WO1998031824A1

WO1998031824A1 - Fluorescent dyes from microorganisms

Info

Publication number: WO1998031824A1
Application number: PCT/AU1998/000023
Authority: WO
Inventors: Philip Bell; Jian Shen
Original assignee: Macquarie Research Ltd
Current assignee: Access Macquarie Ltd
Priority date: 1997-01-21
Filing date: 1998-01-20
Publication date: 1998-07-23
Anticipated expiration: 1999-07-21
Also published as: AUPO470197A0

Abstract

A compound produced by a fungal species, preferably an Epicoccum species, suitable for use as a fluorescent marker for a biomolecule, capable of binding to a biomolecule, and which emits fluorescence in red wavelengths after excitation at blue wavelengths when bound to a biomolecule.

Description

Fluorescent dyes from microorganisms

Technical Field

This invention relates generally to compounds produced by microorganisms which can function as fluorescent dyes, methods of producing the compounds, and uses of compounds in scientific applications.

Background Art

Compounds that fluoresce are essential in many scientific applications. One (amongst many) of these scientific applications is the technique of flow cytometry. For many applications, however, there is a lack of suitable fluorescent dyes. In particular there is a lack of dyes which fluoresce in longer (red) wavelengths when excited with blue light such as the 488 nm laser commonly used in cytometry.

The argon-ion laser is the excitation source in many flow cytometers and confocal laser scanning microscopes, as well as in certain laser scanners. In these instruments, the wavelengths used to excite green, yellow, orange and red fluorescent dyes are limited primarily to the laser^'s 488 nm and 514 nm spectral lines, which severely restricts simultaneous multicoloured detection. For example, when excited at 488 nm or 514 nm. the commonly used Texas Red® fluorophore has a particularly low fluorescence output that is easily obscured by the more intense fluorescein fluorescence in a double- labelling reaction. For these applications it would be useful to have a red fluorescent dye with strong absorption with the spectral lines of the argon- ion laser.

Red fluorescent dyes (each with their own disadvantages) are used extensively in many fields of biological study. Most of these, such as Texas

Red. Tetramethylrhodamine-isothiocyanate or red emitting BODIPY dyes require excitation at green wavelengths such as 542 nm. This limits their utility for flow cytometry as most cytometers are only capable of excitation at blue wavelengths (488 nm). Phycobilloprotein. phycoerythrin, can be excited at 488 nm and does emit in the red wavelengths. This compound, however, has poor stability and a high molecular weight making it unsuitable for cell tracking or labelling of nucleic acid probes.

Microorganisms, and particularly some fungi, are known to produce pigmented or coloured compounds. Most of the coloured compounds produced by microorganisms do not fluoresce and are not suitable as fluorescent dyes in scientific applications. Many coloured compounds produced by fungi have been used as food dyes or as antimicrobial or antitumour agents. For example, the fungus Monascus purpurem is well known for producing a variety of coloured compounds including orange (Monascorubin and rubropunctatin) and purple (monascorubramine and rubropunctamine).

Strains of the yeast genus Saccharomyces are used in some of the largest and oldest biotechnology industries, including baking, brewing, distilling and wine making. Improvements in the performance of the yeast strains used in these processes has come about as a result of the development of yeast with novel genotypes. The methods for producing these improved genotypes include genetic engineering, protoplast fusion, and mutation/selection techniques. However, in many situations, traditional techniques involving mating followed by selection are still effective for strain improvement. In these techniques, spores derived from parental strains of Saccharomyces are isolated, germinated and allowed to mate. The hybrids produced from these matings can be screened to identify novel strains that combine desirable traits. For example in the baking industry, some recent patents describe the development of yeast with improved capacity to leaven a variety of doughs. If parent strains, haploids and hybrids are each capable of growth on the same media, the separation of hybrids from both parents and haploids can be difficult. In laboratory studies of yeast genetics, when two haploid yeast strains with complementary genetic markers and opposite mating types are mixed, they mate, and the hybrids formed can be identified by growth on selective media. Alternatively, hybrids can be physically isolated with a micro-manipulator.

In general, industrial strains of yeast used for baking or brewing lack selectable genetic markers, making identification of hybrids by genetic complementation impossible. Genetic markers can be introduced with mutation into industrial strains, however this is difficult due to polyploidy and undesirable due to possible effects on industrial performance. Furthermore, many industrial strains sporulate at low frequency, and a high proportion of the spores produced are not viable. Due to these problems it is difficult to produce and isolate the large number of new strains required to identify industrial yeast with overall improved characteristics. One method used to overcome this problem has been the introduction of antibiotic resistance markers into yeast to allow the identification of hybrid strains. However, this approach is limited by the small range of suitable markers and the laboriousness of the procedures. In addition the presence of antibiotic resistance markers in industrial yeast is not considered desirable because the product is released live into the environment. There is a need for non- genetic markers suitable to track or select new strains of microorganisms from mating experiments for example.

The present inventors have produced a new compound derived from fungi that is particularly suitable for use as a non-genetic marker in scientific applications.

Disclosure of Invention

In a first aspect, the present invention consists in a compound produced by a fungal species, the compound binds to biomolecules and is suitable for use as a fluorescent dye for biomolecules. Preferably, the compound produced by the fungal species when bound to a biomolecule emits fluorescence after excitation at blue wavelengths, preferably emitting in the red wavelengths.

In a further preferred embodiment of the first aspect of the present invention, the fungal species is an Epicoccum species, preferably Epicoccum nigrum.

In a still further preferred embodiment of the first aspect of the present invention, the blue wavelength is 488 nm.

The binding of the compound to biomolecules may involve direct chemical or physical binding or may be achieved by the use of "linking molecules" known to the art. Biomolecules which may be bound by the compound of the invention when used as a fluorescent dye include, for example, proteins, peptides. sugars, nucleic acids, antibodies, cell surface biomolecules. and cells. Due to the compound^'s fluorescent and biomolecule-binding characteristics, it will be appreciated that the compound will have use in any application where detection of a fluorescent dye attached to a biomolecule is required.

In a second aspect, the present invention consists in a process for producing a compound according to the first aspect of the present invention comprising culturing a fungal species under conditions such that the fungal species produces the compound; and separating the compound from the culture. In a preferred embodiment of the second aspect of the present invention, the fungal species is cultured in the presence of a yeast such that the fungal species produces the compound.

The fungal species is preferably an Epicoccum species, more preferably Epicoccum nigrum, and most preferably Epicoccum nigrum PBl. The present inventors have deposited, under the provisions of the Budapest Treaty, a sample of Epicoccum nigrum PBl that produces a compound according to the present invention. The deposit was made at the Australian Government Analytical Laboratories (AGAL) on 15 January 1998 and was given the Accession Number NM98/00507.

Although the present inventors have isolated and deposited a sample of Epicoccum nigrum PBl that produces a compound suitable for use as a fluorescent dye for biomolecules, it will be appreciated that other fungal species and strains may also produce the same or other compounds suitable for use as fluorescent dyes or markers for biomolecules. Fungi are known to produce coloured pigments and dyes but these known compounds do not fluoresce under ultraviolet light. Epicoccum nigrum PBl is believed by the present inventors to be the first example of a fungal strain that produces a compound suitable as a fluorescent dye for biomolecules. The present inventors have isolated other strains of Epicoccum nigrum and all of these strains produced a similar compound to that of Epicoccum nigrum PBl. The yeast is preferably a Saccharomyces species, more preferably Saccharomyces cerevisiae. A strain that has been found by the present inventors to be particularly suitable is the commercially available strain NCYC 996

The compound can be used as a fluorescent dye when in a crude culture extract or when purified by extraction and separation techniques. It will be appreciated that after the compound is produced by the fungus during culture it can be obtained in substantially pure form by a standard purification technique.

It has been found by the present inventors that during co-culture on semi-solid media of the fungi Epicoccum nigrum and the yeast Saccharomyces cerevisiae, a compound which is suitable as a fluorescent dye is produced by the fungi. This compound becomes fluorescent whereupon it fluoresces in the red wavelengths during excitation in the blue wavelengths after binding to cells. There are several explanations for the production of this new compound by the microorganism. For example, it is possible that the fungi produces a compound that is modified by the presence of yeast cells, or some component produced by the yeast cells, during culture to form a compound that will fluoresce during excitation with blue wavelengths. It may be that a component produced by the yeast cells influences the fungi to produce the compound in its final form. Alternatively, during co-culture there may be alteration in. or depletion of, one or more nutrients by the yeast that causes the fungi to produce the compound. There may even be a combination of some or all of the above occurrences that causes the production of this new compound. It will be appreciated, however, that it should be possible to cause fungi to produce the same or other new compounds which have fluorescent characteristics by modifying cultural conditions of the fungi. This may be achieved by co-culturing in the presence with other microorganisms or by manipulation of nutrients or environmental conditions in the culture.

It will also be appreciated that it should be possible to produce compounds according to the first aspect of the present invention by using techniques other than the influence of yeast cells in culture. For example, if the fungi produces a "pre-cursor", then it will be possible to modify that precursor to its fluorescent form by chemical, physical or enzymatic means. The knowledge that such compounds can be obtained from microorganisms should allow the discovery and production of other compounds suitable for use as fluorescent dyes belonging to the same family or quite distinct compounds with useful characteristics. The compound may also be produced synthetically by direct chemical synthesis, or by modification of intermediate(s) in the biosynthetic pathway used by the fungi.

The compound according to the present invention may also be produced synthetically by chemical means. The knowledge that a new fluorescent compound is produced by fungi may lead to other means of producing the compound apart from culturing the fungi under the required conditions.

The compound according to the present invention has the distinct advantage that it binds to cells and other biomolecules in its fluorescent form so can be used as a means to track the cells or the other biomolecules when labelled with the compound. In a third aspect, the present invention consists in use of the compound according to the first aspect of the present invention as a fluorescent dye in scientific techniques for staining, labelling and/or detecting biomolecules. Examples of the use of the compound according to the first aspect of the present invention include cell tracking dyes for microscopy, membrane fluidity dyes, conjugation with antibodies, conjugation to nucleic acids, cell surface ligand imaging dyes, conjugation to sugars, cytometric analysis, and confocal microscopy. It will be appreciated, however, that the compound would be suitable for any use where fluorescence in the red wavelengths is required, particularly when excited at 488 nm.

In a fourth aspect, the present invention consists in an improved method of fluorescent-labelling a biomolecule comprising causing the compound according to the first aspect of the present invention to bind to the biomolecule such that the biomolecule is fluorescently labelled with the compound.

The biomolecule may include, but not limited to. proteins, peptides, sugars, nucleic acids, antibodies, cell surface biomolecules. and cells. The compound may bind directly to the biomolecule due to a chemical or physical association or may bind to the biomolecule via a linking molecule. If the compound is attached to a ligand specific for the biomolecule. for example an antibody or lectin, then the binding of that ligand to the biomolecule will cause the biomolecule to be fluorescently labelled. In a fifth aspect, the present invention consists in a method of detecting a biomolecule in a sample comprising labelling the biomolecule according to the method of the fourth aspect of the present invention: and detecting the biomolecule in the sample by monitoring or detecting its fluorescence.

The monitoring or detecting of the fluorescence of the labelled- biomolecule may be by any means known to the art. Such means include, but not limited to. microscopy and cytometry.

The compound according to the first aspect of the present invention has several advantages over currently used fluorescent dyes presently available. The compound has been called Beljian Red (BR) by the present inventors and has the following characteristics:

• is water soluble • can be stored at temperatures at or below 4°C

• is easy to use as a cell stain

• is relatively stable in light

• the fluorescent form has wide range of absorbances • has strong fluorescent emission after staining but a low fluorescent emission when free in solution. This ensures low background fluorescence levels. The compound has been found by the present inventors to stain fungal, bacterial and mammalian cells

• stained cells do not substantially leak the fluorescent label to neighbouring cells thus permitting their fate to be followed (i.e. as a cell tracking dye)

• the Stokes shift of cells fluorescently labelled with this dye is such that flow cytometers can detect the emission in a separate channel from green- emitting dyes (e.g. fluorescein) making the fluorescent labelling conferred by the dye ideal for two-colour fluorescence imaging in conjunction with fluorescein.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

In order that the present invention may be more clearly understood, preferred forms will be described with reference to the following examples and accompanying drawings. Brief Description of Drawings

Figure 1 shows a NMR spectrum of Beljian Red. a fluorescent compound according to the first aspect of the present invention: peak 1 is the compound showing benzene ring structures indicating the compound is aromatic, peak 2 is water, and peak 3 is DMSO solvent peak: Figure 2 shows the separation of cells stained using Beljian Red. a fluorescent compound according to the present invention:

Figure 3 shows results of mating of haploid strains of S. cerevisiae. A) Strain SMC19-A stained with CellTracker Green BODIPY: B) Strain X stained with Beljian Red: C) 5 min after the mixing of two strains together under conditions suitable for mating: D) After 16 hours the gated region was used to sort mated cells. All values are in arbitrary units: Figure 4 shows gating region used for isolation of small clusters of mating cells. Linear gains were used for both SSC (Side scatter) and FSC (Forward scatter) parameters to provide sufficient resolution for size discrimination. Values are in arbitrary units; and Figure 5 shows results of rare mating of an industrial polyploid strain with a laboratory haploid strain of S. cerevisiae. Strain X (haploid) stained with Beljian Red and strain Nl (polyploid) stained with CellTracker Green BODIPY. were mixed under conditions suitable for mating. After 16 hours cells from the gated region were sorted twice to enrich for rare mated hybrids. All values are in arbitrary units. Modes for Carrying Out the Invention MATERIALS AND METHODS Beljian Red Dye

Production of dye. Method included the steps of: Streak mixed culture of Saccharomyces cerevisiae NCYC 996 and

Epicoccum nigrum PBl onto semi-solid GYPAgar (Glucose 2% w/v. Peptone 2% w/v. Yeast Extract 1% w/v Agar 2% w/v)..

Streaks should be ideally 1 cm apart and parallel or arranged in a cross formation. Incubate culture at 20°C for several days (3 - 5) until Epicoccum nigrum fungal filaments are approximately 3-4 mm long and start secreting a pale yellow compound.

Transfer culture to 4°C incubator.

Incubate until agar turns bright red. Cut out agar containing high concentration of red dye.

Pool agar containing high concentrations of red dye.

Compress agar until dye containing liquid is extruded from the gel structure.

Centrifuge red liquid for 10 minutes to remove insoluble components. Transfer red coloured supernatant (crude extract) into storage tubes.

This raw extract can be either used directly as a stain or purified to homogeneity.

Cell staining. Add fluorescent dye to cells and incubate at room temperature for 30 minutes. The cells will now be suitable for washing and will be stained by the dye. Purification. Dye extracts from GYP Agar (crude dye extract) were made up to 30% acetonitrile. After centrifugation to remove particles, the extract was loaded onto a S75 Superdex size exclusion column (Pharmacia) and separated using 30% Acetonitrile. 0.116% v/v Ammonia 880. 0.116% v/v trifluoroacetic acid (TFA) in Milli Q water running buffer.

After collecting fractions, the fraction with the highest 508 nm absorbance was selected and the running buffer in the fraction evaporated. The fraction was then further purified on a reversed phase HPLC C18 column using 85% acetonitrile. 0.045% TFA in Milli Q water. The fraction with maximum 508 nm absorbance was selected and further purified on a Mono Q ion-exchange column.

Identification of the fluorescent dye was by NMR and GC/MS. Preliminary NMR results indicate a largely aromatic molecule (Figure 1). Data from NMR and purification show that the compound is largely aromatic and of molecular mass of a few hundred Dalton. The compound is hydrophobic but water soluble and. therefore, probably charged.

The compound, after purification, had an absorbance maximum at 508 nm and a second peak absorbance at 386 nm.

This compound becomes fluorescent after contact with and possibly modification by the bakers yeast, Saccharomyces cerevisiae. The compound is easy to produce, easy to use. and stable to freeze/thaw and storage. The fluorescent form that forms in contact with yeast emits in the longer orange to red wavelengths. Uses of Dye Strains and culture conditions. Studies were performed using three strains of Saccharomyces cerevisiae: X [MATa trpl hisl MAL6T::lαcZ). SMC19-A {MATα MAL2-8^c MAL3 leul SUC3). and an industrial baking strain Nl (Burns Philp Technology and Research Centre culture collection). Yeast strains were grown on a variety of media. Rich medium was composed of. per litre: 20 g glucose (Oxoid. Sydney. Australia): 5g yeast extract (Oxoid): 10 g peptone (Oxoid) and 3 g KH2PO4 (Sigma- Aldrich. Sydney. Australia). 20 g agar (Oxoid). Two types of minimal media were used: maltose minimal indicator medium was composed of 20 g maltose. 6 g Na₂HPO₄ . 6g KH₂PO . 20 g agar. 6.7 g yeast nitrogen base (YNB. Difco. Sydney. Australia) and X-β- Gal at 40 μg per litre: glucose minimal indicator medium was composed of 20 g glucose. 20 g agar. 6.7 g YNB without amino acids supplemented with CSM (Bio. 101 Inc.. Vista. CA) minus histidine. leucine and tryptophan (as per manufacturers instructions) per litre. Cultures were prepared for mating by growth for 18 h in 5 ml rich medium in capped 50 ml centrifuge tubes at 30°C with shaking at 200 rpm. After this culture period cells were washed and resuspended in rich medium to a density of approximately 10⁸/ml.

Staining with Cell-Tracker dyes. Cell Tracker (CT) probes were obtained from Molecular Probes (Molecular Probes Inc.. OR USA). Stock solutions of CT-Green (CMFDA). CT-Green BODIPY. CT-Orange. CT-Yellow- Green and CT-SNARF were made up to 10 inM with DMSO from a freshly opened flame sealed ampoule (99.9 atom % DMSO. Sigma. Sydney). Dye stocks were stored frozen at -50°C in single use aliquots and sealed from moisture or light. After defrosting, any unused dye from each aliquot was discarded. Staining for flow cytometry was performed in 1 ml final reaction volumes consisting of 25 μl of a suspension of overnight yeast culture added to 975 μl YNB. Thus, during staining the cell density was approximately 10⁷ yeast cells/ml. For staining, cells were typically incubated at 30°C for 45 min. in darkness with a working dye concentration of 10 μM. This staining concentration was determined after staining using working dye concentrations ranging from 0.5 μM to 25 μM (data not shown). Unbound dye was removed by centrifugally washing (pelleting for 1 min. at 12.000 x g. removal of accessible supernatant with pipette, re-suspension in 1 ml YNB) three times. To allow time for slow leakage of unbound dye. cells were further incubated for 30 min. at 30°C in YNB in new microfuge tubes followed by three further centrifugal washes. Staining with PKH-26. The PKH-26 (Sigma. Sydney) was stored and used to stain cells as described in the manufacturers instructions. Dye concentrations ranging from 2xl0^"6 to 8xl0^"6 molar were tested.

Staining with Beljian Red. Aqueous dye stocks of the dye according to the present invention (BR) were prepared. Staining reactions were prepared in 150 μl final reaction volumes by pelleting 25 μl cells from overnight yeast cultures and resuspending in 50 μl YNB followed by addition of 100 μl BR stock. Thus, during staining the cell density was approximately 10⁸ yeast cells/ml. Staining reactions were incubated at room temperature (21 - 25°C) for 30 min. Unbound dye was removed by centrifugal washing three times. incubating for 30 min. at 30°C in YNB in new microfuge tubes, followed by three further centrifugal washes. Mating procedure. After staining and washing, cells were resuspended in 500 μl 10 x YNB. The two parents were transferred to a 1.5 ml microfuge tube and vortex mixed. After centrifugation for 1 min. at 12,000 x g, cells were incubated static at 20°C for 16 h in darkness. Flow cytometry. To disrupt aggregates, all samples were vortex mixed for 10 s immediately prior to flow cytometry. The FACSCalibur-Sort flow cytometer (Becton Dickinson, Lane Cove, NSW, Australia) was operated using Isoton II (Coulter Electronics Ltd, Brookvale, NSW, Australia) diluted 1:1000 with filtered (0.2 μm) purified (MilliQ, Millipore, Sydney, Australia) distilled water as the sheath fluid. Coulter ImmunoCheck beads were analysed each day to ensure the cytometer was correctly aligned. The flow rate was adjusted to keep the total data rate below 1000 events per second during analysis or below 300 events per second during sorting. The detection threshold was set in the FSC channel at a level just below that of the lowest yeast cell signals. The excitation light, wavelength 488 nm. was a 15 mW argon-ion laser. Fluorescence was monitored in FLl (CT-BODIPY, CT-Green, CT-Yellow-Green) or FL2 (CT-SNARF, BR, PKH-26). Compensation controls consisted of unlabelled and single dye labelled cells and these were prepared each time staining was carried out for compensation setting. Actual settings used depend on the physiological conditions of cells and dye concentrations and differed for each mating pair. Typical settings are given in Table 1.

TABLE 1. Typical flow cytometer settings used for hybrid selection.

Detector settings

FSC SSC FLl FL2

Voltage E00 300 600 600 Gain Linear, 1 Linear, 1 Log Log

Compensation settin gs

FL1-FL2 FL2-FL1

5% 75% Characterisation of cell type. Sort regions were defined on an FLl vs FL2 dot-plot. To determine the nature of cells sorted from the defined regions, sorted cells were examined using microscopy and regions modified until sorting accuracy was confirmed. The sorter was always set to single cell mode. For sorting rare mating hybrids, the first round of sorting consisted of collecting 20.000 events in BSA coated (FACSCalibur Users Manual. Becton-Dickinson) 50 ml sterile Falcon tubes (Bacto. Sydney. Australia). Sorted cells are recovered in high volume from catcher tube sorters such as the FACSCalibur. Therefore, cells were concentrated by centrifugation at 4.000 x g for 20 min. Supernatant was carefully removed and pellets resuspended into 2 ml YNB. Re-suspended cells were then loaded into the cytometer for a second round of sorting with 50 cells being collected into 50 ml tubes. Cells sorted from this second round were concentrated under sterile conditions (laminar flow cabinet) on 47 mm diameter 0.22 μm filters (Millipore) and mounted on rich medium. Plates were incubated at 30 °C for 48 hours before analysis of colonies.

For common mating, the first round of sorting was performed as above, collecting 20.000 cells into 50 ml tubes and re-suspending in 2 ml YNB. Re- suspended cells were loaded into the cytometer for a second round of sorting with 150 cells being collected directly onto both rich medium and glucose minimal indicator medium in triplicate. Plates were incubated at 30°C for 48 h. Comparison between the number of colonies on the rich medium vs the number on the minimal medium indicated the efficiency of hybrid isolation. As a further confirmation. 100 colonies from the rich medium were picked randomly and patched onto maltose minimal agar. On this medium, hybrids were identified by the unique ability to grow and synthesise β-galactosidase enzyme. This is because strain X has lacZ linked to the MAL6 promoter and integrated into the genome. As a result, lacZ is expressed at high levels on maltose based medium. Microscopy. A Nikon Optiphot II epifluorescence microscope (Nikon.

Sydney. Australia) fitted with x 12 eyepieces and x 20 and x 40 objectives (Fluor20 and Fluor40 respectively) was used for examination of sorted populations. The excitation source was a 50W Hg vapour arc lamp. The appropriate fluorescence filters and bright field optics were used. Fluorescence was visualised using a DM510 filter block with the standard band pass filter swapped for a BA520 nm filter. PCR fingerprinting. To discriminate parent strains from rare mated hybrid strains. PCR fingerprints were obtained using commercially available primers (Yeast Mutilplex PCR primers. Bresatec. Sydney. Australia). Banding patterns were scored visually after agarose gel electrophoresis of PCR products.

RESULTS Initial Experiments

Studies on the use of this compound as a dye in flow cytometry have shown that the compound has exceptionally good characteristics. In particular, the fluorescence of cells labelled with the dye is not detected significantly in the FLl channel of Becton-Dickinson FACScan/Calibur type instruments, but is detected in the FL2 and FL3 channels. In addition, once stained, the compound does not diffuse from stained cells and does not stain neighbouring cells making it ideal for cell tracking. It is likely that the compound will have uses in other scientific applications due to its excellent fluorescent characteristics, low molecular weight, and its moderate water solubility.

A compound according to the present invention was used as a fluorescent dye to stain cells which were subsequently separated by flow cytometry. A scan of the separated cells stained by the compound is shown in Figure 2. The fluorescent dye allowed the differentiation of several populations of cells and their sorting by the cytometer.

Isometric plots of Figure 2 show the number of cells having particular fluorescence and light scatter characteristics. The number of cells is given on the vertical axis (cell number) and the side scatter (SSC) axis is as indicated. Plots A and B show green fluorescence (FLl). plots C and D show orange fluorescence (FL2) and plots E and F show red fluorescence (FL3). Data were collected using the FACSCalibur instrument at Macquarie University. Plots A. C and E are unstained rehydrated high activity dry yeast (HADY) and plots B. D and F are rehydrated HADY stained in distilled water with 5 μl per ml of a crude culture extract containing the compound of the present invention. The fluorescent and SSC axes are logarithmic scale, the cell number axis is linear. Note the great increase in fluorescence for stained cells in channels FL2 and FL3. but not in FLl. Rapid Selection of Novel Yeast Hybrids

Selection of cell tracking dyes. The criteria for selecting the most appropriate dyes were as follows. The two dyes preferably should have different spectral emission properties (e.g. one green, the other one orange) such that labelled cells can be readily discriminated by flow cytometry. Each dye should be bright enough to allow discrimination between parental cell types. Once the yeast are labelled, the dyes should be retained by the cells of the parent strain for the duration of the mating reaction. In addition they should not rapidly leak from one strain to the other. A range of potentially suitable dyes were tested from the "CellTracker" (CT) dye range of Molecular Probes Inc. and the tracking dye, PKH-26. The dye according to the present invention, "Beljian Red", (BR). Three dyes, BR (orange), CT-Green (green) and CT-BODIPY (green), were found to label cells with sufficient fluorescence for discrimination from unlabelled cells. The dyes were retained for up to 25 hours after staining and washing, after which time measurements ceased (Table 2.). After labelling and washing, all three dyes showed little leakage to unstained cells, but two populations (stained and unstained) could still be discriminated. CT-Orange, CT- Yellow-Green, CT- SNARF and PKH-26 were also tested but did not brightly stain yeast cells under any of the incubation conditions trialed.

TABLE 2. Retention of cell tracking dyes by Saccharomyces cerevisiae strain Nl

Time Beljian Red Cell Tracker Green Cell Tracker lapsed (h) BODIPY Green

Mean orange Mean green Mean green fluorescence fluorescence fluorescence

0 590 (59) 600 (83) 500 (154)

4 490 (48) 640 (78) 300 (88)

25 430 (56) 690 (132) 180 (63) Cell Tracker Green BODIPY and Cell Tracker Green were both measured in the green fluorescence Channel (FLl) (525 nm ± 5) and Beljian Red was measured in the orange fluorescence channel (FL2) (575 nm ± 5 nm). All values are in arbitrary fluorescence units and are averages of three experiments with standard deviations in brackets. Unstained cells had typical mean auto-fluorescence values of 8-12 in both FLl and FL2.

Mating of haploids of complementary mating type. Two brightly stained parent strains (Fig. 3A, 3B) were mixed together under conditions suitable for mating. Shortly after mixing (5 min.), two distinct populations of cells were present in the mating reaction on dot-plots of green fluorescence channel (FLl) vs orange fluorescence channel (FL2) (Fig. 3C). Cell sorting and microscopic observation confirmed that one of the populations was fluorescent green (representing strain SMC19-A) and the other population was fluorescent orange (representing strain X). After 16 hours, fluorescence of both these strains had decreased and a third, dual-stained population was detected on dot-plots of FLl vs FL2 (Fig. 3D). Cell sorting and microscopic observation confirmed that this region represented cell clusters containing at least one cell of each parent, including cell pairs displaying 'shmoo^' morphology, characteristic of mating cells. Sorting revealed that the clear majority of events in the dual-stained region were clusters of more than two cells, (actual proportion not determined). This multi-cell agglutination is typical of mating reactions and is commonly used as an indicator of mating type.

Based purely on the FLl vs FL2 fluorescence characteristics, cell clusters could not be precisely distinguished from doublets or mating pairs.

To enrich for the mating pairs and minimise interference from multi-cell clusters, the FLl vs FL2 dot-plot was gated by a region defined on the forward scatter (FSC) vs side scatter (SSC) dotplot (Fig. 4) that included the events of lowest light scatter. To obtain better resolution. FSC and SSC amplifiers were set to linear. This low-scatter population was expected to correspond to the smallest clusters which would have included mated pairs. Cell sorting and microscopic observation of the low-scatter gated dual- stained population confirmed that this gating strategy excluded large clusters and included a large proportion of mating pairs. Hybrid strains were identified by being able to form colonies after sub- culturing onto glucose minimal indicator medium, and by expression of lacZ derived from strain X. Using this method of analysis it was found that prior to sorting 33% of the population were hybrids. After one round of sorting the percentage of hybrids within the mixed population had increased to 70% and after two rounds it had increased to 96%. Rare mating of a polyploid industrial strain with a laboratory haploid. Shortly after mixing, two distinct populations representing the unmated parent strains were detectable in the mating reaction (data not shown). Cell sorting and microscopic observation confirmed that one of the populations was fluorescent green (strain Nl) and the other population was fluorescent orange (strain X). After 16 h a small proportion (< 1%) of cells formed a third "dual-stained" population (Fig. 5). Cell sorting and microscopic observation confirmed that this dual-stained region on the FLl vs FL2 dotplot represented cell clusters containing at least one cell of each parent. This region was sorted twice, and 50 isolates were obtained. Of these 50 isolates, three had PCR fingerprints consistent with being the result of hybridisation between the two parental strains. The three putative rare mated products had the phenotypes of both parents. These included lacZ expression, derived from X, and prototrophy, derived from Nl.

The method presented here was developed for the rapid production and isolation of yeast hybrid strains without the need for genetic markers. In this study, the percentage of yeast hybrids isolated following mating between two strains with opposite mating types was increased from 33% to 96% after two rounds of sorting. The major advantage of the method is that a population highly enriched for hybrids can be produced without the use of genetic markers. This process is useful for production of new strains since isolates selected from the mating reaction are likely to be hybrids.

The present inventors also have demonstrated that this method can be applied to situations where hybrids are only rarely produced such as in 'rare mating'. Rare mating can occur when a diploid or polyploid yeast strain which does not have a mating type spontaneously mutates into a strain with either an a or alpha mating type. The resultant mutant can mate with a haploid or another polyploid strain of opposite mating type to produce a hybrid yeast. In heterothallic strains of yeast, which generally includes the brewing and baking strains, this is a rare event. The reported frequency of spontaneous ma ting-type switching in heterothallic haploid strains is increased by DNA damaging agents due to a gene conversion process. In this study, three rare mated hybrids were identified from 50 isolates sorted from a mating pool of greater than two million cells.

Although the strains used in this study had well defined phenotypes, the process should work equally well for uncharacterised strains using PCR fingerprinting to identify hybrids from the pool of double sorted cells.

Application of this tool for isolating rare mating events should allow the production of novel strains, even in situations where mating is highly inefficient, such as with baking and brewing strains. This could be applied to streamline the production of novel strains that combine the useful characteristics of different industrial parent strains.

In a further application of the ability to identify rare mating events, the new technique could be used for the isolation of interspecific crosses. Such hybrids may have significant industrial applications since it has been reported that some lager strains (Saccharomyces carlsbergensis) are the result of interspecific hybridisation. More generally, the ability to rapidly and efficiently isolate hybrids between two sexual gametes need not only be applied to yeast. It may be possible to apply the techniqvie to other situations where mating is required between organisms which do not have convenient genetic markers. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS:

I. A compound produced by a fungal species, wherein the compound is capable of binding to a biomolecule and is suitable for use as a fluorescent dye for the biomolecule. 2. A compound according to claim 1 such that when bound to a biomolecule. the compound emits fluorescence after excitation at blue wavelengths.

3. The compound according to claim 1 such that when bound to a biomolecule. the compound emits fluorescence in red wavelengths after excitation at blue wavelengths.

4. The compound according to any one of claims 1 to 3 wherein the fungal species is an Epicoccum species.

5. The compound according to claim 4 wherein the fungal species is Epicoccum nigrum. 6. The compound according to claim 5 wherein the fungal species is

Epicoccum nigrum PBl (NM98/00507).

7. The compound according to any one of claims 2 to 6 wherein the blue wavelength is 488 nm.

8. The compound according to claim 7 being Beljian Red (BR) as hereinbefore defined.

9. A process for producing a compound according to any one of claims 1 to 8 . the process comprising culturing the fungal species under conditions such that the fungal species produces the compound: and separating the compound from the fungal culture. 10. The process according to claim 9 wherein the fungal species is cultured in the presence of a yeast such that the fungal species produces the compound.

II. The process according to claim 10 wherein the fungal species is an Epicoccum species and the yeast is a Saccharomyces species. 12. The process according to claim 10 wherein the fungal species is

Epicoccum nigrum and the yeast is Saccharomyces cerevisiae. 13. A method of fluorescent-labelling a biomolecule. the method comprising causing the compound according to any one of claims 1 to 8 to bind to the biomolecule such that the biomolecule is fluorescently labelled by the compound.

14. The method according to claim 13 wherein the biomolecule is selected from the group consisting of proteins, peptides, glycoproteins, sugars, nucleic acids, antibodies, cell surface biomolecules, and cells.

15. The method according to claim 14 wherein the biomolecule is a yeast cell.

16. A method of detecting a biomolecule in a sample including the biomolecule. the method comprising labelling the biomolecule according to the method of any one of claims 13 to 15; and detecting the biomolecule in the sample by monitoring or detecting fluorescence of the bound compound. 17. The method according to claim 16 wherein the monitoring or detecting of the fluorescence of the labelled-biomolecule is by microscopy or cytometry.

18. Use of the compound according to any one of claims 1 to 8 as a fluorescent dye or marker for staining, labelling and/or detecting biomolecules.

19. The use according to claim 18 selected from the group consisting of cell tracking dyes for microscopy, membrane fluidity dyes, conjugation with antibodies, conjugation to nucleic acids, cell surface ligand imaging dyes, conjugation to sugars, cytometric analysis, and confocal microscopy.