
required. A single gene or multiple genes may encode the nickase
and helicase functions imparted by the Mob proteins.
[0155] 5. A sacB Coding Region (Encoding Levansucrase) Under The
Control Of A Suitable Promoter
[0156] It has been demonstrated that the Bacillus subtilis and B.
amyloliquefaciens sacB gene (GenBank.RTM. Accession No.s X02730 and
X52988, respectively), encoding the secreted enzyme levansucrase
(EC 2.4.1.10), confers sucrose sensitivity upon various
Gram-negative bacteria. Other likely Bacillus species which may be
used as a source of the sacB gene include, for example: B. pumilis,
B. brevis, B. licheniformis, B. thurgiensis, B. sphearicus, and B.
stearothenmophilus. The B. subtilis and B. amyloliquefaciens sacB
nucleic acid fragments may be used to isolate genes encoding
homologous proteins from the same or other microbial species.
Isolation of homologous genes using sequence-dependent protocols is
well known in the art. Examples of sequence-dependent protocols
include, but are not limited to: 1.) methods of nucleic acid
hybridization; 2.) methods of DNA and RNA amplification, as
exemplified by various uses of nucleic acid amplification
technologies [e.g., polymerase chain reaction (PCR), Mullis et al.,
U.S. Pat. No. 4,683,202; ligase chain reaction (LCR), Tabor, S. et
al., Proc. Acad. Sci. USA, 82:1074 (1985); or strand displacement
amplification (SDA), Walker, et al., Proc. Natl. Acad. Sci. U.S.A.,
89:392 (1992)]; and 3.) methods of library construction and
screening by complementation.
[0157] For example, sacB genes encoding similar proteins or
polypeptides to B. subtilis and B. amyloliquefaciens could be
isolated directly by using all or a portion of the instant sacB
nucleic acid fragments as DNA hybridization probes to screen
libraries from any desired bacteria using methodology well known to
those skilled in the art. Specific oligonucleotide probes based
upon the instant nucleic acid sequences can be designed and
synthesized by methods known in the art (Maniatis, supra).
Moreover, the entire sequences can be used directly to synthesize
DNA probes by methods known to the skilled artisan such as random
primers DNA labeling, nick translation, end-labeling techniques, or
RNA probes using available in vitro transcription systems. In
addition, specific primers can be designed and used to amplify a
part of or full-length of the instant sequences. The resulting
amplification products can be labeled directly during amplification
reactions or labeled after amplification reactions, and used as
probes to isolate full length DNA fragments under conditions of
appropriate stringency.
[0158] Alternatively the instant B. subtilis and B.
amyloliquefaciens sacB sequences may be employed as hybridization
reagents for the identification of homologs. The basic components
of a nucleic acid hybridization test include a probe, a sample
suspected of containing the gene or gene fragment of interest, and
a specific hybridization method. Hybridization methods are well
defined; see, for example, Maniatis (supra).
[0159] The expression of the sacB gene requires a suitable
promoter. Initiation control regions or promoters, which are useful
to drive expression of the sacB gene in the desired host cell are
numerous and familiar to those skilled in the art. Virtually any
promoter capable of driving these genes is suitable for the present
invention. Preferred promoters for the present invention are, but
not limited to: CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH,
ADC1, TRP1, URA3, LEU2, ENO, and TPI (useful for expression in
Saccharomyces); AOX1 (useful for expression in Pichia); and lac,
ara, tet, trp, IP.sub.L, IP.sub.R, T7, tac, and trc (useful for
expression in Escherichia coli), as well as the amy, apr, npr
promoters and various phage promoters (useful for expression in
Bacillus). Additionally, the deoxyxylulose phosphate synthase or
methanol dehydrogenase operon promoter (Springer et al., FEMS
Microbiol Lett, 160:119-124 (1998)), the promoter for
polyhydroxyalkanoic acid synthesis (Foellner et al., Appl.
Microbiol. Biotechnol., 40:284-291 (1993)), promoters identified
from native plasmids in methylotrophs (EP 296484), Plac (Toyama et
al., Microbiology, 143:595-602 (1997); EP 62971), Ptrc (Brosius et
al., Gene, 27:161-172 (1984)), and promoters associated with
antibiotic resistance [e.g., kanamycin (Springer et al., FEMS
Microbiol Lett, 160:119-124 (1998); Ueda et al., Appl. Environ.
Microbiol., 57:924-926 (1991)) or tetracycline (U.S. Pat. No.
4,824,786)] are suitable for expression in C1 metabolizers. Most
preferred in the present invention is the Bacillus npr promoter,
whose native function in Bacillus is to drive expression of the
neutral protease (npr) gene.
[0160] Termination control regions may also be derived from various
genes native to the preferred hosts. Optionally, a termination site
may be unnecessary, however, it is most preferred if included.
[0161] It would be readily apparent to one skilled in the art that
a variety of alternative markers, other than sacB, could be
utilized in the construction chromosomal integration vectors having
positive selection attributes, without significantly altering the
spirit or scope of the present positive selection method for C1
metabolizing bacteria. For a review of other genes and the
construction of positive selection vectors generally see: Maniatis,
supra; Alexeyev, Mikhail F., BioTechniques, 26(5):824-828 (1999);
Matin et al., Anal. Biochem., 278(1):46-51 (2000); and Bramucci et
al., WO 97/16558.
[0162] 6. A Replacement Nucleotide Sequence Of Interest, Having
Homology to a Chromosomal Nucleotide Sequence of Interest
[0163] The particular strategy to be used for selection of
double-crossover mutants (i.e., the one-step or two-step selection
process) affects the construction of the re-NSI that is cloned into
the chromosomal integration vector. As discussed previously, the
one-step selection process requires the presence of a genetic
selectable marker within re-NSI. In contrast, the two-step
selection process is limited only such that re-NSI is modified with
respect to chr-NSI by the addition, substitution, or deletion of at
least one nucleotide.
[0164] One factor to consider regardless of the specific type of
re-NSI generated is the overall homology between the re-NSI and the
chr-NSI. Typically the re-NSI and chr-NSI will have at least two
regions of homology facilitating homologous recombination. These
regions will be at least about 10 bp in length where at least about
50 bp in length is preferred and wherein lengths of up to about
2000 bp are suitable.
[0165] When preparing a mutant re-NSI for use in the selection
processes described herein, it is preferable to have regions
homologous to the chr-NSI flanking (both 5' and 3') the site of
mutation. More preferably, a 1 kB region of homology is preferred
on both sides of the addition, substitution, or deletion. In
contrast, re-NSI is not expected to be limited in length, beyond
the limitations inherent to homologous recombination. Thus, it is
contemplated that the present invention is suitable for
recombination of re-NSI fragments that range in size from about
0.10-20 kB; more preferred from about 0.5-10 kB; and most
preferably from about 1-5 kB.
[0166] Re-NSI may, for example, represent a portion of a gene, the
complete coding sequence of a gene, a series of genes or an operon,
a promoter, a non-coding sequence of DNA, or any combination
thereof. The nucleic acids may be derived from any source including
microbial sources (e.g., bacterial, yeast, fungi or algae), as well
as from plant, animal or human sources and may have medical,
veterinary, environmental, nutritional or industrial significance.
Additionally, re-NSI's or mutant re-NSI's can be produced
synthetically.
[0167] Replacement Nucleotide Sequences Of Interest for One-Step
Selection
[0168] Insertion of a genetic selectable marker within the re-NSI
for use in the one-step selection process can be readily generated
using methods well-known to one of skill in the art (e.g., ligation
of selectable markers into specific restriction sites, use of
overlap PCR methods to insert selectable markers into the re-NSI,
or in vitro transposition to randomly insert into the re-NSI).
[0169] In a preferred embodiment, the present method employs a
method of in vitro transposition to disrupt the re-NSI (this
approach is referred as "targeted insertion"). In vitro
transposition involves the use of a transposable element in
combination with a transposase enzyme. For a discussion of
transposase use and function see Haren et al. (Annu. Rev.
Microbiol., 53:245-281 (1999)). When the transposable element or
transposon is contacted with a nucleic acid fragment in the
presence of the transposase, the transposable element will randomly
insert into the nucleic acid fragment. The technique is useful for
rapid disruption of a gene, when the specific site of disruption
within the gene is not relevant; nonetheless, it is possible to
quickly determine the site of disruption on the basis of the
sequence of the transposable element.
[0170] Kits for in vitro transposition are commercially available.
For example, see: 1.) The Primer Island Transposition Kit
(available from Perkin Elmer Applied Biosystems, Branchburg, N.J.),
based upon the yeast Ty1 element (including the AT2 transposon);
2.) The Genome Priming System (available from New England Biolabs,
Beverly, Mass.), based upon the bacterial transposon Tn7; and 3.)
the EZ::TN Transposon Insertion Systems (available from Epicentre
Technologies, Madison, Wis.), based upon the Tn5 bacterial
transposable element).
[0171] Although not available commercially, several other in vitro
transposition reactions have been reported in the literature. A DNA
sequencing tool was developed based on the bacteriophage Mu in
vitro transposition reaction (Haapa et. al., Genome Research,
9:308-315 (1999)). Similarly, a tool for DNA sequencing and
mutagenesis was developed based on the bacterial transposon Tn552
in vitro transposition system (Griffin et. al., Nucleic Acid
Research, 27(19):3859-3865 (1999)). The mariner transposable
element Himar1 also has an in vitro transposition reaction used for
mutagenesis and DNA sequencing (Lampe et. al., Proc. Natl. Acad.
Sci., 96:11428-11433 (1999)).
[0172] Thus, transposons suitable in the present invention include,
but are not limited to those based upon: the yeast Ty1 element, the
bacterial transposon Tn7, EZ::TN, bacteriophage Mu, bacterial
transposon Tn552, and the mariner transposable element Himar1. A
number of transposons and methods of identifying and isolating
transposons are reviewed by Dyson, Paul J. (Methods Microbiol. 29
(Genetic Methods for Diverse Prokaryotes):133-167 (1999)). Although
these specific transposon systems have been developed for use in in
vitro systems, it is contemplated that many of the transposon
systems, currently only available for in vivo transposition, may be
modified and developed for in vitro work. With appropriate
development and characterization, these in vivo transposon systems
will also be suitable within the context of the present
invention.
[0173] Although any commercially available in vitro transposition
system can be used as a mutagenizing tool, the Tn7-based in vitro
transposition system (New England Biolabs, Beverly, Mass.) is
preferred. In addition to being able to customize the Tn7-based
element with an appropriate selectable or screenable genetic marker
for the microorganism under study, the insertion of the Tn7-based
transposon into any DNA target molecule renders that molecule
"immune" to further insertions by the Tn7-based element (Stellwagen
and Craig, The EMBO Journal, 16(22):6823-6834 (1997)). The genetic
marker is used to identify transformants having the transposon
disrupted re-NSI. The genetic marker may be associated with the
transposon in any fashion; however, it is particularly suitable if
the genetic marker is located between the ends of the transposon.
Most common are antibiotic resistance markers encoding, e.g.,
ampicillin-resistance, kanamycin-resistance,
tetracycline-resistance, etc. Also suitable are genetic markers
encoding metal resistance, substrate-utilization, and genes
encoding fluorescent and bioluminescent proteins (e.g., green
fluorescent proteins, Lux genes), as well as lacZ, gfp, cat, galK,
inaZ, luc, luxAB, bgaB, nptll, phoA, uidA, and xylE.
[0174] Markers may be used singly or in association with other
agents to identify allelic exchange. For example, tetracycline can
also be used in combination with detergents for the detection of
cells that have lost the non-homologous region of the chromosomal
integration vector (Maloy and Nunn, J. Bact., 145(2):1110-1112
(1981)).
[0175] Targeted insertion may be performed directly upon an
isolated re-NSI. The library of DNA fragments containing insertions
may then be cloned into the chromosomal insertion vector. Or, the
re-NSI may be first cloned into the chromosomal integration vector
and subjected to the in vitro transposition reaction. In either
case, the chromosomal integration vector is then used in homologous
recombination to remove the chr-NSI from the bacterial host and
insert the re-NSI.
[0176] Replacement Nucleotide Sequences Of Interest for Two-Step
Selection
[0177] Generation of a mutant re-NSI (wherein the mutation does not
constitute a genetic selectable marker, as discussed above for the
one-step selection method) containing a mutation (e.g. addition,
substitution, or deletion of at least one nucleotide with respect
to the chr-NSI) can be accomplished using numerous techniques known
to a skilled artisan in the field of molecular biology. In all
cases, the advantage of the two-step selection methodology as
compared to that for the one-step selection method is that the
double-crossover mutant thus generated is "markerless". This
enables subsequent mutations to be created using the same technique
(i.e., since there is no need for a different selectable marker
corresponding to each mutation created).
[0178] Although not intended to be limiting, deletions and
additions may be generated by the use of restriction endonucleases,
in vitro transposition reactions, or PCR methodologies, as is well
known to one of skill in the art.
[0179] Substitutions may be generated by mutagenesis of the re-NSI.
Two suitable approaches include error-prone PCR (Leung et al.,
Techniques, 1:11-15 (1989); Zhou et al., Nucleic Acids Res.,
19:6052-6052 (1991); and Spee et al., Nucleic Acids Res.,
21:777-778 (1993)) and in vivo mutagenesis. The principal advantage
of error-prone PCR is that all mutations introduced by this method
will be within the re-NSI, and any change may be easily controlled
by changing the PCR conditions. Alternatively, in vivo mutagenesis
may be employed using commercially available materials such as E.
coli XL1-Red strain, and the Epicurian coli XL1-Red mutator strain
from Stratagene (La Jolla, Calif.; Greener and Callahan,
Strategies, 7:32-34 (1994)). This strain is deficient in three of
the primary DNA repair pathways (mutS, mutD, and mutT), resulting
in a mutation rate 5000-fold higher than that of wild-type. In vivo
mutagenesis does not depend on ligation efficiency (as with
error-prone PCR); however, a mutation may occur at any region of
the vector and the mutation rates are generally much lower.
[0180] It is also contemplated that it may be desirable to replace
a wild type gene of interest (i.e., chr-NSI) in the C1 metabolizing
bacteria with a mutant enzyme (i.e., re-NSI) that has been
constructed using the method of "gene shuffling" (U.S. Pat. No.
5,605,793; U.S. Pat. No. 5,811,238; U.S. Pat. No. 5,830,721; and
U.S. Pat. No. 5,837,458). The method of gene shuffling is
particularly attractive due to its facile implementation, and high
rate of mutagenesis. The process of gene shuffling involves the
restriction of a gene of interest into fragments of specific size
in the presence of additional populations of DNA regions of both
similarity to, or difference to, the gene of interest. This pool of
fragments is then denatured and reannealed to create a mutated
gene. The mutated gene is subsequently screened for altered
activity.
[0181] Any wild type C1 genomic sequence may be mutated and
screened for altered or enhanced activity by this method. The
sequences should be double stranded and can be of various lengths
ranging from about 50 bp to about 10 kB. The sequences may be
randomly digested into fragments ranging from about 10 bp to about
1000 bp, using restriction endonucleases well known in the art
(Maniatis, supra). In addition to the full-length sequences,
populations of fragments that are hybridizable to all or portions
of the sequence may be added. Similarly, a population of fragments
that are not hybridizable to the wild type sequence may also be
added. Typically these additional fragment populations are added in
about a 10- to 20-fold excess by weight as compared to the total
nucleic acid. Generally this process will allow generation of about
100 to 1000 different specific nucleic acid fragments in the
mixture. The mixed population of random nucleic acid fragments are
denatured to form single-stranded nucleic acid fragments and then
reannealed. Only those single-stranded nucleic acid fragments
having regions of homology with other single-stranded nucleic acid
fragments will reanneal. The random nucleic acid fragments may be
denatured by heating. One skilled in the art could determine the
conditions necessary to completely denature the double-stranded
nucleic acid. Preferably, the temperature is from about 80.degree.
C. to about 100.degree. C. The nucleic acid fragments may be
reannealed by cooling. Preferably the temperature is from about
20.degree. C. to about 75.degree. C. Renaturation can be
accelerated by the addition of polyethylene glycol ("PEG") or salt.
The salt concentration is preferably from about 0 mM to about 200
mM. The annealed nucleic acid fragments are next incubated in the
presence of a nucleic acid polymerase and dNTP's (i.e., dATP, dCTP,
dGTP and dTTP). The nucleic acid polymerase may be the Klenow
fragment, the Taq polymerase or any other DNA polymerase known in
the art. The polymerase may be added to the random nucleic acid
fragments prior to annealing, simultaneously with annealing or
after annealing. The cycle of denaturation, renaturation and
incubation in the presence of polymerase is repeated for a desired
number of times. Preferably the cycle is repeated from about 2 to
about 50 times, more preferably the sequence is repeated from about
10 to about 40 times. The resulting nucleic acid is a larger
double-stranded polynucleotide of from about 50 bp to about 100 kB
and may be screened for expression and altered activity by standard
cloning and expression protocols (Maniatis, supra).
[0182] One factor to consider during the preparation of a re-NSI
for use in the two-step selection strategy concerns the placement
of the mutation within the re-NSI. Specifically, the re-NSI is
first inserted into the chromosome by integration of the
chromosomal integration vector (a single-crossover event). The
second crossover event that occurs can result in either a mutant or
wildtype sequence in the chromosome, since the single-crossover
contains two copies of the nucleotide sequence of interest. In
order to increase the percentage of segregants that retain the
re-NSI, as opposed to reverting to the wildtype encoded by the
chr-NSI, it is desirable to "center" the mutation with respect to
the flanking DNA that has homology to the chr-NSI. For example, if
a point mutation was perfectly centered within a re-NSI, about 50%
of the segregants would be expected to retain the mutation in the
chromosome (thus producing a 1:1 ratio of double-crossover mutants
to wild-type cells). However, it is contemplated that the present
invention is suitable for homologous recombination wherein the
mutation (i.e., insertion, deletion, or substitution) is positioned
within about 5 to about 95% of the total length of re-NSI; more
preferred when positioned within about 25 to about 75% of the total
length of re-NSI; and most preferred when positioned within about
45 to about 55% of the total length of re-NSI.
Transformation of C1 Metabolizing Bacteria
[0183] Techniques for the transformation of C1 metabolizing
bacteria are not well developed, although general methodology
mimics that utilized for other bacteria and well known to those of
skill in the art.
[0184] Electroporation has been used successfully for the
transformation of: Methylobacterium extorquens AM1 (Toyama, H., et
al., FEMS Microbiol Lett. 166:1-7 (1998)), Methylophilus
methylotrophus AS1 (Kim, C. S., and T. K. Wood. Appl Microbiol
Biotechnol 48: 105-108 (1997)), and Methylobacillus sp. strain 12S
(Yoshida, T., et al., Biotechnol. Lett., 23: 787-791 (2001)).
Extrapolation of specific electroporation parameters from one
specific C1 metabolizing utilizing organism to another may be
difficult, however, as is well to known to one of skill in the
art.
[0185] Bacterial conjugation, relying on the direct contact of
donor and recipient cells, is frequently more readily amenable for
the transfer of genes into C1 metabolizing bacteria.
Simplistically, this bacterial conjugation process involves mixing
together "donor" and "recipient" cells in close contact with one
another. Conjugation occurs by formation of cytoplasmic connections
between donor and recipient bacteria, with direct transfer of newly
synthesized donor DNA into the recipient cells. As is well known in
the art, the recipient in a conjugation is defined as any cell that
can accept DNA through horizontal transfer from a donor bacterium;
the donor in conjugative transfer is a bacterium that contains a
conjugative plasmid, conjugative transposon, or mobilizable
plasmid. The physical transfer of the donor plasmid can occur in
one of two fashions, as described below: [0186] 1. In some cases,
only a donor and recipient are required for conjugation. This
occurs when the plasmid to be transferred is a self-transmissible
plasmid that is both conjugative and mobilizable (i.e., carrying
both tra genes and genes encoding the Mob proteins). In general,
the process involves the following steps: 1.) Double-strand plasmid
DNA is nicked at a specific site in onT; 2.) A single-strand DNA is
released to the recipient through a pore or pilus structure; 3.) A
DNA relaxase enzyme cleaves the double-strand DNA at onT and binds
to a release 5' end (forming a relaxosome as the intermediate
structure); and 4.) Subsequently, a complex of auxiliary proteins
assemble at onT to facilitate the process of DNA transfer. [0187]
2. Alternatively, a "triparental" conjugation is required for
transfer of the donor plasmid to the recipient. In this type of
conjugation, donor cells, recipient cells, and a "helper" plasmid
participate. The donor cells carry a mobilizable plasmid or
conjugative transposon. Mobilizable vectors contain an onT, a gene
encoding a nickase, and have genes encoding the Mob proteins;
however, the Mob proteins alone are not sufficient to achieve the
transfer of the genome. Thus, mobilizable plasmids are not able to
promote their own transfer unless an appropriate conjugation system
is provided by a helper plasmid (located within the donor or within
a "helper" cell). The conjugative plasmid is needed for the
formation of the mating pair and DNA transfer, since the plasmid
encodes proteins for transfer (Tra) that are involved in the
formation of the pore or pilus.
[0188] Examples of successful conjugations involving C1
metabolizing bacteria include the work of: [0189] Stolyar et al.
(Mikrobiologiya, 64(5): 686-691 (1995)), wherein Inc P-1
incompatibility group plasmids were transferred by conjugation from
Eschenchia coli and Pseudomonas putida into Methylomonas rubra 15sh
and Methylococcus thermophilus 111p; [0190] Motoyama, H. et al.
(Appl. Micro. Biotech., 42(1): 67-72 (1994)), wherein genes coding
for the L-threonine biosynthetic enzymes were transferred by
conjugation into the Gram-negative obligate methylotroph,
Methylobacillus glycogenes; [0191] Lloyd, J. S. et al. (Archives of
Microbiology, 171(6): 364-370 (1999)), wherein broad-host range
plasmids containing heterologous soluble methane monooxygenase
(sMMO) genes were transferred into Methylocystis parvus OBBP and
Methylomicrobium album BG8; and [0192] Odom, J. M. et al. (WO
02/18617), wherein the broad-host range plasmid pBHR1 containing
carotenoid genes of Pantoea stewartii were transferred into
Methylomonas sp. 16a. Screening Methods-SacB
[0193] Methods of screening in microbiology are discussed at length
in Brock, supra. In one preferred embodiment, a one-step selection
process permits the identification of double-crossover mutations in
C1 metabolizing bacterial cells by applying positive selection
pressure. In this embodiment, the positive selection vector
comprises a first genetic selectable marker, a sacB marker, and a
second genetic selectable marker that disrupts the sequence of the
re-NSI. For selection, cells that have undergone a double-crossover
event are selected by plating onto medium that permits selection
according to the sacB marker on the chromosomal integration vector
and the second genetic selectable marker disrupting the re-NSI.
This selection strategy efficiently identifies only
double-crossover events, as it relies on two selectable and
screenable markers. First, the sacB marker is able to select
against all single-crossover mutants, since SacB expression (which
occurs when the entire chromosomal integration vector, comprising
the vector plus the re-NSI, merely integrates into the host's
genome) enables conversion of sucrose into levan, which is toxic to
most Gram-negative bacteria. Those bacterial cells possessing a
double-crossover, in contrast, do not possess the sacB gene within
their chromosome; the lethal marker is present on the
non-replicating chromosomal plasmid integration vector. Secondly,
the second genetic selectable marker disrupting the re-NSI ensures
allelic exchange has occurred between the chr-NSI and the re-NSI
introduced into the cell on the chromosomal integration vector. To
confirm the double-crossover event, the cells may be transferred to
media containing the positive selection vector's first genetic
selectable marker (and optionally containing the second genetic
selectable marker); those cells unable to grow have undergone
allelic exchange.
[0194] In an alternative preferred embodiment, a two-step selection
process permits the identification of double-crossover mutations in
C1 metabolizing bacterial cells by applying positive selection
pressure. Using this strategy, the positive selection vector should
comprise a first genetic selectable marker and a sacB marker.
Selection involves first growing the transformants on media
containing the first genetic selectable marker, to identify those
cells that have undergone a single-crossover (i.e., wherein the
entire chromosomal integration vector has integrated into the host
cell's genome). Then, the selection pressure is removed and a
second crossover event may occur. Selection for allelic exchange
mutants requires growth of the cells on sucrose, since SacB
expression will be lethal to all single-crossover mutants.
Differentiation between allelic exchange mutants containing the
wildtype and mutant allele is then possible using standard
molecular techniques (e.g., PCR), well known to one of skill in the
art.
C1 Metabolizing Bacterial Hosts
[0195] In one embodiment, heterologous host cells for use with the
present positive selection methodologies are C1 metabolizing
bacteria that are able to use single carbon substrates (i.e., those
substrates lacking carbon to carbon bonds) as a sole energy source.
Many C1 metabolizing microorganisms are known in the art which are
able to use a variety of single carbon substrates. Single carbon
substrates useful in the present invention include, but are not
limited to: methane, methanol, formate, formaldehyde, formic acid,
methylated amines (e.g., mono-, di- and tri-methyl amine),
methylated thiols, carbon dioxide, and various other reduced carbon
compounds which lack any carbon-carbon bonds. In another
embodiment, the single carbon substrates are methane and/or
methanol.
[0196] All C1 metabolizing microorganisms are generally classified
as methylotrophs. Methylotrophs may be defined as any organisms
capable of oxidizing organic compounds that do not contain
carbon-carbon bonds. However, facultative methylotrophs, obligate
methylotrophs, and obligate methanotrophs are all various subsets
of methylotrophs. Specifically: [0197] Facultative methylotrophs
have the ability to oxidize organic compounds which do not contain
carbon-carbon bonds, but may also use other carbon substrates such
as sugars and complex carbohydrates for energy and biomass.
Facultative methylotrophic bacteria are found in many environments,
but are isolated most commonly from soil, landfill and waste
treatment sites. Many facultative methylotrophs are members of the
.beta. and .gamma. subgroups of the Proteobacteria (Hanson et al.,
Microb. Growth C1 Compounds., [Int. Symp.], 7.sup.th (1993), pp
285-302. Murrell, J. Collin and Don P. Kelly, Eds. Intercept:
Andover, UK; Madigan et al., Brock Biology of Microorganisms,
8.sup.th ed., Prentice Hall: UpperSaddle River, N.J. (1997)).
[0198] Obligate methylotrophs are those organisms that are limited
to the use of organic compounds that do not contain carbon-carbon
bonds for the generation of energy. [0199] Obligate methanotrophs
are those obligate methylotrophs that have the distinct ability to
oxidize methane.
[0200] For the purposes of the present invention herein, suitable
facultative methylotrophic bacteria include, but are not limited
to: Methylophilus, Methylobacillus, Methylobacterium,
Hyphomicrobium, Xanthobacter, Bacillus, Paracoccus, Nocardia,
Arthrobacter, Rhodopseudomonas, and Pseudomonas. Exemplary
methanotrophs are included in, but not limited to, the genera
Methylomonas, Methylobacter, Methylococcus, Methylosinus,
Methylocyctis, Methylomicrobium, and Methanomonas, although this is
not intended to be limiting.
[0201] Of particular interest in the present invention are high
growth obligate methanotrophs having an energetically favorable
carbon flux pathway. For example, Applicants have discovered a
specific strain of methanotroph having several pathway features
that make it particularly useful for carbon flux manipulation (U.S.
Pat. No. 6,689,601; hereby incorporated by reference). This
particular strain possessing an energetically favorable carbon flux
pathway was used as a "model" organism in the present application
and is known as Methylomonas 16a (ATCC PTA 2402).
Applications for Positive Selection Methods
[0202] Knowledge of the sequence of the present chromosomal
integration vector and methodologies for one-step and two-step
selection of double-crossover C1 metabolizing mutant bacteria as
disclosed herein will be useful in manipulating various genetic
pathways in any C1 metabolizing organism, particularly in
methanotrophs. As is well known to those of skill in the art,
efforts to genetically engineer a microorganism for high-level
production of a specific product frequently require substantial
manipulation to the native host machinery and biosynthetic
pathways.
[0203] In the past, metabolic engineering of methanotrophs has
remained relatively undeveloped due to the general lack of
efficient genetic engineering tools, as compared to other
industrial bacteria such as E. coli and the yeasts. Most
methanotrophic molecular biology has focused on the engineering of
the methane monooxygenase, such that it is directed toward more
useful co-metabolic products (and wherein methane is not directly
incorporated into the product molecule). In contrast, pathway
engineering for net synthesis of carbon-containing compounds from
methane requires both: 1.) the ability to inactivate genes already
present in the methanotroph; and 2.) the ability to introduce
foreign genes into the organism, to create new metabolic
capabilities. In this way, new metabolic networks can be
constructed to produce products that could not be made by the
genetic engineering of methane monooxygenase alone.
[0204] The present invention provides a method to make mutations in
specific genes and/or competing pathways when the nucleotide
sequence of the gene to be altered is known. This permits reduction
or elimination of the expression of certain genes in the target
pathway or in competing pathways that may serve as competing sinks
for energy or carbon. The gene alterations can be "tagged" with a
genetic selectable marker; or alternatively, the mutations can be
markerless, thus allowing subsequent mutations to be introduced
into the chromosome using the same methodology and genetic markers
for selection.
[0205] In an alternative embodiment (or in addition to the
selective reduction or elimination of specific genes), it may be
desirable to introduce multiple copies of a gene into a specific
site of the C1 metabolizing host genome, to promote that gene's
enhanced biological activity. In this instance, the gene could be
foreign or endogenous to the cell. This methodology would be
especially useful for industrial processes, where it is frequently
undesirable to require constant selection pressure for maintenance
of a specific expression plasmid. Additionally, the ability to
target the insertion event to specific genes within the host
chromosome is advantageous since the controlled insertion event
will not unknowingly disrupt other essential genes and pathways of
the host organism.
[0206] Once a key genetic pathway has been identified and
sequenced, it may also be necessary to optimize specific genes to
increase the output of the pathway. For example, a gene encoding a
rate-limiting enzyme within the host organism may be subjected to a
"gene shuffling" reaction or error-prone PCR reaction. Upon
identification of a mutant gene having enhanced biological activity
for the rate-limiting reaction, it may be desirable to insert this
mutated gene containing point mutations into the chromosome of the
C1 metabolizing host in place of the wildtype gene.
[0207] As such, the positive selection methods described herein are
expected to be useful to create allelic exchange mutations in a
variety of metabolic pathways within methanotrophic hosts. Useful
metabolic pathways include, but are not limited to: glycolytic
pathways for the production of C.sub.3 and C.sub.2 molecules,
pentose phosphate pathways for the production of C.sub.3, C.sub.4,
C.sub.5, C.sub.6 and C.sub.7 sugars, aromatic amino acid
biosynthesis pathways for the production of a variety of aromatics,
pathways leading to the intra- and extra-cellular production of
polysacharrides, and isoprenoid biosynthesis pathways.
[0208] Finally, one skilled in the art will recognize that the use
of a two-step selection strategy allows the construction of defined
unmarked mutants with changes that are usually undetectable, such
as point mutations or in-frame deletions. These "markerless"
mutants may offer significant advantages during industrial
development of a C1 metabolizing host bacterium, in terms of
required regulatory approvals. For example, isoprenoids are
actively made by many methanotrophs and metabolic engineering of
this pathway offers the ability to produce a wide variety of
carotenoid compounds. Using the positive selection methods
described herein, it is possible to create allelic exchange
mutations in genetically engineered hosts expressing carotenoid
compounds that will be useful in various food and feed
applications, in which the presence of antibiotic resistance genes
would be undesirable.
DESCRIPTION OF PREFERRED EMBODIMENTS
[0209] Methylomonas sp. 16a is a C1 metabolizing bacteria that is
capable of efficiently using either methanol and/or methane as a
carbon substrate. Methylomonas is also metabolically versatile in
that it contains multiple pathways for the incorporation of carbon
from formaldehyde into 3-carbon units, and is capable of genetic
exchange with donor species such as Escherichia coli via bacterial
conjugation. Despite these advantageous qualities, the utility of
this organism as a microbial platform of choice for the production
of a variety of materials was hampered prior to the development of
a method for the generation and identification of defined mutations
within the organism by homologous recombination. Previous attempts
to create mutations via a double-crossover event were unsuccessful,
largely due to the organism's slow growth rate, difficulties
associated with identification of a suitable chromosomal
integration vector, and the rarity of double-crossover events.
Furthermore, no prior studies had confirmed the ability of
Methylomonas sp. 16a to undergo homologous recombination.
[0210] The present study was initiated to determine if the sacB
gene could be employed as a positive selectable marker in C1
metabolizing bacterium, such as Methylomonas. Thus, the effect of
sucrose on the growth of Methylomonas sp. 16a was first examined by
growing the wildtype bacteria on media containing 1%, 2.5%, 5%, and
10% sucrose. Since sucrose did not appear to interfere with normal
growth, work aimed at the development of a positive selection
vector based on sacB was pursued.
[0211] Although a variety of metabolic pathways within Methylomonas
sp. 16a could have been chosen as a model pathway to investigate
the utility of the positive selection methods described herein, it
was desirable to focus work on the native C.sub.30 carotenoid
biosynthetic pathway of Methylomonas sp. 16a since it was
anticipated that creation of particular allelic exchange mutations
within this pathway would result in a non-pigmented bacterial
strain. The transformation from wildtype (pink-pigmented) host to a
transformed non-pigmented host would provide visual confirmation of
allelic exchange.
[0212] A carotenoid biosynthetic operon has been identified within
the genomic sequence of Methylomonas sp. 16a, which encodes three
genes (FIG. 3): [0213] The first gene (designated crtN1; SEQ ID
NO:1) encodes a putative diapophytoene dehydrogenase with the
highest BLAST hit to a diapophytoene dehydrogenase from
Heliobacillus mobilis (34% identity and 58% similarity). [0214] The
middle gene (designated ald; SEQ ID NO:2) encodes a putative
aldehyde dehydrogenase with the highest BLAST hit to a betaine
aldehyde dehydrogenase from Arabidopsis thaliana (33% identity and
50% similarity). [0215] The third gene (designated crtN2; SEQ ID
NO:3) also encodes a putative diapophytoene dehydrogenase with the
highest BLAST hit to a hypothetical protein of phytoene
dehydrogenase family from Staphylococcus aureus (51 % identity and
67% similarity). The interaction between the proteins encoded by
these three genes in Methylomonas sp. 16a is not well understood at
this time.
[0216] The ability of the one-step selection process to identify
allelic exchange mutants was first investigated herein, by creating
a re-NSI wherein the native crtN2 gene of Methylomonas was
disrupted via in vitro transposition using the EZ::TN.TM.
<Kan-2> transposon Insertion Kit (Epicentre Technologies;
Madison, Wis.). Following triparental conjugation to mobilize the
chromosomal integration vector pGP704::sacB::crtN2::EZ::TN.TM.
<Kan-2> into Methylomonas, it was found that crtN2 mutants
resulting from allelic-exchange events were readily identified by
the one-step selection process.
[0217] Subsequently, allelic-exchange mutants wherein the native
Methylomonas ald(crtN1) gene was deleted from the genome were also
identified using the two-step selection process. The
allelic-exchange mutants generated contained a major deletion
within the ald gene (all but .about.80 nucleotides were removed)
and a small deletion within the carboxyl terminus of the crtN1 gene
(.about.30 nucleotides remained). Specifically, two DNA fragments
flanking ald(crtN1) were prepared by PCR and ligated together
within the chromosomal integration vector pGP704::sacB to create a
markerless re-NSI. Upon mobilization of the
pGP704::sacB::.DELTA.a/d(crtN1) construct into Methylomonas,
application of the two-step selection strategy permitted isolation
of transformant clones lacking the ald gene and lacking any
residual selective markers.
[0218] The results described herein and in the following Examples
clearly illustrate the utility of sacB, as a means to facilitate
experiments designed to identify allelic-exchange events in C1
metabolizing bacteria using positive selection.
EXAMPLES
[0219] The present invention is further defined in the following
Examples. It should be understood that these Examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only. From the above discussion and these Examples,
one skilled in the art can ascertain the essential characteristics
of this invention, and without departing from the spirit and scope
thereof, can make various changes and modifications of the
invention to adapt it to various usages and conditions.
General Methods
[0220] Standard recombinant DNA and molecular cloning techniques
used in the Examples are well known in the art and are described by
Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A
Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring
Harbor, N.Y. (1989) (Maniatis); by T. J. Silhavy, M. L. Bennan, and
L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor
Laboratory: Cold Spring Harbor, N.Y. (1984); and by Ausubel, F. M.
et al., Current Protocols in Molecular Biology, published by Greene
Publishing Assoc. and Wiley-Interscience (1987).
[0221] The meaning of abbreviations is as follows: "sec" means
second(s), "min" means minute(s), "hr" means hour(s), "d" means
day(s), ".mu.L" means microliter(s), "mL" means milliliter(s), "L"
means liter(s), ".mu.M" means micromolar, "mM" means millimolar,
"M" means molar, "mmol" means millimole(s), ".mu.mole" mean
micromole(s), "g" means gram(s), ".mu.g" means microgram(s), "ng"
means nanogram(s), "U" means unit(s), "bp" means base pair(s), and
"kB" means kilobase(s).
Molecular Biology Techniques:
[0222] Methods for agarose gel electrophoresis were performed as
described in Maniatis (supra). Polymerase Chain Reactions (PCR)
techniques were found in White, B., PCR Protocols: Current Methods
and Applications, Humana: Totowa, N.J. (1993), Vol. 15.
Media and Culture Conditions:
[0223] General materials and methods suitable for the maintenance
and growth of bacterial cultures are found in: Experiments in
Molecular Genetics (Jeffrey H. Miller), Cold Spring Harbor
Laboratory: Cold Spring Harbor, N.Y. (1972); Manual of Methods for
General Bacteriology (Phillip Gerhardt, R. G. E. Murray, Ralph N.
Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G.
Briggs Phillips, eds), American Society for Microbiology:
Washington, D.C., pp 210-213; or, Thomas D. Brock in Biotechnology:
A Textbook of Industrial Microbiology, 2.sup.nd ed. Sinauer
Associates: Sunderland, Mass. (1989). All reagents and materials
used for the growth and maintenance of bacterial cells were
obtained from Aldrich Chemicals (Milwaukee, Wis.), DIFCO
Laboratories (Detroit, Mich.), Gibco/BRL Life Technologies
(Gaithersburg, Md.), or Sigma Chemical Company (St. Louis, Mo.),
unless otherwise specified.
Example 1
Growth of Methylomonas 16a
[0224] Example 1 summarizes the standard conditions used for growth
of Methylomonas sp. 16a (ATCC# PTA-2402), as described in U.S. Pat.
No. 6,689,601.
Methylomonas Strain and Culture Media
[0225] The growth conditions described below were used throughout
the experimental Examples for treatment of Methylomonas 16a, unless
conditions were specifically described otherwise.
[0226] Methylomonas 16a is typically grown in serum stoppered
Wheaton bottles (Wheaton Scientific, Wheaton, Ill.) using a
gas/liquid ratio of at least 8:1 (i.e., 20 mL of Nitrate liquid
"BTZ" media in 160 mL total volume). The standard gas phase for
cultivation contained 25% methane in air, although methane
concentrations can vary ranging from about 5-50% by volume of the
culture headspace. These conditions comprise growth conditions and
the cells are referred to as growing cells. In all cases, the
cultures were grown at 30.degree. C. with constant shaking in a
Lab-Line rotary shaker unless otherwise specified.
[0227] Ammonium and Nitrate Medium for Methylomonas 16a
[0228] Methylomonas 16a typically grows in a defined medium
comprised of only minimal salts; no organic additions such as yeast
extract or vitamins are required to achieve grow. This defined
medium known as BTZ medium (also referred to herein as "ammonium
liquid medium") was comprised of various salts mixed with Solution
1 as indicated below (Tables 2 and 3). Alternatively, the ammonium
was replaced with 10 mM sodium nitrate, where specified. Solution 1
provides the composition for a 100 fold concentrated stock solution
of trace minerals. TABLE-US-00003 TABLE 2 Solution 1* Conc. MW (mM)
g per L Nitriloacetic acid 191.10 66.90 12.80 CuCl.sub.2 .times.
2H.sub.2O 170.48 0.15 0.0254 FeCl.sub.2 .times. 4H.sub.2O 198.81
1.50 0.30 MnCl.sub.2 .times. 4H.sub.2O 197.91 0.50 0.10 CoCl.sub.2
.times. 6H.sub.2O 237.90 1.31 0.312 ZnCl.sub.2 136.29 0.73 0.10
H.sub.3BO.sub.3 61.83 0.16 0.01 Na.sub.2MoO.sub.4 .times. 2H.sub.2O
241.95 0.04 0.01 NiCl.sub.2 .times. 6H.sub.2O 237.70 0.77 0.184
*Mix the gram amounts designated above in 900 mL of H.sub.2O,
adjust to pH=7, and add H.sub.2O to an end volume of 1 L. Keep
refrigerated.
[0229] TABLE-US-00004 TABLE 3 Ammonium liquid medium (BTZ)** Conc.
MW (mM) g per L NH.sub.4Cl 84.99 10 0.85 KH.sub.2PO.sub.4 136.09
3.67 0.5 Na.sub.2SO.sub.4 142.04 3.52 0.5 MgCl.sub.2 .times.
6H.sub.2O 203.3 0.98 0.2 CaCl.sub.2 .times. 2H.sub.2O 147.02 0.68
0.1 1 M HEPES (pH 7) 238.3 50 mL Solution 1 10 mL **Dissolve in 900
mL H.sub.2O. Adjust to pH=7, and add H.sub.2O to give 1 L. For agar
plates: Add 15 g of agarose in 1 L of medium, autoclave, cool
liquid solution to 50.degree. C., mix, and pour plates.
Example 2
Construction of a Positive-Selective Suicide Vector for
Methylomonas sp. 16a
[0230] The construction of chromosomal mutations within the
Methylomonas genome required the use of suicide vectors. Thus, a
modified version of the conditional replication vector pGP704 was
created, comprising a npr-sacB cassette.
pGP704 as a Vector Backbone for the C1 Chromosomal Integration
Vector
[0231] The plasmid pGP704 (Miller and Mekalanos, J. Bacteriol.,
(170): 2575-2583 (1988); FIG. 4) was chosen as a suitable vector
backbone for the C1 chromosomal integration vector, since it could
be used as a vehicle to transfer replacement nucleotide sequences
of interest into Methylomonas sp. 16a via conjugation. Plasmid
pGP704 is a derivative of pBR322 that is AmP.sup.R but has a
deletion of the pBR322 origin of replication (oriE1). Instead, the
plasmid contains a cloned fragment containing the origin of
replication of plasmid R6K. The R6K origin of replication (oriR6K)
requires the .PI. protein, encoded by the pir gene. In E. coli, the
.PI. protein can be supplied in trans by a prophage (.gamma. pir)
that carries a cloned copy of the pir gene. The pGP704 plasmid also
contains a 1.9 kB BamHl fragment encoding the mob region of RP4.
Thus, pGP704 can be mobilized into recipient strains by transfer
functions provided by a derivative of RP4 integrated in the
chromosome of E. coli strain SM10 or SY327. Once the plasmid is
transferred, however, it is unable to replicate in recipients that
lack the .PI. protein (e.g., recipients such as Methylomonas and
other C1 metabolizing bacteria). This inability permits homologous
recombination to occur between nucleotide sequences of interest on
pGP704 and the intact chromosomal nucleotide sequences of
interest.
[0232] Thus, on the basis of the above characteristics, the pGP704
vector backbone met the following conditions for a chromosomal
integration vector suitable C1 metabolizing bacteria: 1.) it was
conditional for replication, thus allowing selection for
integration into the chromosome; 2.) it possessed at least one
selectable marker; 3.) it had an origin of transfer that was
expected to be suitable for C1 metabolizing bacteria; 4.) it
possessed mobilization genes; and 5.) it contained a variety of
unique cloning sites. Other alternative chromosomal integration
vectors having the characteristics above are expected to be
suitable for use in the present invention, as described herein.
[0233] Plasmid pGP704 did not, however, permit easy detection and
identification of clones that had undergone allelic exchange. Thus,
pGP704 was modified to permit the positive selection of
double-crossover events within Methylomonas and other C1
metabolizing bacteria.
Cloning of the npr-sacB Cassette
[0234] Plasmid pBE83 contained a Bacillus amyloliquifaciens sacB
gene under the control of the neutral protease (npr) promoter (gift
from V. Nagarajan, E.I. du Pont de Nemours and Co., Inc.). The
npr-sacB cassette was PCR amplified from pBE83 using DNA primers
Drdl/npr-sacB and Tthlll/npr-sacB. The DNA primers were constructed
to include unique restriction sites at each terminus of the PCR
product, to facilitate subsequent cloning (as indicated by the
underlined sequences below): TABLE-US-00005 DrdI/npr-sacB:
5'-GACATCGATGTCGAATTCGAGCTCGGTACCGATC-3' (SEQ ID NO:4)
TthIII/npr-sacB: 5'-GACCTCGTCGCTGTTATTAGTTGACTGTCAGC-3' (SEQ ID
NO:5)
[0235] The PCR reaction mixture was composed of the following: 10
.mu.L of 10.times. PCR buffer; 16 .mu.L (4 .mu.L each) of dNTPs
(320 mM stock); 1 .mu.L Methylomonas chromosomal DNA (.about.500
ng/.mu.L); 8 .mu.l MgCl.sub.2 (25 mM); 0.5 .mu.L Taq polymerase (5
U/.mu.L); 1 .mu.L Drdl/npr-sacB primer (.about.36 nmoles); 1 .mu.L
Tthlll/npr-sacB primer (.about.35 nmoles); and 71 .mu.L sterile
nanopure water. The PCR protocol was the performed on a 9600
GeneAmp PCR System (Perkin Elmer), according to the thermocycling
parameters below: [0236] 1 cycle: 94.degree. C. (5 min); [0237] 1
cycle: 94.degree. C. (5 min), 60.degree. C. (2 min), 72.degree. C.
(3 min); [0238] 35 cycles: 94.degree. C. (1 min), 60.degree. C. (2
min), 72.degree. C. (3 min); [0239] 1 cycle: 94.degree. C. (1 min),
60.degree. C. (2 min), 72.degree. C. (10 min); and [0240] Hold
-4.degree. C. (.infin.). Afterward, the PCR product was ligated
into the pCR2.1TOPO vector per the manufacturer's instructions
(Invitrogen, Carlsbad, Calif.). The ligation mixture was
transformed into TOP10 One Shot.TM. calcium chloride competent
cells and transformants were screened as recommended by
Invitrogen.
[0241] Plasmid DNA was isolated from positive clones (white
colonies in a blue/white screen) using the QIAprep.RTM. Spin
Mini-prep Kit (Qiagen, Valencia, Calif.) and the DNA was digested
according to the manufacturer's instructions with restriction
endonucleases Drdl and Tthlll (New England Biolabs; Boston, Mass.).
Initially, this PCR product was to be inserted into pGP704 digested
with Drdl and Tthlll; however, there were difficulties in cloning
the Drdl/Tthlll PCR product.
[0242] A modified cloning strategy was adopted, such that the PCR
reaction of above was "repeated" using the PFU polymerase
(Stratagene; LaJolla, Calif.). Specifically, the PCR reaction and
protocol were performed exactly as described above, with the
exception that PFU polymerase and buffers from Stratagene were
used. A PCR product having flush or blunt ends was produced. This
PCR product was ligated directly into the Xcal site of pGP704. The
ligation mixture was transformed into calcium chloride competent E.
coli SY327 cells (Miller, V., L., and Mekalanos, J., J., Proc.
Natl. Acad. Sci., 81(11):3471-3475 (1984)).
[0243] The transformants were screened using the Drdl/npr-sacB and
Tthlll/npr-sacB PCR primers (SEQ ID NOs: 4 and 5, respectively) to
identify vectors containing the npr-sacB insert. The PCR products
were analyzed on a 0.8% agarose gel. Plasmid DNA was isolated from
cells containing the pGP704::sacB vector.
Example 3
Construction of pGP704::sacB::crtN2::EZ::TN.TM. <Kan-2>
[0244] The native Methylomonas gene encoding crtN2 was disrupted by
EZ::TN.TM. <Kan-2> (Epicenter Technologies, Madison, Wis.) to
generate an appropriate replacement nucleotide sequence of interest
(i.e., re-NSI) in the chromosomal integration vector pGP704::sacB.
This construct, pGP704::sacB::crtN2::EZ::TN.TM. <Kan-2> was
then utilized in Examples 4 and 5 to test the homologous
recombination abilities of Methylomonas sp. 16a, using the one-step
selection strategy to identify allelic exchange mutants.
PCR Amplification and Cloning of the crtN2 DNA Fragment into
pGP704::sacB
[0245] DNA primers crtN#2-ctg 288+1 kB/Bg/ll and crtN#2-ctg
288+1kB/Xbal were used to amplify a .about.3.1 kB DNA fragment from
Methylomonas sp. 16a chromosomal DNA, comprising SEQ ID NO:3
flanked by approximately 1 Kb of DNA. TABLE-US-00006 crtN#2-ctg 288
+ 1kB/Bg/II: 5'-AGATCTTTCCGGTCATGCTGGAGTTGGG-3' (SEQ ID NO:6)
crtN#2-ctg 288 + 1kB/XbaI: 5'-TCTAGATTGCAGCTCAAGCGATTCGG-3' (SEQ ID
NO:7)
[0246] As described in Example 2, the underlined sequences
represent restriction endonuclease recognition sites; additionally,
the PCR reactions and cloning protocol were the same as described
in Example 2. The PCR fragment generated above was cloned in the
pCR2.1 TOPO vector and then transformed into calcium chloride
competent E. coli TOP10 One Shot.TM. cells (Invitrogen, Carlsbad,
Calif.). Following screening for positive clones, plasmid DNA was
isolated from several positive clones using the QIAprep.RTM. Spin
Mini-prep Kit and the DNA was digested with restriction
endonucleases Bglll and Xbal according to manufacturer's
instructions. The restriction digestion products were separated on
a 0.8% agarose gel. The crtN2+1kB DNA fragment (.about.3.1 kB) was
excised from the agarose gel and the DNA was extracted from the
agarose using the QiaQuick.RTM. Gel Extraction Kit (Qiagen).
[0247] The purified crtN2+1 kB DNA fragment was ligated into
pGP704-sacB (from Example 2) digested with Bg/ll and Xbal. After an
overnight incubation at room temperature, the ligation mixture was
transformed into calcium chloride competent E. coli SY327 (.gamma.
pir+) cells (Miller, V., L., and Mekalanos, J., J., Proc. Natl.
Acad. Sci., 81(11):3471-3475 (1984)). The transformation mixture
was plated onto BTZ medium containing 25 .mu.g/mL Amp. E. coli
transformants containing the appropriate insert DNA fragment were
detected using PCR amplification with DNA primers crtN#2-ctg 288+1
kB/Bg/ll (SEQ ID NO:6) and crtN#2-ctg 288+1 kB/Xbal (SEQ ID NO:7).
Plasmid DNA was purified from positive clones using the
QIAprep.RTM. Spin Mini-prep Kit (Qiagen) and was used as the
"target DNA" in an in vitro transposition reaction.
The in vitro Transposition Reaction
[0248] The crtN2 gene was disrupted via in vitro transposition
using the EZ::TN.TM. <Kan-2> transposon Insertion Kit
(Epicentre Technologies; Madison, Wis.). The transposon donor DNA
used in the transposition reactions was the EZ::TN.TM. Transposon
(a linear DNA fragment), which carries the gene that confers
resistance to kanamycin between the ends of the transposable
element. As per the manufacturer's instructions, 0.2 .mu.g of the
target DNA was incubated with molar equivalents of the EZ::TN.TM.
<Kan-2> transposon and 1 U of the EZ::TN.TM. <Kan-2>
transposase. The reaction mixture was incubated for 2 hrs at
37.degree. C. The transposition reaction was stopped by adding 1
.mu.L of the 1.times. Stop Solution and incubating the mixture for
10 min at 70.degree. C.
[0249] PCR amplification using gene-specific primers crtN#2-ctg
288+1 kB/Bg/ll (SEQ ID NO:6) or crtN#2-ctg 281+1 kB/Xbal (SEQ ID
NO:7) and transposon-specific primers KAN-2FP-1 (SEQ ID NO:8) and
KAN-2RP-1 (SEQ ID NO:9) were used to determine the location of the
EZ::TN.TM. <Kan-2> transposon within the crtN2 gene. One
clone was identified, in which the EZ::TN.TM. <Kan-2>
transposon was located near the middle of the crtN2 gene. This
crtN2:: EZ::TN.TM. <Kan-2> construct in pGP704:sacB served as
the donor plasmid in a tri-parental mating reaction (see Example
4).
Example 4
Triparental Conjugation of pGP704:sacB:crtN2::EZ::TN.TM.
<Kan-2> into Methylomonas sp. 16a
[0250] The pGP704::sacB::crtN2::EZ::TN.TM. <Kan-2> construct
from Example 3 was transferred into Methylomonas sp. 16a, to test
the homologous recombination abilities of the organism. A
triparental conjugation was performed, using the following:
Methylomonas sp. 16a (recipient), E. coli SY327
pGP704:sacB:crtN2::EZ::TN.TM. <Kan-2> (donor; from Example
3), and E. coli pRK2013 (helper; ATCC No. 37159).
Theory of the Conjugation
[0251] The mobilization of vector DNA into Methylomonas occurs
through conjugation (tri-parental mating). The pGP704::sacB vector
used to make chromosomal mutations in Methylomonas has a R6K origin
of replication, which requires the FI protein. This vector can
replicate in E. coli strain SY327, which expresses the .PI.
protein. However, this protein is not present in the Methylomonas
genome. Therefore, once the vector DNA has entered into
Methylomonas, it is unable to duplicate itself. If the vector also
contains a DNA segment that shares homology to a region of the
Methylomonas genome, the vector can be integrated into the host's
genome through homologous recombination. The homologous
recombination system of Methylomonas appears to be similar to that
of other Gram-negative organisms.
[0252] In the case of Methylomonas, the mobilizable plasmid
(pGP704::sacB) was used to transfer re-NSI into this bacterium. The
conjugative plasmid (pRK2013), which resided in a strain of E.
coli, facilitated the DNA transfer.
Growth of Methylomonas sp. 16a
[0253] The growth of Methylomonas sp. 16a for tri-parental mating
initiated with the inoculation of an -80.degree. C. frozen stock
culture into 20 mL of BTZ medium containing 25% methane. The
culture was grown at 30.degree. C. with aeration until the density
of the culture was saturated. This saturated culture was in turn
used to inoculate 100 mL of fresh BTZ medium containing 25%
methane. The 100 mL culture was grown at 30.degree. C. with
aeration until the culture reached an OD.sub.600 between 0.7 to
0.8. To prepare the cells for the tri-parental mating, the
Methylomonas sp. 16a cells were washed twice in an equal volume of
BTZ medium. The Methylomonas cell pellets were re-suspended in the
minimal volume needed (approximately 200 to 250 .mu.L).
Approximately 40 .mu.L of the re-suspended Methylomonas cells were
used in each tri-parental mating experiment.
Growth of the Escherichia coli Donor and Helper Cells
[0254] Isolated colonies of the E. coli donor (containing
pGP704::sacB::crtN2::EZ::TN.TM. <Kan-2> ) and helper
(containing conjugative plasmid pRK2013) cells were used to
inoculate 5 mL of LB broth containing 25 .mu.g/.mu.L Kan; these
cultures were grown overnight at 30.degree. C. with aeration. The
following day, the E. coli donor and helper cells were mixed
together and incubated at 30.degree. C. for .about.2 hours.
Subsequently, the cells were washed twice in equal volumes of fresh
LB broth to remove the antibiotics.
Tri-Parental Mating: Mobilization of the Donor Plasmid into
Methylomonas Strain 16a
[0255] Approximately 40 .mu.L of the re-suspended Methylomonas
cells were used to re-suspend the combined E. coli donor and helper
cell pellets. After thoroughly mixing the cells, the cell
suspension was spotted onto BTZ agar plates containing 0.05% yeast
extract. The plates were incubated at 30.degree. C. for 3 days in a
jar containing 25% methane.
[0256] Following the third day of incubation, the cells were
scraped from the plate and re-suspended in BTZ broth. The entire
cell suspension was plated onto several BTZ agar plates containing
25 .mu.g/.mu.L Kan. The plates were incubated at 30.degree. C. in a
jar containing 25% methane until colonies were visible (.about.4-7
days).
[0257] Individual colonies were streaked onto fresh BTZ+25
.mu.g/.mu.L Kan agar plates and incubated at 30.degree. C. in the
presence of 25% methane. Cells growing on the BTZ+Kan agar plates
were used to inoculate BTZ+Kan medium in bottles containing 25%
methane. The bottles were incubated overnight at 30.degree. C. with
aeration. Then, to rid the cultures of E. coli cells that were
introduced during the tri-parental mating, the cultures were
inoculated into BTZ medium which contained nitrate (15 mM) as the
nitrogen source and methanol (200 mM) as the carbon source, and
incubated overnight at 30.degree. C. with aeration. The cultures
were monitored for E. coli killing by plating onto LB agar
plates.
Example 5
Evaluation of Methylomonas Transconjugants Containing
pGP704:sacB:crtN2::EZ::TN.TM. <Kan-2>
[0258] Following the mobilization of the
pGP704::sacB::crtN2::EZ::TN.TM. <Kan-2> construct into
Methylomonas sp. 16a, the one-step selection strategy to applied as
described below to identify allelic exchange mutants (FIG. 1).
Preliminary Screening for Allelic Exchange Mutants
[0259] Cultures free of E. coli cells (from Example 4) were
transferred back into BTZ+Kan medium and grown overnight at
30.degree. C. with aeration. Cells were plated onto BTZ+Kan,
BTZ+Amp (35 .mu.g/.mu.L), and BTZ+Kan+Sucrose (5%) agar plates. The
ability of cells to grow on the BTZ +Kan +Sucrose agar plates
suggested that the cells had undergone a double-crossover event,
wherein the transposon-disrupted crtN2 gene (crtN2::EZ::TN.TM.
<Kan-2> ) had replaced the intact crtN2 gene in the
Methylomonas chromosome.
[0260] To confirm the allelic exchange, individual colonies were
streaked onto BTZ+Kan, BTZ+Amp, and BTZ+Kan+Sucrose agar plates.
Those clones identified as double-crossover mutants (i.e., those
where crtN2 appeared to be disrupted with the EZ::TN.TM.
<Kan-2> transposon) were able to grow on the BTZ+Kan and
BTZ+Kan+Sucrose agar plates, but not on the BTZ+Amp agar
plates.
Confirmation of Allelic Exchange Mutants
[0261] PCR methodology was used to confirm the construction of the
Methylomonas crtN2 mutant using transposon-specific and outside
primers (OP). OP primers were homologous to regions outside of the
gene-specific primers. Thus, PCR reactions were performed using the
following combination of primers: TABLE-US-00007 Primer Set #1:
OP#1crtN#2; (SEQ ID NO:10) KAN-2-FP-1; (SEQ ID NO:8) and
KAN-2-RP-1. (SEQ ID NO:9) Primer Set #2: OP#2crtN#2; (SEQ ID NO:11)
KAN-2-FP-1; (SEQ ID NO:8) and KAN-2-RP-1. (SEQ ID NO:9)
[0262] A PCR fragment of .about.2.6 kB was obtained when Primer Set
#1 was used in the PCR reaction. In addition, a PCR fragment of
.about.2.8 kB was detected for the PCR reaction using Primer Set
#2. Since neither OP#1crtN#2 (SEQ ID NO:10) nor OP#2crtN#2 (SEQ ID
NO:11) primer sequences were present on the integration vector, PCR
products should only be detected if re-NSI had integrated into the
Methylomonas genome at the appropriate site. Thus, these PCR
reactions confirmed the integration of the transposon-disrupted
crtN2 gene into the genome of Methylomonas and the utility of the
one-step selection method for identification of allelic exchange
mutants.
Example 6
Construction of pGP704::sacB::.DELTA.ald(crtN1)
[0263] The native Methylomonas ald gene (SEQ ID NO:2), normally
located between the crtN1 (SEQ ID NO:1) and crtN2 (SEQ ID NO:3)
genes within a single carotenoid gene cluster (FIG. 3), was deleted
from the operon structure to generate an appropriate replacement
nucleotide sequence of interest (i.e., re-NSI) in the chromosomal
integration vector pGP704::sacB. Specifically, two DNA fragments
flanking ald were prepared by PCR (i.e., crtN1/ald deletion
fragment #1 and crtN1/ald deletion fragment #2) and ligated
together within pGP704::sacB. The crtN1/ald deletion fragment #1
also caused the deletion of .about.10 amino acids from the carboxyl
terminus on the CrtN1 protein. The resulting construct,
pGP704::sacB::.DELTA.aid(crtN1), was then utilized in Examples 7
and 8 to test the homologous recombination abilities of
Methylomonas sp. 16a, using the two-step selection strategy to
identify allelic exchange mutants in which ald(crtN1) had been
deleted from the Methylomonas sp. 16a genome.
PCR Amplification and Cloning of the .DELTA.ald(crtN1) DNA Fragment
into pGP704::sacB
[0264] For amplification of crtN1/ald deletion fragment #1
(.about.1.1 kB in size) and crtN1/ald deletion fragment #2
(.about.1.1 kB in size), the following DNA primers were used, along
with Methylomonas sp. 16a chromosomal DNA as template.
TABLE-US-00008 TABLE 4 Primers Utilized for Cloning of the
.DELTA.ald DNA fragment Deletion Fragment Forward Primer Reverse
Primer crtN1/ald deletion Bg/II/ald (deletion) SphI-NotI/ fragment
#1 #1: ald (deletion) #1: 5'-AGATCTTTGCAACGGG 5'-CATGCGCGGCCGCCC
TATTCGACGAAGG-3' CGATGTTTCTGGGAAATC (SEQ ID NO:12) AGC-3' (SEQ ID
NO:13) crtN1/ald deletion NotI/ald (deletion) #2: SphI/ald
(deletion) #2: fragment #2 5'-GCGGCCGCAATCAG 5'-GCATGCGTTGCGGA
CAAGCGCTGCAAGC-3' TACAGCCTGTCC-3' (SEQ ID NO:14) (SEQ ID NO:15)
**Underlined sequences represent restriction endonuclease
recognition sites.
The methodology used for PCR reactions and cloning into E. coli
TOP10 One Shot.TM. cells (Invitrogen) were the same as previously
described in Example 3.
[0265] Several colonies from the transformation were screened for
the proper insert DNA fragments using the QIAprep.RTM. Spin
Mini-prep Kit for plasmid isolation. The crtN1/aid deletion
fragment #1 was digested with Bglll and Sphl and the crtN1/ald
deletion fragment #2 was digested with Notl and Sphl. The
restriction digestion products were separated on an 0.8% agarose
gel, subsequently excised from the agarose gel, and the DNA was
extracted using the QiaQuick.RTM. Gel Extraction Kit (Qiagen).
[0266] Purified crtN1/ald deletion fragment #1 was ligated into
pGP704::sacB digested with Bglll and Sphl. Following overnight room
temperature incubation, the ligation mixture was used to transform
calcium chloride competent E. coli SY327 cells (Miller, V., L., and
Mekalanos, J., J., supra (1984)). The transformation mixture was
plated onto LB+Amp (25 .mu.g/mL) agar plates. PCR amplification
using PCR primers Bglll/ald (deletion)#1 (SEQ ID NO:12) and
Sphl-Notl/ald (deletion)#1 (SEQ ID NO:13) was used to detect E.
coli transformants containing the correct DNA insert fragment.
Plasmid DNA was purified from positive clones using the
QIAprep.RTM. Spin Mini-prep Kit (Qiagen) and the resulting vector
was digested with Notl and Sphl.
[0267] Purified crtN1/ald deletion fragment #2 was ligated into the
linearized pGP704::sacB::crtN1/ald deletion fragment #1 vector
described above. After overnight incubation at room temperature,
the ligation mixture was transformed into E. coli SY327 cells and
transformants were selected on LB+Amp (25 .mu.g/mL) agar plates.
PCR amplification using PCR primers Bg/ll/ald (deletion)#1 (SEQ ID
NO:12) and Sphl/ald (deletion)#2 (SEQ ID NO:15) was used to detect
E. coli transformants containing the appropriate DNA insert
fragment. Subsequently, E. coli cells containing
pGP704::sacB::.DELTA.aid were streaked onto LB+Amp (25 .mu.g/mL)
agar plates to obtain isolated colonies.
Example 7
Triparental Conjunction of pGP704::sacB::.DELTA.ald(crtN1) into
Methylomonas sp. 16a
[0268] The pGP704::sacB::.DELTA.ald(crtN1) construct from Example 6
was transferred into Methylomonas sp. 16a via triparental
conjugation. Specifically, the following were used as recipient,
donor, and helper, respectively: Methylomonas sp. 16a, E. coli
SY327 pGP704::sacB::.DELTA.ald(crtN1) (from Example 6), and E. coli
pRK2013. The mating was performed as described in Example 4;
however, the cell suspension was plated onto BTZ agar plates
containing Amp (35 .mu.g/.mu.L) instead of BTZ+Kan agar plates.
[0269] Isolated colonies were streaked onto BTZ+35 .mu.g/.mu.L Amp
agar plates and grown 1-2 days at 30.degree. C. in a jar containing
25% methane. These cells were used to inoculate bottles containing
20 mL of BTZ+25% methane. After overnight growth, 5 mL of the
culture was concentrated by centrifugation using a tabletop
centrifuge. The cells were inoculated into 20 mL of BTZ liquid
medium containing nitrate (10 mM), methanol (200 mM), and 25%
methane and grown overnight at 30.degree. C. with aeration. Cells
from the BTZ (NO.sub.3) cultures were again inoculated into BTZ+25%
methane and grown overnight growth at 30.degree. C. with aeration.
The cultures were monitored for E. coli killing by plating onto LB
agar plates.
Example 8
Evaluation of Methylomonas Transconjugants Containing
pGP704::sacB::.DELTA.ald(crtN1)
[0270] Following the mobilization of the
pGP704:sacB::.DELTA.ald(crtN1) construct into Methylomonas sp. 16a,
the two-step selection strategy was applied as described below to
identify allelic exchange mutants (FIG. 2).
Preliminary Screening for Allelic Exchange Mutants
[0271] Cultures free of E. coli cells were passaged several times
into fresh media (1 mL of culture into 20 mL of fresh BTZ medium),
to increase the probability of occurrence of a second crossover
event. Subsequently, cells were plated onto BTZ+Sucrose (5%) agar
plates. Those cells growing on plates containing sucrose had lost
the integration vector, which contained the sacB gene. However, the
loss of the vector sequences could be due to the second crossover
event occurring either on the same or opposite side of the
ald(crtN1) gene deletion that was present on the insert DNA. If the
second crossover event had occurred on the same side of the
ald(crtN1) gene deletion as the first crossover event, the wildtype
ald gene would be regenerated. In contrast, if the second crossover
event occurred on the opposite side of the ald(crtN1) gene deletion
as the first crossover event, the deletion of the ald(crtN1) gene
would be established in the Methylomonas genome.
Confirmation of Allelic Exchange Mutants
[0272] Chromosomal DNA was purified from three cultures that had
grown on the sucrose plates using the MasterPure DNA Purification
Kit (Epicentre). PCR amplification using PCR primers
Bg/ll/ald(deletion)#1 (SEQ ID NO:12) and Sphl/ald(deletion)#2 (SEQ
ID NO:15) was used to distinguish between cells that contained the
intact ald(crtN1) gene (wherein the expected size of the PCR
product was .about.3.7 kB) and those that contained the ald(crtN1)
gene deletion (wherein the expected size of the PCR product was
.about.2.1 kB). Two of the three cultures produced a PCR product
that was 2.1 kB, while the third culture appeared to have the
wildtype gene present. Thus, these two PCR reactions confirmed the
deletion of the ald(crtN1) gene in the genome of Methylomonas.
Additionally, these results clearly demonstrated the utility of the
two-step selection method for identification of allelic exchange
mutants that are markerless, wherein the targeted chromosomal
mutation does not require that an antibiotic resistance marker be
left in the chromosome of the Methylomonas host.
Sequence CWU 1
1
15 1 1536 DNA Methylomonas sp.16a 1 atggccaaca ccaaacacat
catcatcgtc ggcgcgggtc ccggcggact ttgcgccggc 60 atgttgctga
gccagcgcgg cttcaaggta tcgattttcg acaaacatgc agaaatcggc 120
ggccgcaacc gcccgatcaa catgaacggc tttaccttcg ataccggtcc gacattcttg
180 ttgatgaaag gcgtgctgga cgaaatgttc gaactgtgcg agcgccgtag
cgaggattat 240 ctggaattcc tgccgctaag cccgatgtac cgcctgctgt
acgacgaccg cgacatcttc 300 gtctattccg accgcgagaa catgcgcgcc
gaattgcaac gggtattcga cgaaggcacg 360 gacggctacg aacagttcat
ggaacaggaa cgcaaacgct tcaacgcgct gtatccctgc 420 atcacccgcg
attattccag cctgaaatcc tttttgtcgc tggacttgat caaggccctg 480
ccgtggctgg cttttccgaa aagcgtgttc aataatctcg gccagtattt caaccaggaa
540 aaaatgcgcc tggccttttg ctttcagtcc aagtatctgg gcatgtcgcc
gtgggaatgc 600 ccggcactgt ttacgatgct gccctatctg gagcacgaat
acggcattta tcacgtcaaa 660 ggcggcctga accgcatcgc ggcggcgatg
gcgcaagtga tcgcggaaaa cggcggcgaa 720 attcacttga acagcgaaat
cgagtcgctg atcatcgaaa acggcgctgc caagggcgtc 780 aaattacaac
atggcgcgga gctgcgcggc gacgaagtca tcatcaacgc ggattttgcc 840
cacgcgatga cgcatctggt caaaccgggc gtcttgaaaa aatacacccc ggaaaacctg
900 aagcagcgcg agtattcctg ttcgaccttc atgctgtatc tgggtttgga
caagatttac 960 gatctgccgc accataccat cgtgtttgcc aaggattaca
ccaccaatat ccgcaacatt 1020 ttcgacaaca aaaccctgac ggacgatttt
tcgttttacg tgcaaaacgc cagcgccagc 1080 gacgacagcc tagcgccagc
cggcaaatcg gcgctgtacg tgctggtgcc gatgcccaac 1140 aacgacagcg
gcctggactg gcaggcgcat tgccaaaacg tgcgcgaaca ggtgttggac 1200
acgctgggcg cgcgactggg attgagcgac atcagagccc atatcgaatg cgaaaaaatc
1260 atcacgccgc aaacctggga aacggacgaa cacgtttaca agggcgccac
tttcagtttg 1320 tcgcacaagt tcagccaaat gctgtactgg cggccgcaca
accgtttcga ggaactggcc 1380 aattgctatc tggtcggcgg cggcacgcat
cccggtagcg gtttgccgac catctacgaa 1440 tcggcgcgga tttcggccaa
gctgatttcc cagaaacatc gggtgaggtt caaggacata 1500 gcacacagcg
cctggctgaa aaaagccaaa gcctga 1536 2 1593 DNA Methylomonas sp.16a 2
atgacgacga tagcagccgt ctccccactg gatggccgct tgctgggaca ttttccagtc
60 agcaagccgg cgctcattca gcaacagctg acaaaatccc gccgcgccgc
cctgctttgg 120 cgcgagctgc cggtcacgga acgggtcaaa cgcctgtcgc
ccttgaaaaa acagctgctg 180 gataacctgg acagactctg cgaaaccatc
cgcctcagca ccggcaaggt tcgcaccgag 240 gccttgctgg gggaaattta
tccggtgctg gatttactgg cgtattacca aaagcgggcg 300 ccgcggattc
tacgcacgcg cgccgtgtcc acctcgccgt tcgcgtttcc ggccgccacc 360
gcccgcatcg aacgccgccc ttacggcgtg gtcgcggtga tctcgccatg gaattacccg
420 tttcacctga gcgtcgcccc gctgctgacc gctttgctgg ccggcaatgc
ggtaatcctg 480 aaaccctccg aactctgctt gccggtcggt cagttgatcg
tcgatttgtt cgccacgctg 540 gatttgccgg acgggttggt gcaatgggtc
atcggcgacg gccaaaccgg cgcggaactg 600 atagacgccc gccccgatct
ggtgtttttc accggcggcc tgcagaccgg tcgggcggtc 660 atgcaacgcg
ccgcccggca tccgattccg gtcatgctgg agttgggcgg taaagacacc 720
atgctggtgc tggccgacgc cgacctcaag cgcgccagcg ctgccgcgct gtacggcgcg
780 ttttgcaata gcggccaagt ctgcgtctcg gtcgaacgtc tgtacgtgca
acaagcctgt 840 tttgcggaat tcctggccat gctgctgaag ggcctgtcca
agctcaaggt cggccatgac 900 ccgcacggcg atgtgggagt gatgacgtcc
gcccggcaaa tcgacatcgt ccaggcccat 960 tacgaggacg ccatcgccca
gggcgccaag gcctccggcc cgctgctgcg cgacggcaat 1020 gtcgtgcaac
ccgtggtgct ttgggacgtg caccacggca tgaaggtcat gcgcgaggaa 1080
accttcggtc cgttgctgcc ggtcatgccg ttcagcgacg aagccgaggc catcaagctc
1140 gccaacgaca gcgatctggg tctaaacgcc agcatctgga gccaggatat
aatcaaggcc 1200 gagcgccttg ctggacaact agatgtcggc aactgggcga
tcaacgacgt attgaaaaac 1260 gtgggccatt ccggcctgcc cttcggcggc
gtcaagcaaa gcgggtttgg ccgttatcac 1320 ggcgccgaag gcttgctgaa
cttcagctac ccggtatcgg gcctgaccaa tcgcagccgc 1380 ttgcccaaag
aacccaactg gttcccttac agcgcatcag gctatgaaaa tttcaagggt 1440
ttcctcgatt ttatctacgg cgaagactcg atgctgcagc gcggtcgccg caatcagcaa
1500 gcgctgcaag ccttccgcga gttttccatt ttcgattgga cacaacgctg
gcaaaacctg 1560 aaactgctgt tttcttggac acgggatgac taa 1593 3 1494
DNA Methylomonas sp.16a 3 atgaactcaa atgacaacca acgcgtgatc
gtgatcggcg ccggcctcgg cggcctgtcc 60 gccgctattt cgctggccac
ggccggcttt tccgtgcaac tcatcgaaaa aaacgacaag 120 gtcggcggca
agctcaacat catgaccaaa gacggcttta ccttcgatct ggggccgtcc 180
attttgacga tgccgcacat ctttgaggcc ttgttcacag gggccggcaa aaacatggcc
240 gattacgtgc aaatccagaa agtcgaaccg cactggcgca atttcttcga
ggacggtagc 300 gtgatcgact tgtgcgaaga cgccgaaacc cagcgccgcg
agctggataa acttggcccc 360 ggcacttacg cgcaattcca gcgctttctg
gactattcga aaaacctctg cacggaaacc 420 gaagccggtt acttcgccaa
gggcctggac ggcttttggg atttactcaa gttttacggc 480 ccgctccgca
gcctgctgag tttcgacgtc ttccgcagca tggaccaggg cgtgcgccgc 540
tttatttccg atcccaagtt ggtcgaaatc ctgaattact tcatcaaata cgtcggctcc
600 tcgccttacg atgcgcccgc cttgatgaac ctgctgcctt acattcaata
tcattacggc 660 ctgtggtacg tgaaaggcgg catgtatggc atggcgcagg
ccatggaaaa actggccgtg 720 gaattgggcg tcgagattcg tttagatgcc
gaggtgtcgg aaatccaaaa acaggacggc 780 agagcctgcg ccgtaaagtt
ggcgaacggc gacgtgctgc cggccgacat cgtggtgtcg 840 aacatggaag
tgattccggc gatggaaaaa ctgctgcgca gcccggccag cgaactgaaa 900
aaaatgcagc gcttcgagcc tagctgttcc ggcctggtgc tgcacttggg cgtggacagg
960 ctgtatccgc aactggcgca ccacaatttc ttttattccg atcatccgcg
cgaacatttc 1020 gatgcggtat tcaaaagcca tcgcctgtcg gacgatccga
ccatttatct ggtcgcgccg 1080 tgcaagaccg accccgccca ggcgccggcc
ggctgcgaga tcatcaaaat cctgccccat 1140 atcccgcacc tcgaccccga
caaactgctg accgccgagg attattcagc cttgcgcgag 1200 cgggtgctgg
tcaaactcga acgcatgggc ctgacggatt tacgccaaca catcgtgacc 1260
gaagaatact ggacgccgct ggatattcag gccaaatatt attcaaacca gggctcgatt
1320 tacggcgtgg tcgccgaccg cttcaaaaac ctgggtttca aggcacctca
acgcagcagc 1380 gaattatcca atctgtattt cgtcggcggc agcgtcaatc
ccggcggcgg catgccgatg 1440 gtgacgctgt ccgggcaatt ggtgagggac
aagattgtgg cggatttgca ataa 1494 4 34 DNA Artificial Sequence Primer
DrdI/npr-sacB 4 gacatcgatg tcgaattcga gctcggtacc gatc 34 5 32 DNA
Artificial Sequence Primer TthIII/npr-sacB 5 gacctcgtcg ctgttattag
ttgactgtca gc 32 6 28 DNA Artificial Sequence Primer crtN#2-ctg288
+1kb/BglII 6 agatctttcc ggtcatgctg gagttggg 28 7 26 DNA Artificial
Sequence Primer crtN#2-ctg 288 + 1kb/XbaI 7 tctagattgc agctcaagcg
attcgg 26 8 25 DNA Artificial Sequence Primer KAN-2FP-1 8
acctacaaca aagctctcat caacc 25 9 25 DNA Artificial Sequence Primer
KAN-2RP-1 9 gcaatgtaac atcagagatt ttgag 25 10 21 DNA Artificial
Sequence Primer OP#1crtN#2 10 tcgtcgattt gttcgccacg c 21 11 25 DNA
Artificial Sequence Primer OP#2crtN#2 11 gccctatctt tttttatatt
gtgtt 25 12 29 DNA Artificial Sequence Primer BglII/ald(deletion)#1
12 agatctttgc aacgggtatt cgacgaagg 29 13 37 DNA Artificial Sequence
Primer SphI-NotI/ald (deletion)#1 13 gcatgcgcgg ccgccccgat
gtttctggga aatcagc 37 14 28 DNA Artificial Sequence Primer NotI/ald
(deletion) #2 14 gcggccgcaa tcagcaagcg ctgcaagc 28 15 26 DNA
Artificial Sequence Primer SphI/ald (deletion) #2 15 gcatgcgttg
cggatacagc ctgtcc 26
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