Endophyte Ergot Alkaloid Synthetic Compounds, Compounds

17 Pages·2016·1.1 MB·English

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Plant Pathology Faculty Patents Plant Pathology
1-1-2002
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WWhhiicchh EEnnccooddee TThheerreeffoorr aanndd RReellaatteedd MMeetthhooddss
Christopher L. Schardl
University of Kentucky, [email protected]
Jinhung Wang
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Part of the Plant Pathology Commons
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Schardl, Christopher L. and Wang, Jinhung, "Endophyte Ergot Alkaloid Synthetic Compounds, Compounds
Which Encode Therefor and Related Methods" (2002). Plant Pathology Faculty Patents. 3.
https://uknowledge.uky.edu/plantpath_patents/3
This Patent is brought to you for free and open access by the Plant Pathology at UKnowledge. It has been accepted
for inclusion in Plant Pathology Faculty Patents by an authorized administrator of UKnowledge. For more
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US006335188B1
(12) United States Patent (16) Patent N6.= US 6,335,188 B1
Schardl et a1. (45) Date of Patent: Jan. 1, 2002
(54) ENDOPHYTE ERGOT ALKALOID (56) References Cited
SYNTHETIC COMPOUNDS, COMPOUNDS
WHICH ENCODE THEREFOR AND PUBLICATIONS
RELATED METHODS
Gebler, et al., 114 J. of theAmerican Chemical Society 7354
(75) Inventors: Christopher L. Schardl; Jinghung (1992)
Wang, both of Lexington, KY (Us) Tsai, et al., 216 Biochem & Biophys Res Comm 119 (1995).
(73) AssigneeZ University of Kentucky Research Tudzynskl, et al., 261 Molec Gen Genet 133 (1999).
Foundation, Lexington, KY (Us) Gebler et al., 296 Archives of Biochemistry and Biophysics
308 (1992).
( * ) Notice: subjecpto any (glisglaimeéi the fiermgf TudZynski,et al., 103 Mycological Research 1044 (1999).
gusenct lisi?gnbe 021; as Juste un er ArntZ et a1. (1997), Current Genetics, vol. 31(4):357—360,
~ ~ ~ y y ' 1997.*
(21) Appl. NO.Z 09/518,657 * Cited by examiner
(22) Filed: Mar. 3, 2000
Primary Examiner—Rebecca E. Prouty
Related US. Application Data Assistant Examiner—Manjunath N. Rao
(60) Provisional application No. 60/125,490, ?led on Mar. 22, (74) Attorney, Agent, Or Firm—MCDerInOtt, Will & Emery
1999' (57) ABSTRACT
(51) Int. Cl.7 ......................... .. C12P 21/06; C12N 15/09;
C12N 9/00; C12N 1/20; C12N 5/00 The present invention provides, inter alia, dmaW nucleic
(52) US. Cl. ....................... .. 435/193; 435/691; 435/183; acid Sequences and the proteins for Which they encode Also
435/2523; 435/2541; 435/25411; 435/3201; provided are methods for the utilization of knockout mutants
435/410; 435/419; 536/232; 536/236; of the sequences Which are useful for engineering ergot
536/237; 536/2374 alkaloid-de?cient fungal symbionts (endophytes) of plants.
(58) Field Of Search ............................... .. 435/193, 252.3, Other methods and materials related to these Sequences are
435/419, 6, 69.1, 183, 254.1, 254.11, 320.1, also provided
410, 23.1; 536/232, 23.6, 23.7, 23.74;
800/295, 298 19 Claims, 4 Drawing Sheets
U.S. Patent Jan. 1, 2002 Sheet 1 0f 4 US 6,335,188 B1
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U.S. Patent Jan. 1, 2002 Sheet 4 0f 4 US 6,335,188 B1
Figure 4
endophyte in V . endophyte infection of ,
seed embryo \I in seed K host ovule /&
/————> I: ‘ .~ é <——— <—— germinating
endophyte ascospores on
asexual "1 seed'm sexual cycle/ srass?oret
cycle/ horizontal ___;/
vertical propagation transmrssion 3555;,”
of plant with (meiotic spores)
transmission ’ ‘ - erected
emigpsyete ‘speatia
\ (_/"—' slpleyntrnaantsiiae rtso ' ' ‘. =
fun at stroma stmma.
sgn'ounds 0' oPpos'le mature stroma on
in?orescence maung type ?ag-leaf sheath
in?orescence with _
endophyte in "oral and arrests as K karyogarny
merislems development & meiosis
Alternative life cycles of Epichloe and Neotyphodium species in host grasses. The
Neotyphodium (formerly Acremonium) endophytes have only the asexual/vertical
transmission life cycle. Thus, unlike Epichloe species, the Neotyphodium species cannot
transmit contagiously; that is, they cannot transmit from an infected plant of one maternal
lineage to an uninfected plant of a different maternal lineage. However, the Neotyphodr'um
species and some Epichloe species transmit vertically at extremely high frequency, such
that 98%-] 00% of seeds produced on an endophyte-containing mother plant also contain
endophyte and, upon germination, give rise to endophyte-containing plants. Also, there
are standard methods whereby seedlings or other plant material that lacks endophyte
can be inoculated with mycelium of wild type or transformed endophyte to give rise
to endophyte-containing plants; and such plants will pass on the endophyte in the nonnal
fashions shown here depending on whether the endophyte is compatible with that host
grass and what is (are) its normal transmission mode(s) [Latch et al., [07 Annals of
Applied Biology 17 (1985)][Tsai et al., 22 Current Genet 399 (l992)].
US 6,335,188 B1
1 2
ENDOPHYTE ERGOT ALKALOID More recently, a C. purpurea dmaW sequence Was disclosed
SYNTHETIC COMPOUNDS, COMPOUNDS in TudZynski et al., 261 Molec Gen Genet 133 (1999), and
WHICH ENCODE THEREFOR AND is 62% identical at the DNA level to the present sequence.
RELATED METHODS Citation of the above documents is not intended as an
admission that any of the foregoing is pertinent prior art. For
This application claims priority to US. Provisional eXample, in some instances above, the publication Was less
Patent Application Serial No. 60/125,490, Which Was ?led than one year before the ?ling date of this patent application.
on Mar. 22, 1999. All statements as to the date or representation as to the
The present invention Was funded in part by USDA NRI contents of these documents is based on subjective charac
grant 95-37303-1678; the US. Government may have cer 10 teriZation of information available to the applicant at the
tain rights in this invention. time of ?ling, and does not constitute an admission as to the
accuracy of the dates or contents of these documents.
FIELD OF THE INVENTION
SUMMARY OF THE INVENTION
The present invention relates to sequences that encode
dimethylallyl-diphosphate:L-tryptophan dimethylallyltrans 15 It is therefore an object of the present invention to provide
sequences useful to engineer ergot alkaloid-de?cient
ferase (“DmaW” or “dimethylallyltryptophan synthase”), an
symbionts, thus ergot-alkaloid-de?cient plants.
enZyme present in some grass endophytes, and that catalyZes
the formation of 4- y,y-dimethylallyltryptophan. This It is a further object to provide methods to engineer ergot
enZyme activity is the ?rst committed step in the production alkaloid-de?cient endophytes.
of ergot alkaloids, including those With clavine and ergoline 20 It is yet another object to provide ergot alkaloid-de?cient
ring structures. Such alkaloids include clavines, lysergic seeds.
acid, lysergic acid amides, and ergopeptines. The sequences It is yet another object to provide plants With ergot
encode a DmaW from fungi that are symbionts of commer alkaloid-de?cient endophytes.
cially signi?cant grasses.
It is also an object of the invention to provide materials
25
such as vectors for engineering ergot alkaloid-de?cient
BACKGROUND OF THE INVENTION endophytes.
Certain fungal species eXist as symbiotic and integral It is also an object of the invention to provide enZymes
parts of grasses and are passed from generation to generation useful in ergot alkaloid synthesis.
of plants, but many are not passed from plant to plant eXcept 30 Also, it is an object to use the present nucleic acid
by transmitting in seeds of maternal plant lineages. Repre compounds to determine the potential or lack of potential of
sentatives of these fungi are the Neotyphoa'ium species symbiont strains to produce ergot alkaloids.
(“Neotyphodium” and sometimes “Acremonium”, for Other objects Will be apparent from the present disclo
eXample, N. coenophialum) and Epichloe species (eg E.
sure.
festucae and E. typhina), Which are symbionts and integral
35 De?nitions:
parts of many grass cultivars. These fungi, termed For the purposes of the present application, the folloWing
“endophytes”, are seed-transmissible at extremely high effi
terms have the folloWing meanings. All other terms have the
ciency. Their symbioses With host grasses are characteriZed meaning as speci?cally recogniZed in the art.
by mutual bene?ts to the hosts and symbionts. Bene?ts to
“Allelic variant” is meant to refer to a full length gene or
the grass hosts include protection from insects and
40 partial sequence of a full length gene that occurs at essen
vertebrates, and resistance to Water stress (drought). Anti
tially the same locus (or loci) as the referent sequence, but
insect activities are mainly due to pyrrolopyraZine and Which, due to natural variations caused by, for eXample,
pyrroliZidine alkaloids produced by the endophytes. Anti
mutation or recombination, has a similar but not identical
vertebrate activities are mainly due to indole alkaloids, sequence. Allelic variants typically encode proteins having
including the ergot alkaloids (clavines, lysergic acid and its
45 similar activity to that of the protein encoded by the gene to
derivatives, and ergopeptines).
Which they are being compared. Allelic variants can also
Tall fescue is groWn on over 14 million hectares as an comprise alterations in the 5‘ or 3‘ untranslated regions of the
important forage, turf and conservation grass; most of the gene (e.g., in regulatory control regions).
tall fescue groWn in the US. contains ergot-alkaloid “Fragment” is meant to refer to any subset of the referent
producing endophytes. The anti vertebrate activity of the 50 nucleic acid sequence.
ergot alkaloids, Which manifests as “tall fescue toXicosis” in “Knockout construct” means a DNA sequence Which has
cattle and other livestock, causes losses estimated at more been altered via any knoWn means, for eXample, deletion,
than $600 million per year. insertion, point mutation or rearrangement, so as to alter or
In 1992, Gebler and Poulter puri?ed the DmaW enZyme eliminate the function of the naturally-occurring sequence
from Claviceps sp. ATCC 26245 to a single protein band 55 product, but not so as to alter the ability of the DNA
observable by SDS-PAGE electrophoresis, and fragmented sequence to recombine With the naturally-occurring
the protein With CNBr. Gebler et al., 114 Journal of the sequence.
American Chemical Society 7354 (1992). The three resulting “Knockout mutants” are cells, embryos, fungi or plants in
fragments Were puri?ed and their N-termini sequenced. In Which a naturally-occurring dmaW sequence has been
research by one of the inventors of the present invention, 60 replaced through genetic engineering With a knockout
there Was disclosed a sequence of a dmaW gene (herein construct, so as to result in a ergot alkaloid-de?cient
dmaW) from C. fusiformis AT CC 26245 organism from phenotype, especially a dimethylallyl-diphosphate:L
Which the sequence Was identi?ed Was mis-named C. pur tryptophan dimethylallyltransferase- de?cient phenotype.
purea by the supplier to the ATCC, and Was actually C. “Proteins” means any compounds Which comprise amino
fusiformis.) Tsai et al., 216 Biochem & Biophys Res Comm 65 acids, including peptides, polypeptides, fusion proteins, etc.
119 (1995). The C. fusiformis sequence from that research is Moreover, for the purposes of the present invention, the
58% identical to the present sequences at the DNA level. term “a” or “an” entity refers to one or more of that entity;
US 6,335,188 B1
3 4
for example, “a protein” or “a nucleic acid molecule” refers expressed protein. For example, substitution of valine for
to one or more of those compounds or at least one com leucine, arginine for lysine, or asparagine for glutamine may
pound. As such, the terms “a” (or “an”), “one or more” and not cause a change in functionality of the polypeptide.
“at least one” can be used interchangeably herein. It is also Lastly, a nucleic acid sequence homologous to the exempli
to be noted that the terms “comprising”, “including”, and ?ed nucleic acid molecules (or allelic variants or degener
“having” can be used interchangeably. Furthermore, a com ates thereof) can have approximately 85%, preferably
pound “selected from the group consisting of” refers to one approximately 90%, and most preferably approximately
or more of the compounds in the list that folloWs, including 95% sequence identity With a nucleic acid molecule in the
mixtures (i.e., combinations) of tWo or more of the com sequence listing.
pounds. According to the present invention, an isolated, or 10 Stringent hybridiZation conditions are determined based
biologically pure, protein or nucleic acid molecule is a on de?ned physical properties of the gene to Which the
compound that has been removed from its natural milieu. As nucleic acid molecule is being hybridiZed, and can be
such, “isolated” and “biologically pure” do not necessarily de?ned mathematically. Stringent hybridiZation conditions
re?ect the extent to Which the compound has been puri?ed. are those experimental parameters that alloW an individual
An isolated compound of the present invention can be 15 skilled in the art to identify signi?cant similarities betWeen
obtained from its natural source, can be produced using heterologous nucleic acid molecules. These conditions are
molecular biology techniques or can be produced by chemi Well knoWn to those skilled in the art. See, for example,
cal synthesis. Sambrook, et al., 1989, Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Labs Press, and Meinkoth, et
BRIEF DESCRIPTION OF THE DRAWINGS 20 al., 1984, Anal. Biochem. 138, 267-284.
It is knoWn in the art that there are commercially available
FIG. 1 schematically depicts the ?rst step of the herein
computer programs for determining the degree of similarity
described indirect gene replacement.
betWeen tWo nucleic acid sequences. These computer pro
FIG. 2 schematically depicts the second step of the
grams include various knoWn methods to determine the
herein-described indirect gene replacement.
25 percentage identity and the number and length of gaps
FIG. 3 schematically depicts the herein-described betWeen hybrid nucleic acid molecules. Preferred methods
homologous gene replacement. to determine the percent identity among amino acid
FIG. 4 schematically depicts the alternative life cycles of sequences and also among nucleic acid sequences include
Epichloe and Neotyphodium species in host grasses. analysis using one or more of the commercially available
30 computer programs designed to compare and analyZe
DETAILED DESCRIPTION OF THE nucleic acid or amino acid sequences. These computer
INVENTION programs include, but are not limited to, GCGTM (available
from Genetics Computer Group, Madison, Wis), DNAsisTM
The present invention provides, inter alia, isolated nucleic
(available from Hitachi SoftWare, San Bruno, Calif.) and
acid molecule encoding a DmaW sequence, Wherein said
35 MacVectorTM (available from the Eastman Kodak Company,
nucleic acid molecule comprises a nucleic acid sequence
NeW Haven, Conn.). A preferred method to determine per
selected from the group consisting of:
cent identity among amino acid sequences and also among
(a) a nucleic acid sequence Which has more than 63% nucleic acid sequences includes using the Compare function
identity to a sequence selected from the group consist by maximum matching Within the program DNAsis Version
ing of SEQ ID NO 1 and SEQ ID NO 3, and Wherein 40 2.1 using default parameters.
said identity can be determined using the DNAsis In one embodiment of the present invention, a preferred
computer program and default parameters; dmaW nucleic acid molecule includes an isolated nucleic
(b) a nucleic acid molecule selected from the group acid molecule Which is at least about 50 nucleotides, or at
consisting of: a nucleic acid molecule Which encodes a least about 150 nucleotides, and Which hybridiZes under
DmaW amino acid sequence selected from the group 45 conditions Which preferably alloW about 25% base pair
consisting of: SEQ ID NO 2; SEQ ID NO 4; a protein mismatch, more preferably under conditions Which alloW
encoded by an allelic variant of SEQ ID NO 1; and a about 20% base pair mismatch, more preferably under
protein encoded by an allelic variant of SEQ ID NO 3. conditions Which alloW about 15% base pair mismatch,
Allelic variants, fragments (including a portion of a more preferably under conditions Which alloW about 10%
molecule) and homologues are, by de?nition of “nucleic 50 base pair mismatch and even more preferably under condi
acid molecule”, included Within this and other embodiments. tions Which alloW about 5% base pair mismatch With a
Included Within the scope of the present invention, With nucleic acid molecule selected from the group consisting of
particular regard to the nucleic acids above, are allelic SEQ ID NO 1 and SEQ ID NO 3.
variants, degenerate sequences and homologues. Allelic Another embodiment of the present invention includes a
variants are expected to be found in nature. The present 55 nucleic acid molecule comprising at least about 150 base
invention also includes variants due to laboratory pairs, Wherein the nucleic acid molecule hybridiZes, in a
manipulation, such as, but not limited to, variants produced solution comprising 1X SSC and 0% formamide, at a
during polymerase chain reaction ampli?cation or site temperature of about 56° C., to a nucleic acid sequence
directed mutagenesis. It is also Well knoWn that there is a selected from the group consisting of: SEQ ID NO 1 and
substantial amount of redundancy in the various codons 60 SEQ ID NO 3. Also preferred are fragments of any of such
Which code for speci?c amino acids. Therefore, this inven nucleic acid molecules.
tion is also directed to those nucleic acid sequences Which Additional preferred dmaW nucleic acid molecules of the
contain alternative codons Which code for the eventual present invention include an isolated nucleic acid molecule
translation of the identical amino acid. Also included Within Which is at least about 50 nucleotides, or at least about 150
the scope of this invention are mutations either in the nucleic 65 nucleotides, comprising a nucleic acid sequence that is
acid sequence or the translated protein Which do not sub preferably at least about 65% identical, more preferably
stantially alter the ultimate physical properties of the about 70% identical, more preferably about 75% identical,
US 6,335,188 B1
5 6
more preferably about 80% identical, more preferably about Would be a useful knockout construct. Moreover, excision of
85% identical, more preferably about 90% identical and the coding region or replacing the coding region With a
even more preferably about 95% identical to a nucleic acid antibiotic (i.e. hygromycin) resistance gene Would be useful.
sequence selected from the group consisting of SEQ ID NO FIGS. 1 through 3 describe examples of such manipulations.
1 and SEQ ID NO 3. Also preferred are fragments of any of 5 For example, the folloWing seeds, embryos or plants With
such nucleic acid molecules. Percent identity may be deter
endophyte transformed With knockout constructs are con
mined using the Compare function by maximum matching
sidered Within the present invention. Particularly preferred
Within the program DNAsis Version 2.1 using default
are: forage, turf and conservation grasses. These include, for
parameters.
example, tall fescue (Festuca arundinacea), meadoW fescue
Vectors Which comprise the above molecules are Within
10 (Festuca pratensis), and red fescue (Festuca rubra), Which
the scope of the present invention, as are endophytes and
are common turf, conservation (to hold soil and reclaim strip
other fungi transformed With the above sequences as are mines) and forage grasses in the US. and WorldWide. Also
plants having endophytes transformed With the above
used for these purposes, are the ryegrasses such as perennial
sequences. Vectors may be obtained from various commer ryegrass (Lolium perenne). All these have endophytes and
cial sources, including Clontech Laboratories, Inc. (Palo 15 most such endophytes produce ergot alkaloids. In particular,
Alto, Calif), Stratagene (La Jolla, Calif), Invitrogen
tall fescues are most preferred. HoWever, any seed, embryo
(Carlsbad, Calif.), NeW England Biolabs (Beverly, Mass.)
or plant Which comprise endophytes Which produce ergot
and Promega (Madison, Wis).
alkaloid is Within the scope of the present invention. Of
Preferred vectors are those Which are capable of transfer
course, those in the art recogniZe that any seed, embryo or
ring the molecules disclosed herein into fungal cells. A
20 plant With endophyte transformed With knockout constructs
vector Which provided for either an early or late promoter in
Which are useful for producing plants for biomass are Within
conjunction With the present sequences Would be useful in
the scope of the present invention.
certain circumstances. For instance, the folloWing promoters
Transformation of cells With the nucleic acid molecules of
Would be useful in early expression of the present
the present invention can be accomplished according to
sequences: 25 knoWn procedures. The folloWing procedures are Well
glyceraldehyde-3-phosphate dehydrogenase gene pro knoWn: electroporation [Tsai et al., 22 Current Genetics 399
moter [Jungehiilsing et al., 25 Current Genetics 101 (1992)], treatment of Wall-less fungal cells With vector DNA
(1994)]. plus CaCl2 and polyethyleneglycol [Yelton et al., 81 Pro
trpC promoter [Yelton et al., 82 Proceedings of the ceedings of the National Academy of Sciences of the United
National Academy of Sciences of the United States of 30 States of America 1470 (1984)], and biolistics [Armaleo et
America 834 (1985)]. al., 17 Current Genetics 97 (1990)]. In addition, fungi have
beta-tubulin gene promoter [Tsai et al., 22 Current Genet been transformed using vector-containing bacterial strains,
ics 399 (1992)]. namely Agrobacterium tumefaciens [Gouka et al., 17 Nature
These are fungal promoters that are knoWn to Work in Biotechnology 598 (1999)]. The transformed cells are also
endophytes or (for the glyceraldehyde-3-phosphate dehy 35 Within the scope of this invention.
drogenase gene promoter) the related fungus Claviceps The transformed cells may be groWn into a fungal myce
purpurea. lium (thallus), Which in turn gives rise to spores. Fungal
In order to then constitutively express the sequences mycelium and spores are propagated inde?nitely. In
described above, the construct optionally contains, for addition, transformed fungal endophyte can be introduced
example, a beta-tubulin promoter according to the procee 40 into grass plants. The current preferred method to introduce
dures in Tsai et al., 22 Current Genetics 399 (1992). the fungus into plant is by inoculation of seedling meristems
Moreover, the most commercially signi?cant use of the [Latch and Christensen, 107 Annals of Applied Biology 17
present invention is in the construction of “knockout (1985)]. Another knoWn method is inoculation and regen
mutants” using the above sequences, or knoWn sequences, eration of plant tissue culture [Johnson et al., 70 Plant
for design and construction of DmaW-de?cient mutants. In 45 Disease 380 (1986)].
other Words, the present invention is informative to those Once introduced into a plant the endophyte Will remain
skilled in the art as to their usefulness in making the inde?nitely and propagate inside all plant propagules includ
naturally-occurring sequence inactive. For example, the ing tillers, stolons, and seeds (unless procedures are under
above sequences can be mutated by any means, i.e., deletion, taken to eliminate live fungal mycelium in the grass, for
insertion, point mutation, rearrangement, etc, so long as the 50 example by long storage of seeds at ambient temperature).
mutated version or sequences nearby in replicatable DNA of In any grass breeding Where the female plant possesses the
the fungus (e.g. chromosome) retains the ability to recom endophyte the seeds Will almost all possess the identical
bine. The mutated version of the sequence is then introduced endophyte, and those seeds Will give rise to plants With that
into cells of a preferred line via routine methods (i.e. endophyte [Siegel et al., 74 Phytopathology 932 (1984)]. In
biolistic processes, electroporation, treatment of Wall-less 55 this Way a grass variety With transformed endophyte can be
cells With vector, Agrobacterium-mediated transformation, developed, propagated, and planted for forage, pasture, turf,
etc.). DmaW-de?cient mutants of the preferred line Would revegetation, or soil conservation.
then be selected and propagated. These “knockout” mutant Therefore, also provided are methods for constructing
embryos, seeds and plants are Within the scope of the present sequences With the ability to knockout the above sequences,
invention, as are the knockout constructs, i.e. sequences and 60 comprising one of the folloWing techniques: inserting a
vectors. foreign piece of DNA into one of the disclosed sequences;
In particular, sequences near the active site of enZyme deleting a piece of DNA from one of the disclosed
function, and the site itself, Would be preferred targets. sequences; or creating a mutation such that the DmaW
Moreover, sequences Which are conserved among related activity is eliminated.
organisms are also preferred targets. It is contemplated that 65 Also provided are antisense constructs and methods to
a modi?cation of the present invention such that the start inhibit translation or accumulation of mRNA transcripts of
codon has been eliminated, or replaced With a stop codon, the disclosed sequences, so as to either eliminate or reduce
US 6,335,188 B1
7 8
the amount of sequence product. The procedures for anti There are also provided recombinant cells comprising the
sense inhibition for mRNA are described in US. Pat. No. nucleic acid molecules and/or proteins herein described.
5,554,743, Which patent is expressly incorporated by refer Proteins Which Would result from expression of the
ence into this application. Alternatively, the present inven nucleic acid molecules herein disclosed are preferred, With
tion could be used to design riboZymes Which speci?cally the proteins Which Would result from expression of the
cleave dmaW mRNA. exempli?ed nucleic acid molecules being most preferred. It
Also provided in the present invention are methods to is understood that proteins Which Would result from expres
express or overexpress the dmaW sequences described sion of allelic variants of the exempli?ed sequences, as Well
herein, and using the DmaW in pharmaceutical processes.
as proteins Which Would result from the expression of
Ergot alkaloids produced in fungal fermentation or chemi
cally modi?ed ergot alkaloids from fungal fermentation are 10 nucleic acid molecules Which hybridiZe under stringent
hybridiZation conditions to the nucleic acid molecules exem
Well knoWn pharmaceuticals. The dmaW gene can be intro
pli?ed are Within the scope of the present invention as Well.
duced into an ergot alkaloid-producing fungal strain, for
Lastly, an amino acid sequence substantially homologous to
example of C. purpurea, thus increasing the copy number
and potentially the expression of the DmaW protein. Utili a referent dmaW-encoded protein Will have at least 85%
Zation of a constitutive promoter such as for beta-tubulin 15 sequence identity, preferably 90%, and most preferably 95%
[Tsai et al., 22 Current Genetics 399 (1992)] may help sequence identity With the amino acid sequence of a referent
increase expression of the gene and, thus, of the protein. dmaW-encoded protein or a peptide thereof. For example, an
Because DmaW catalyZes the rate limiting step of ergot amino acid sequence is substantially homologous to dmaW
alkaloid synthesis in C. purpurea [Lee et al., 177 Archives of encoded protein if, When aligned With dmaW-encoded
Biochemistry & Biophysics 84 (1976)], its increased expres 20 protein, at least 85% of its amino acid residues are the same.
sion may increase ergot alkaloid production. The loW level SEQ ID NO 2 and SEQ ID NO 4 are the most preferred
of sequence identity betWeen dmaW from different genera proteins.
(for example betWeen dmaW sequences of Neotyphodium DmaW homologs can be the result of natural allelic
and Claviceps) Will reduce the problem knoWn as quenching variation or natural mutation. DmaW homologs of the
or cosuppression Which limits production of gene products 25 present invention can also be produced using techniques
in fungi and plants [Cogoni et al., 65 Antonie Van Leeu knoWn in the art including, but not limited to, direct modi
Wenhoek International Journal of Microbiology 205 (1994)]. ?cations to the protein or modi?cations to the gene encoding
Overexpression can be as skill of the art, in particular, the protein using, for example, classic or recombinant
according to the procedures described in US. Pat. No. nucleic acid techniques to effect random or targeted
5,477,001. mutagenesis.
30
Also provided in the present invention are methods to
identify Neotyphodium or Acremonium or Epichloe fungi EXAMPLE
that lack dmaW and, therefore, are unlikely to produce ergot
Example 1
alkaloids. The cloned Neotyphodium genes can be used for
standard Southern blot hybridiZation, by anyone skilled in 35 Obtaining dmaW sequence from N. coenophialum
the art, to screen these related fungi for the presence of
The folloWing degenerate primers Were used to amplify
homologous genes. In addition, degenerate primers can be
genomic DNA fragments from N. coenophialum by poly
used for polymerase chain reaction under conditions
merase chain reaction (PCR). A (+) means they correspond
described to amplify segments of dmaW from fungi in
family Clavicipitaceae or from Neotyphodium or Acremo in sequence to a portion of mRNA, thus read 5‘—3‘ on the
40
nium species. The ampli?ed segments can be analyZed by gene, and a (—) indicates they are reverse complements of a
gel electrophoresis and sequence analysis by anyone skilled portion of the mRNA and read 3‘—5‘ on the gene: thus, each
PCR involved a combination of a (+) and a (—) primer.
in the art. Fungi that lack DmaW can then be introduced into
grass plants and thereby incorporated into breeding lines as dmaWdeg(+)1: GAR CAR MGN YTN TGG TGG CA
described above. dmaWdeg(+)2: GGN AT H TTY AAR AAR CAY AT
45
Transformation of plant endophytes With these sequences dmaWdeg(-)3: AR NGT CCA NAR RTC YTC CAT
Would be according to knoWn procedures as described dmaWdeg(-)4: TA NAC YTG NGG YTC NGG CAT
above. Plants can be groWn according to knoWn procedures. N=A,G,C or T; Y=C or T; R=A or G; M=A or C; H=A,
Lastly, the present sequences are useful to identify related T or C.
sequences, such as those from Balansia, Balansiopsis, 50 Four PCR reactions Were performed to amplify fragments
Echinodothis, Atkinsonella, Myriogenospora, from each fungus, using four different primer combinations,
Neotyphodium, and Parepichloe, or natural or induced dmaWdeg(+)1 and dmaWdeg(-)3, dmaWdeg(+)1 and
mutants. For example, screening could be by Southern blot dmaWdeg(-)4, dmaWdeg(+)2 and dmaWdeg(-)3,
hybridiZation analysis of genomic DNA or by polymerase dmaWdeg(+)2 and dmaWdeg(-)4. Each 50 microL reaction
chain reaction using DNA oligonucleotide primers targeted 55 mixture contained 200ng fungal genomic DNA template,
to dmaW or the locus that contains it. 200 microM each deoxyribonucleotide triphosphate (dATP,
The present invention also provides isolated proteins dGTP, dTTP and dCTP), 25 mM each primer, 1 X PCR
encoded by a dmaW sequence, Wherein said proteins com buffer (Perkin-Elmer), and 2.5 units Taq DNA polymerase
prise an amino acid sequence selected from the group (AmpliTaq Gold from Perkin-Elmer). Reactions Were held
consisting of:
60 at 93 ° C. for 9 min and 95° C. for 3 min, then subjected to
(a) an amino acid sequence Which has more than 68% 35 cycles of the folloWing pro?le: 94° C. for 45 s, 53° C. for
identity to an amino acid sequence selected from the 45 s, 72° C. for 80 s in a Perkin-Elmer model 2400 Thermal
group consisting of SEQ ID NO 2 and SEQ ID NO 4 Cycler. After completing these temperature cycles the reac
and Wherein said identity can be determined using the tions Were incubated 5 min at 72° C., then analyZed by
DNAsis computer program and default parameters; and 65 agarose gel electrophoresis.
(b) an amino acid sequence Which is encoded by a nucleic The resulting ampli?ed genomic fragments Were cloned
acid sequence of claim 1. and used as probes of a cosmid library (and thus obtained