WO1998050056A1

WO1998050056A1 - Anti-fungal protein extracts from seeds of marigold

Info

Publication number: WO1998050056A1
Application number: PCT/US1998/009118
Authority: WO
Inventors: John A. Greaves; Friedhelm Brinkhaus; Steven J. Ziegenfuss
Original assignee: Kemin Industries, Inc.
Priority date: 1997-05-06
Filing date: 1998-05-05
Publication date: 1998-11-12
Also published as: AU7285098A

Abstract

The present invention describes the method of extraction and the effect of a crude protein extract from the seed tissue of the botanical species Tagetes, on the survival of two economically important fungi Penicillium oxalicum and Aspergillus ficum.

Description

ANTI-FUNGAL PROTEIN EXTRACTS FROM

SEEDS OF MARIGOLD

This application claims the benefit of Provisional Appl. 60/045,695, filed 05/06/98.

Background ofthe Invention

1. Field ofthe Invention

The present invention relates to a botanical extract of protein from marigold (Tagetes

sp.) that has anti-fungal activity. More specifically the present invention relates to proteins

having anti-fungal activity against fungi Penicillium sp. and Aspergillus sp. The present

invention describes the method of extraction and the effect of a crude protein extract from the

seed tissue of the botanical species on the survival of two economically important fungi

Penicillium oxalicum and Aspergillus ficum. The present invention includes a method for the

inhibition of molding in flour products such as bagels, breads, cakes and the like. The present

invention also includes use of genes that code for the proteins having the anti-fungal activity

that are transformed and expressed in grain products such that the resultant grain has anti-fungal

activity.

2. Background ofthe Prior Art

There are several synthetic chemicals such as organic acids which can be used to control

Penicillium sp. and Aspergillus sp. in feeds. However, these chemicals are not without there

own concerns on the health of animals and humans. Also, secondary plant products contained

in an essential oil fraction have been isolated from many botanical species, among them

Tagetes sp., and are known to exhibit strong antimicrobial as well as anti-fungal activities. Broekaert et al, 1996 demonstrated the use of a range of specific peptides derived from

botanical species belonging to the Brassicaceae, Compositae and Leguminosae families including Raphanus, Brassica, Sinapis, Arabidopsis, Dahlia, Cnicus, Lathyrus, Clitoria,

Amaranthus, Capsicum, Briza and related species on several fungal organisms and on gram-

positive bacteria. To our knowledge there are no documented examples of these natural, plant- derived proteins or peptides which have a significant biocidal effect on fungal organisms. In

addition, there are no reports on antimicrobial or anti-fungal proteins characterized in Tagetes

sp. The present invention describes novel proteins from botanical sources and their effect on

more important fungi. Summary ofthe Invention

The present invention describes the method of extraction and the effect of a crude

protein extract from the seed tissue of a botanical species on the survival of two economically

important fungi Penicillium sp. and Aspergillus sp. Both fungi are extremely common in

animal feed and production animals and are of grave concern in animal agriculture. Transmitted

through the food chain, both fungal organisms cause food spoilage and control of these

organisms is a public health issue. They also cause economic losses in production animal

agriculture through their effects on the gastro-intestinal tracts of monogastric animals such as

poultry and swine. The anti-fungal proteins are derived from the seeds of marigold (Tagetes

sp.).

The proteins extracted from the seeds of such botanical species can be used to control

Penicillium sp. and Aspergillus sp. in animal feeds and human foods. Furthermore, the proteins

and the genes that code for them can be sequenced and manipulated via genetic engineering techniques for expression in production microorganisms via fermentation. In addition, the genes can be expressed in the seeds of economically important crops such as maize, soybean,

sunflower, wheat, barley, sorghum, canola, sugarbeet, rice, cotton, and tobacco for inclusion of

such anti-fungal proteins in the downstream processing and use of these grains in animal feed

and human food. Production of proteins via genetic engineering of microbes and plants

followed by fermentation or agronomic production is now common place and established

practice. Overproduction of secondary plant products via pathway engineering, on the other

hand, is still technically a challenge. Thus there remains a need to meet this challenge.

Detailed Description ofthe Invention

The present invention relates to a crude protein extract, the purified protein(s) and the

gene(s) encoding for the protein that are extracted from marigold. The protein inhibits the activity of Penicillium sp. and Aspergillus sp. The protein extract also inhibits E. coil,

Salmonella typhimurium and Listeria monocytogenes.

The present invention is protein(s), and the gene encoding such proteins, which inhibit

the growth ofthe fungal organisms. These proteins are naturally occurring in the seeds of

marigold. These proteins can be used in the raw form, as a crude protein extract, and applied to

animal feed or human foodstuff (particularly those foodstuff that are high in Penicillium

oxalicum and Aspergillus ficum). The proteins can be purified and applied to feed for animals

and foodstuff for humans. Alternatively, the protein or the extract can be applied directly to

places in which fungal organisms multiply.

The genes encoding for these proteins can be transformed into bacterium, fungi, yeasts,

plants or mammals (prokaryotic or eukaryotic cells) and the protein can be harvested and applied directly to the material harboring fungi. Additionally, the proteins can be produced

within the cells ofthe material that carries the fungus. For example, the protein can be produced in flour products. Flour products include rice flour and corn flour and wheat flour and oats flour

and rye flour and the like. One ofthe primary bread molding organism is Penicillium sp. Since the crude extracted protein is heat stable it could be added directly into the batter ofthe food

product to inhibit the growth ofthe fungi in the resultant food product. Alternatively the

purified protein can be added or the grain from which the flour was made can express the

protein. The protein can also be produced in the grains of material in animal foods. In chicken feed the protein can be expressed in the petals or seeds ofthe marigold flower and supplied as

marigold meal (that is supplied for its lutein) as a feed additive. A number of cereal grains

could be transformed so that the protein is expressing in the seed which is harvested.

Alternatively, the gene product could be expressed in the green portion ofthe plant. This allows

less fungal activity in material that is ensilaged or dried and fed to the animals such as forage

materials. The crude extract or the purified protein(s) can be supplied into silage inoculant that

is employed in the ensilaging process. The transgenic corn, soybean , canola, peanut, oat,

wheat, barley, rice seed can have the protein expressed therein and thus when the grain is

prepared for animal feed or for preparation of human food products the protein is released from

the seed and is active in decreasing the fungi present in the grain, food or feed product.

Our environment is full of fungi that may cause allergic diseases. Mainly agricultural

workers have suffered form allergic diseases ofthe respiratory tract which are caused by the

spores of genera Penicillium, Aspergillus which are present in the open air. Thus the gene

product expressed in the green plant material and the grain and the protein extract applied to grain and forage materials will provide less spores and thus less allergic reactions in humans.

There may be other pharmaceutical uses of the present invention.

Transgenic corn, soybean , canola, peanut, oat , wheat, barley, rice, and other

transformable fruits and vegetables and plants can have the protein expressed in tissue other

then the seed and thus when the fruit, vegetable or other plant tissue or forage is prepared for

feeding the fungal activity is decreased. This is particularly useful for the preparation of silage

from corn and sorghum.

Additionally, the present invention has usefulness in the pharmaceutical field and the

fields of veterinary science. Clearly if the fungal which are present in such items as grains and

flours and can produce toxins such as aflatoxins in animals the mammal can ingest the protein to decrease the fungi present.

The following methods of protein extract ofthe anti-fungal material is described.

The crude protein extract of the present invention is run on a protein gel to separate the

proteins in the extract. The proteins are then purified and each protein is tested against the

listed fungi for the inhibition effects. The protein and or proteins with the most inhibiting

effects are sequenced by known sequencing methods. The methods to do this type of

experiment and the procedures involving gene sequencing and vector production are clearly

outlined in Current Protocols in Molecular Biology published by John Wiley and Sons, New

York (1995). This manual or the short protocols of this manual can be found in most

biotechnology labs and in libraries.

Transformation Methods - are means for integrating new genetic coding sequences into

the target organism's genome by the incorporation of these sequences into an organism through man's assistance.

Using the sequence of the purified protein the DNA encoding for such protein could be isolated and usedj via known procedures, to transform a suitable host organism such that the

protein is produced by the recombinant host in commercially useful amounts. The protein encoding DNA could be reversed engineered using codons that are acceptable and recognizable

to the host. Or the gene could be isolated by screening nucleic acid libraries of species which

produce the protein. Oligonucleotide probes that are complementary to a polynucleotide encoding a portion of the protein, for examples a N-terminus sequence can be employed to

locate the gene. Probes can be employed, in a known manner, to screen a genomic or cDNA

encoding for library or to synthesis polymerase chain reaction (PCR) probes for the

amplification ofthe cDNA encoding for isolated from an RNA which translated into the protein

of the present invention. Such cDNA could then be cloned into a suitable expression vector for

the selected host and transformed into a host organism. One of the typical host organisms is E.

coil. Thus the host would have to be selected to be capable of producing the protein without the

protein harming the host.

The vector for transformation would preferably comprise a nucleotide sequence that

corresponds to the protein amino acid sequence that maybe optimized for the selected host. The

vector would also be designed with regard to codon selection, the initiation of translation, the

promoter and the targeting sequences if needed to maximize the expression of recoverable

amounts of the protein. Vectors for hosts such as plants, algae, insect, animal, yeast, fungi and

fungal and humans are commercially available from companies such as Novagon, and a number

of other sources. Vectors for different hosts are described in the in Current Protocols In Molecular Biology and in a number of commercial catalogs.

There are a large number of known methods to transform plants. However, certain types

of plants are more amenable to transformation than are others. Tobacco is a readily

transformable plant and its transformation is well published. Most dicots can be transformed by

the methods used to transform tobacco. Monocots are transformed by different methods which are also widely published and thus the basic steps of transforming plants including monocots

are known in the art.

The steps for transformation of a monocot such as maize are concisely outlined in U~S.

patent number 5,484,956 "Fertile Transgenic Zea mays Plants Comprising Heterologous DNA Encoding Bacillus Thuringiensis Endotoxin" issued January 16, 1996 and in U.S. patent

number 5,489,520 "Process of Producing Fertile Zea mays Plants and Progeny Comprising a

Gene Encoding Phosphinothricin Acetyl Transferase" issued February 6, 1996.

Plant cells such as maize can be transformed by a number of different techniques. Some

of these techniques which have been reported on and are known in the art include maize pollen

transformation (See University of Toledo 1993 U.S. Patent No. 5,177,010); Biolistic gun

technology (See U.S. Patent No. 5,484,956); Whisker technology (See U.S. Patents No.

5,464,765 and 5,302,253); Electroporation; PEG on Maize; Agrobacterium (See 1996 article on

transformation of maize cells in Nature Biotechnology, Volume 14, June 1996) along with

numerous other methods which may have slightly lower efficiency rates than those listed.

Some of these methods require specific types of cells and other methods can be

practiced on any number of cell types. The use of pollen, cotyledons, meristems and ovum as

the target issue can eliminate the need for extensive tissue culture work. However, the present state ofthe technology does not provide very efficient use of some of this material.

Generally, cells derived from meristematic tissue are useful for transformation as they -ire very regenerable. Zygotic embryos can also be used. The method of transformation of

meristematic cells of cereal is taught in the PCT application W096/04392. Any of the various cell lines, tissues, plants and plant parts can and have been transformed by those having

knowledge in the art. Methods of preparing callus from various plants are well known in the art

and specific methods are detailed in patents and references used by those skilled in the art.

Cultures can be initiated from most of the above identified tissue. The only true

requirement ofthe transforming material is that it can form a transformed plant. The DNA used for transformation of these plants clearly may be circular, linear, double

or single stranded. Usually, the DNA is in the form of a plasmid. The plasmid usually contains

regulatory and/or targeting sequences which assists the expression of the gene in the plant. The

methods of forming plasmids for transformation are known in the art. Plasmid components can

include such items as: leader sequences, transit polypeptides, promoters, terminators, genes,

multiple gene copies can be used, introns, marker genes, etc. The structures of the gene

orientations can be sense, antisense, partial antisense, or partial sense.

The regulatory promoters employed can be constitutive such as CaMv35S (usually for

dicots) and polyubiquitin for monocots or tissue specific promoters such as CAB promoters,

etc. The prior art includes but is not limited to octopine synthase, nopaline synthase, CaMvl9S,

mannopine synthase promoters. These regulatory sequences can be combined with introns,

terminators, enhancers, leader sequences and the like in the material used for transformation.

The isolated DNA is then transformed into the plant. The improvements in transformation technology are beginning to eliminate the need to regenerate plants from cells.

Since 1986, the transformation of pollen has been published and recently the transformation of

plant meristems have been published. The transformation of ovum, pollen, and seedlings meristem greatly reduce the difficulties associated with cell regeneration of different plants or

genotypes within a plant. Duncan, from at least 1985-1988 produced literature on plant

regeneration from callus. Somatic embryogenesis has been performed on various maize tissue

which was once considered unusable for this purpose. The prior art clearly teaches the

regeneration of plants from various monocot and dicot tissues.

The most common method of transformation is referred to as gunning or microprojectile

bombardment. This Biolistic process has small gold coated particles coated with DNA shot into the transformable material. Techniques for gunning DNA into cells, tissue, callus,

embryos, and the like are well known in the prior art.

After the transformation of the plant material is complete, the next step is identifying

the cells or material which has been transformed. In some cases, a screenable marker is

employed such as the beta-glucuronidase gene of the uidA locus of E. coli. Then, the

transformed cells expressing the colored protein are selected for either regeneration or further

use. In many cases, the transformed material is identified by a selectable marker. The putatively

transformed material is exposed to a toxic agent at varying concentrations. The cells which are

not transformed with the selectable marker that provides resistance to this toxic agent die. Cells

or tissues containing the resistant selectable marker generally proliferate. It has been noted that

although selectable markers protect the cells from some of the toxic affects of the herbicide or

antibiotic, the cells may still be slightly effected by the toxic agent by having slower growth rates. If the transformed material was cellular then these cells are regenerated into plants. The

plants from either the transformation process or the regeneration process or crossed to either

such plants or a progeny of such plants are transgenic plants.

The protein was extracted from the plant seeds via the following process.

EXAMPLE 1: Preparation of protein extract from marigold seeds.

Aqueous extraction was followed by ammonium sulfate precipitation in the interval of

30 to 70% relative saturation, heat precipitation at 800° C and dialysis for 3 days. The detailed

methods are described below.

Five hundred grams of marigold seeds were ground in a coffee mill and extracted for 2

hr in 2 1 of extraction buffer at 4° C. The extraction buffer contained 10.0 mM NaH₂PO , 15.0

mM Na₂HP0 , 100.0 mM KC1, 2.0 mM EDTA (ethylenediaminetetraacetic acid) disodium salt,

2.0 mM Thiourea and 1.0 mM PMSF (phenylmethylsulfonyl fluoride) (dissolved in MeOH). The homogenate was squeezed through cheesecloth and centrifuged for 30 min. at 7000 x g.

The pellet was discarded and the supernatant was retained. The supernatant was brought to 30%

relative saturation with ammonium sulfate and precipitated overnight. The precipitate was

removed by centrifugation at 7000 x g for 30 minutes. The supernatant was adjusted to 70%

collected by centrifugation at 7000 x g for 30 min. The pellet was re-suspended in 400 ml water

and heat precipitated in water bath for 30 minutes at 80° C. The precipitate was collected by

centrifugation at 7000 x g and the supernatant was dialyzed against distilled water for 3 days

using cellulose ester dialysis tubing with a molecular weight cut-off of 1000 daltons. The water

was changed 3 times on day 1, and once each day for days 2 and 3. The dialyzed supernatant was then frozen to -60 C and freeze-dried.

EXAMPLE 2: Anti-fungal activity assay. This method was repeated for the marigold extracts. A spectrophotometrical method was used where the optical density of fungal cultures was determined when grown in microtiter

plates.

For appropriate dilution ofthe protein extracts from marigold, 0.1 gram ofthe protein

extract was blended with 10.0 grams dextrose to achieve a homogenous, free-flowing mixture.

One gram ofthe protein extract/dextrose preparations were then dissolved in 2 ml of saline

solution and serially diluted in sterile saline. Twenty μl ofthe appropriately diluted solution were then added to the inoculated wells ofthe microtiter plate to achieve 25 ppm, 250 ppm, or

2500 ppm treatment rates.

One hundred μl of a 10² spore suspension in a 0.89% w/v sterile saline solution of either

the Penicillium or the Aspergillus organisms was added to 0.1 ml of potato dextrose broth at

half strength (commercially available from Difco in Michigan) formed by adding 12 grams of

potato dextrose broth (PDB) per liter of H₂0. Thus the 0.1 ml of the spore suspension and the

0.1 ml PDB was added to the wells of a microtiter plate. Twenty four wells per organism per

treatment rate were used and 24 further wells were used as a control series. For the controls the

half strength PDB (made as above) and the 0.1 ml of spore suspension without the anti-fungal

extract protein extract were added to the wells.

The microtiter plates were then incubated 48 hours at 28° C and the optical density of

the wells determined spectrophotometrically at 405 or 410 nm. As a blank sterile half strength

PDB was used. EXAMPLE 3 : Anti-fungal activity ofthe protein extracts from marigold. The anti-fungal potency ofthe protein extracts from marigold was evaluated against two

fungal organisms Penicillium and the Apergillus. Potency was measured as described in Example 2. The two fungal organisms were tested at three different treatment levels, 25 ppm,

250 ppm and 2500 ppm and each treatment level was replicated 24 times. After 48 hr

incubation, growth of fungi was measured as optical density. The results in percentage ofthe

optical density ofthe control as mean values for the 24 replicates at the 25 ppm and 2500 ppm

treatment levels are presented in Tables 1 and 2. The student's two tailed t-test was conducted

to test for statistical significance with P < 0.01 indicating statistical significance.

Table 1 : Inhibition of Penicillium by 25 ppm of protein extracts from marigold of control.

Pen=Penicillium, Mar=Marigold extract

Table 2: Inhibition of Apergillus by 2500 ppm of protein extracts from marigold of control.

Asp=AspergiIlus, Mar=Marigold extract

The level of crude protein extracts to acheive a maximum inhibition effect under the

conditions of the experimental protocal was observed at 25 ppm for Penicillium sp. with in excess of 80% inhibition and at 2500 ppm for Aspergillus sp. acheiving near 80 % inhibition.

The results ofthe fungal study clearly show that there is a dose response to three ofthe

treatments with significant inhibition.

EXAMPLE 4: Isolation of fungal inhibiting protein from marigolds.

Marigolds produce the desired proteins in the seed ofthe plant. The present experiment

is adapted to increase the production of that protein. The inhibition ofthe fungal by the protein

can be improved by the introduction ofthe gene that encodes for the desired protein into marigold. Additionally, the protein can be accumulated in larger seeds such as the tobacco seed.

A plasmid adapted form a plasmid such as pATCC1616, ATCC accession No. 40806, can have

the phytoene dehydrogenase-4H encoding gene removed and the selected gene inserted by

known restriction site technology. Alternatively, other starting plasmid material can be

purchased for this purpose.. The plasmid is characterized by the ability to be maintained in

Agrobacteruim tumefaciens, which is used to infect the tobacco or a number of other dicots.

The plasmid also has the right and left borders ofthe sequence ofthe T-DNA, and a promoter

associated with the Kanamycin resistance gene in the presence of that antibiotic. The plasmid is

transformed into Agrobacterium tumefaciens strain LBA4404 (CLONTECH, Inc.) according to

standard protocols. The tobacco leaf disc are transformed with agrobacteruim using the method

of Horsch etal., Science, 227:1229-1231(1985). The selectable marker gene gives resistance to

the herbicide norfiurazon (Sandoz; 0.8 micrograms per milliliter). The plant cells that are

transformed do not die in the present ofthe herbicide. The transgenic plants are grown from the transformed cells and the seeds are harvested. Then five hundred grams ofthe tobacco seed

are ground in a coffee mill and extracted according to the procedure above The plasmid used for transformation can be improved by the use of a promoter that targets the seed. Examples of

seed promoters are known in the art are the maize zein storage promoter. Alternatively, the protein can be produced constitutively through out the plant with the use of the 35S CaMV

promoter for example.

A similar transformation of yeast or bacterial hosts can be performed. The starting

plasmid would include a promoter recognized by the host and the selectable marker is often an

antibiotic. The protein product is lysed from the cell. To produce large quantities a fermentation process for producing the protein can be used.

Although the invention has been described with respect to a preferred

embodiment thereof, it is to be also understood that it is not to be so limited since changes and

modifications can be made therein which are within the full intended scope of this invention as

defined by the appended claims.

Claims

We claim:

1. A protein extracted from Tagetes sp. which inhibits fungi.

2. A protein extract as defined in claim 1 which inhibits fungi from the group including

Penicillium sp. and Aspergillus sp.

3. A protein extract as defined in claim 1 which inhibits fungi of species Penicillium oxalicum.

4. A protein extract as defined in claim 1 which inhibits fungi of Aspergillus ficum.

5. An animal feed composition having anti-fungal activity, comprising a feed to which the

protein extract of claim 1 is added in amounts effective to reduce populations of fungi in the

animal.

6. A method of reducing populations of fungi in an animal, comprising the steps of adding

the protein extract of claim 1 to a feed and orally administering the treated feed to the animal to

in an amount effective to reduce the populations of fungi in the animal.

7. A method as defined in claim 6, wherein the protein extract is added in an amount of between about 0.01 ppm and about 10,000 ppm by weight ofthe untreated feed.

8. A grain for use in feed, comprising grain produced on a plant which has been transformed

to express the protein of claim 1 in the grain.

9. An animal feed composition having anti-fungal activity, comprising the grain of claim 8.

10. A grain as defined in claim 8, wherein said plant is selected from the group including

maize, soybean, sunflower, wheat, barley, sorghum, canola, sugarbeet, rice, cotton, and tobacco.

11. A method of producing a protein which inhibits fungi, comprising transformation of a microorganism to express the protein extract of claim 1 , growing the microorganism and

recovering the protein extract produced by the microorganism.

12. The method of claim 12, wherein the microorganism is selected from the group including

bacteria, yeasts and fungi.