FUNGAL GENOME INITIATIVE

GENOMICS FOR FILAMENTOUS FUNGI


During 1996, following closely on the announcement that a multinationalteam had completed the sequencing of the entire genome of the yeastSaccharomyces cerevisiae, two workshops were held to discuss plans forinitiating projects on the genomics of filamentous fungi. At theseworkshops it was decided that the time had come to initiate majorinitiatives in filamentous fungal genomics, beginning with the model species,Aspergillus nidulans and Neurospora crassa. This White Paper summarizessome of the reasons for starting fungal mapping and sequencing initiatives now.

A primer in mycology

Fungi are a large and diverse group of eukaryotes characterized by theirabsorptive mode of nutrition. Modern taxonomists place fungi in their ownkingdom, on equal footing with pl! ants and animals, sometimes called "TheFifth Kingdom." Fungi lead a special way of life that includes sporeformation and the efficient secretion of extracellular enzymes. Most fungiare filamentous, aerobic and terrestrial. They are the most resilient ofeukaryotes, capable of undergoing severe desiccation, living at extreme pHs,and surviving other environmental stresses. Being biochemically versatile, fungi produce a wide array of acids and degradative enzymes to support theirabsorptive life style, as well as an astonishing array of low molecularweight primary and secondary metabolites. Many of these metabolites haveindustrial and pharmaceutical applications.

Fungi lack the embryological development that characterizes plants andanimals, but they do undergo developmental cycles that culminate in theformation of reproductive structures. For species that can be cultured inthe laboratory on defined media, experimental manipulation of these life! cycles facilities research on genetics, physiology, and de! velopment. Thebest studied fungus is the yeast Saccharomyces cerevisiae. It is not onlyeconomically important because of its use in baking, brewing, andfermentation processes, it is also one of the best understood species on theplanet earth and the premier eukaryotic model organism.

Fungal taxonomy is based on morphology, largely of reproductivestructures. Approximately 100,000 species have been described, more thanany other group of microorganisms. The total number of fungal species,including those as yet undescribed, is estimated at 1.5 million. The so called"true fungi" (Eumycota) consist of five subdivisions divided into "lower"and "higher" groups depending on whether or not the cells have cross walls.Several notorious plant pathogens, such as the agent of the Irish potatofamine, are lower fungi. Yeast, most of the important industrial species,as well as familiar macrofungi such as mushrooms and truffles, are allhigher fungi

Th! e largest single taxonomic unit for fungi is called "the Ascomycotina"based on the formation of a sexual structure called the ascus. The ascuscontains the products of meiosis within a single morphological unit. Theascus facilitates elegant studies in classical genetics, especiallyconcerning recombination. Not surprisingly, in addition to the yeastS. cereviase, three other ascomycetes are important genetic models,two filamentous species -- Aspergillus nidulans and Neurospora crassa --and the fission yeast Schizosaccharomyces pombe. The genetic versatilityof these fungal models remains unsurpassed. Many fundamental discoveriesin biology have come from studying ascomycetes, including the Nobel Prizewinning research on N. crassa ("one gene, one enzyme") that heraldedin the era of molecular biology.

Economic impact

Fungi play a vital role in recycling nutrients in the biosphere. Theyare the major decomposer species in terrestrial habit! ats. Fungi can ramifythrough substrates, literally digest! ing their way along by secretingextracellular enzymes while their filamentous growth form facilitatesmechanical penetration of potential food sources.

Fungi possess the most efficient battery of depolymerizing enzymes ofall living things. From the human perspective, this has both good and badeconomic consequences. On the positive side, fungal enzymes drive theearth's carbon cycle, recycling ligninocellulosic remains. Moreover, manyof the oldest biotechnological processes are based on fungal catalyticpower: baking, brewing, wine fermentation, and the koji process areexamples of the way people have used fungal enzymes since antiquity. Morerecently, commercial production of glucoamylases, pectinases, proteases,lipases, cellulases, have been turned into major industries used in themanufacture of sweeteners, in the preparation of fruit juices, and in breadand cheese making. On the negative side, fungal enzymes damage standingtimber, finish! ed wood products, cotton fibers, and many other humanartefacts such as fuels, paints, glues, drugs, and electrical equipment.They compete with insects and rodents as the major destroyers of foods andfeeds. Fungi attack standing crops as plant pathogens (rusts, blights,smuts) and cause equal damage to harvested foods and feeds as storagecontaminants (rots, molds, mildews). They cause untold billions of dollarsof damage to agricultural crops each year. Less obviously, and in thepositive ledger, most plant life depends on symbiotic fungal rootassociations (mycorrhyzae) for proper nutrition and growth.

Fungal secondary metabolites are similarly Janus faced. Some fungalproducts are exploited as antibiotics (e.g., penicillin), immune responsesuppressants (e.g., cyclosporin A), blood pressure lowering agents (e.g.,mevalonin), hemorrhage and migraine control drugs (e.g., ergot alkaloid)and other pharmaceuticals. Others are among the most toxic nat! uralproducts. Mushroom poisons are the best known, but ! mold toxins(mycotoxins) do far more damage. A single mycotoxin, aflatoxin, on a singlecrop, corn, costs growers and producers in the U.S.A. over $100 millionannually. Worldwide losses are staggering.

Returning to the positive side, many fungi are also used directly as foodsand food flavoring agents. Mushroom cultivation is a significant and growingcomponent of the specialty food market. Asian food fermentations such akoji, soy sauce, oncham, and tempeh are a significant component of the dietof millions of people. Additionally, a number of fungal primary metabolitesare commercially exploited. Citric acid is the most important organic acidproduced by industrial fermentation. It is widely used as an acidulant insoft drinks and many foods, as a component of effervescent tables, as abuffer, and emulsifying agent.

Finally, fungi also cause disease. In addition to being the major plantpathogens, several hundred species are of medical and! veterinary importance.These range from relatively minor dermatological conditions, such asathletes foot, to incurable systemic mycoses. Patients after cancerchemotherapy and organ transplantation, suffering from AIDS, or with otherforms of immunocompromised status, are particularly susceptible to lifethreatening fungal infections.

Although antifungal drugs now constitute a billion dollar industry,most of them have undesirable side effects. There is a crying need forbetter therapeutic drugs to treat medical mycoses. Similarly, there is aneed for better fungicides to prevent against biodeterioration and for usein agriculture against plant pathogens. Existing fungicides are nonspecificand frequently toxic, thereby imposing not only a financial burden onsociety but an environmental one as well.

Genomics

A genome is the complete set of genetic material of an organism. Todate, fifty viruses, three bacteria, and one yeast have ! completed genomicDNA sequences. Projects are nearing com! pletion for several other bacteria,notably Escherichia coli, and are underway for the worm, the fly, themalarial protozoan, the mouse, a mustard plant, and Homo sapiens.Projects are initiated or contemplated for numerous other organisms,especially prokaryotes.

The yeast community has taken the lead in fungal genomics. Thissequencing project was funded largely by the European Union and involved aconsortium of 92 laboratories, including five big centers working along sideof the small groups. Data were assembled and annotated at a centrallocation at the Max Planck Institute. The yeast genome consists of 16chromosomes encompassing 12,067,266 base pairs (excluding repeats). Much ofwhat was learned confirmed what was already known: yeast chromosomes aregene-rich and intron poor. A surprising degree of apparent geneticredundancy was found. Most surprising was the number of new genesdiscovered. Over 60% of the open reading frames were new. Bec! ause meaningdoes not automatically leap out of DNA sequence data, it will requireconcerted experimental effort at the biochemical level to assign functionsto these unknown genes. "Functional genomics" is the new buzzword.

"Yeast is the unicellular human and everybody's favorite fungus,"quipped Mark Adams at the Oklahoma State University meeting. The yeastscientific community has had great success in gathering political andfinancial support. This aphoristic sound bite captures why: Saccharomyceshas been promoted not only as a model for studying human disease, but alsoas a model for all fungi.

Without down playing the significant scientific and technicalachievement, it is apparent that yeast is NOT an adequate paradigm forfilamentous fungi. Filamentous fungi have more genes and bigger genomes.Yeasts do not have the developmental, catabolic or anabolic capabilities offilamentous fungi nor do they have the ecological range. Yeasts d! o not formsecondary metabolites. The genes controlling t! hese functions can only bestudied in organisms that possess them. The yeast genome will provide aninvaluable starting place, which will make other fungal sequencing projectseasier, but it is not a substitute for these projects.

Filamentous Fungal Genomics

The technology exists for large scale sequencing. The current philosophyis to tackle the largest section of DNA that can be managed; filamentousfungal chromosomes are in the right size range.

If there is a basic filamentous fungal repertoire of genes (which surelythere is), it can be accessed only by studying filamentous species. At theOklahoma State University meeting, A. nidulans was selected as the targetfilamentous genome. At the New Orleans meeting, the target was expanded toinclude N. crassa. Why these organisms? What do Aspergillus and Neurosporahave to contribute to finding out the function of filamentous fungal genes?One of their big selling points is their m! ore than fifty years of classicalgenetics. Detailed genetic maps and hundreds of mutants are available forboth species. Few systems have been so well characterized with respect tobiological function. It will be easier to assign meaning to sequence datain the context of existing biochemical, morphological and genetic data.Moreover, we have already seen how the molecular analytical tools (i.e.,transformation, gene replacement) developed in these model systems spreadrapidly to industrial and pathogenic species, closing much of thetraditional gulf between basic and applied fungal studies. Similar benefitscan be expected from the DNA sequencing projects. Having a double projectwill also allow each organism to be a control for the other. Where there isconservation of gene order (synteny) it should be possible to acceleratecertain portions of the projects.

Neurospora has received the widest scientific attention of anyfilamentous fungus. Classic! al genetics has provided us with a detailedgenetic map. ! The synergism of classical and molecular genetics has extendedthe flexibility of this system in the molecular era. Moreover, there arealready existing projects in Neurospora genomics. The Neurospora project atthe University of New Mexico is sequencing expressed genes, and has foundthat about half of the genes discovered are new. A physical map is beingcompleted at the Fungal Genome Resource at the University of Georgia.

A. nidulans is also an important genetic model. Additionally, othermembers of the genus Aspergillus that biosynthesize a variety of economicallyimportant primary and secondry metabolites including citric acid, mevalonin,and aflatoxin. A. nidulans has also been developed as a model for studyingpenicillin biosynthesis and possesses most of the aflatoxin gene cluster.Aspergilli secrete large amounts of their own proteins ("homologousproteins") such as glucoamylase and are used widely in industry. Twospecies, Aspergillus niger an! d Aspergillus oryzae, are classified as "GRAS"(Generally Regarded As Safe") by the Food and Drug Administration.Sophisticated chemical engineering technology has been developed to handlelarge scale Aspergillus fermentations, making them attractive as potentialhosts for the production of economically valuable mammalian proteins.However, when target genes are fused into the genomes of filamentous fungi,the capacity to secrete such "heterologous" proteins is usually low.Further engineering is required in order for these species to meet theirpromise as production hosts.

Justification

DNA sequence information is only worth something when you can makesense of it. In effect, the classical genetics studies in A. nidulansand N. crassa have prepared the ground for efficient transfer to functionalgenomics.

Fungal genomics in not merely a technical feat of interest to basicbiologists, although without a doubt studying gene functi! ons in multiplesystems will be useful in fundamental rese! arch. Filamentous fungi offerunique opportunities for commercial exploitation. There is sound reason tobelieve that drug discovery programs based on genomics will be fruitful andhence profitable. If we understand how these organisms biosynthesize theirproducts and secrete them into their environment, we will be in a betterposition to engineer strain improvements. If we catalogue the biochemicalrepertoire of degradative enzymes, we can devise ways to harness thiscatalytic power. If we understand how fungi control their developmentalcycles, we will be in a better position to cultivate hitherto uncultivatableexotic food fungi such as truffles and chanterelles. We could engineerprotease free production strain for the production of heterologous mammalianproteins, we could engineer mycotoxin-free production strains for the foodindustry, and so forth.

Understanding fungal metabolism might also allow us to control unwantedfungal growth. If we kn! ew how pathogenic species colonize, sicken, and killtheir hosts, we would have a better chance of defending against theirattack. If we knew how toxin producing strains synthesized their poisons,we could delete these pathways. The potential for breakthoughs in medicine,agriculture, industry, and basic science is enormous.

Work plan and milestones

The total number of researchers working on filamentous fungi isrelatively small, and we are potentially divided. Success will requirenumerous scientists at university, industrial and government laboratoriesworking together; support from several governments and numerous companies;as well as complex coordination with data centers. Among the issues thatneed to be resolved and the mechanisms that need to be put in place:

1. Consensus building. The Aspergillus community and the Neurosporacommunity have both initiated funding efforts that will seed people to dopilot projects, and have cr! eated U. S. coordinating committees. However,we need an! open forum for soliciting the support of the entire mycologicalcommunity to back the Aspergillus and Neurospora projects. The AsilomarConference in March, 1997, can provide such a forum.

. 2. Governance. A long term organizational plan and coordinating bodyneed to be put into place. Although the Aspergillus community and theNeurospora community in the USA have already formed coordinating committees,we lack a "meta" committee with international input. We recommend theformation of an International Coordinating Committee on Filamentous FungalGenomics with multinational membership drawn from scientists who work onmany different species.

3. Communication. A method for keeping the community of fungalbiologists up to date about the sequencing projects is also essential.Other genomics projects have demonstrated that issues concerning datarelease, and the level of annoation and "finishing" of sequence data priorto release, can generate co! ntroversy. Moeover, it is recommended that weestablish both print and electronic mechanisms for open communication tosupplement standard releases to GenBank. A web site should be establisheddevoted to Filamentous Fungal Genomics. The Fungal Genetics Newsletter andthe newly refurbished Academic Press Journal Fungal Genetics & Biology(formerly Experimental Mycology) can also serve as outlets.

4. Establishing sequencing centers. We should build on the existinginfrastructure. Mapping projects are underway at Oklahoma State Univesityand the University of Georgia. The University of New Mexico has an activecDNA sequencing project for Neurospora. The Southern Regional Research Center,a branch of the Agricultural Research Center located in New Orleans, LA, hasexpressed interest in establish a new center for filamentous fungalgenomics. It is therefore recommended that there be four sequencing groupswith efforts at the Univ. of Georgia and the Univ! ersity of New Mexicofocused on Neurospora and efforts at ! Oklahoma State University and theSouthern Regional Research Center focused on Aspergillus. Pilot projectscan be functioning at all four Centers during 1997.

It will be important to establish a system for prioritizing,coordination and ensuring that different sequencing centers do notduplicate their efforts. For example, if the sequencing center at theSouthern Regional Research center becomes a reality, it might make sense forthem to the chromosome of A. nidulans that contains the sterigmatocystingene cluster, while keeping in close contact with Tom Adams group at TexasA&M.

5. Funding. Even if everyone agrees that it is important to sequence afilamentous genome, and that we should support a doubleAspergillus-Neurospora project, where is the money going to come from?Despite the trememndous importance for industry, we cannot expect industryto foot the entire bill. In the United States, NSF is already supportingseveral Neurospora projec! ts. Additional funding might be available fromDoD, DOE, and the USDA. The Wellcome Trust is another potential source.There was a general consensus at the New Orleans meeting that we areunlikely to get substantial new funding from NIH at this time. At theStillwater meeting, it was decided not to approach the European Union untilafter data from the Aspergillus chromosome IV pilot project were in hand.Nor have formal plans been put in place for approaching Japanesegovernmental agencies. This White Paper is intended to provide a modeldocument for use in different fund raising efforts.

6. Patenting. Given the large potential for developing products,diagnostics, and valuable production strains, fungal biologists must be verysensitive to issues relating to data sharing and patents. We should neitherunderestimate nor exaggerate the potential pitfalls. DNA sequence data perse will not yield profitable commercial ventures. Or, as Nigel Dunn-! Colemanput it: DNA data are only worth something if you a! re smart enough to dosomething with them." Nevertheless, I policy needs to be formulated andimplemented.

7. Other species. A. nidulans and N. crassa are models. Just as yeast isnot an adequate model for all filamentous fungi, these filamentous speciesare not adequate models for all fungi. Mapping projects should be initiatedin additional species, especially those representing important pathogens,both plant and animal. Additionally, taxonomic groups outside of theascomycetes should be targeted. Fungal biologists should create a "wishlist" and prioritize it.

Summary

In summary, molecular mycologists are in the enviable position of beingable to provide an immediate business-linked economic rationale for ourproposed genomics program. In addition to the the potential for commercialexploitation, as producers of enzymes, primary metabolites, secondarymetabolites and as whole cells, filamentous fungi offer the opportunity toan! swer fundamental questions in biology about development, evolution, andregulation. Information gleaned from fungal genomics also has potentialapplication in plant pathology and the treatment of medical mycoses. If thepast history of new technologies is any guide, we can predict that manyoutcomes, perhaps the most scientifically interesting, therapeuticallysuccessful, and commercially profitable, cannot be foreseen.

First draft by

J. W. Bennett, Jan. 1997