Biological Resource Centers Provide Data and Characterized Living Material for Industrial Biotechnology

Introduction

Validated microbiological material is the foundation of the coming bioeconomy, and these materials are typically held in stasis in a dedicated collection of microbial germplasm. ¹ These collections have a variety of essential functions and exist across a range of scales, including private, institutional, public, and patent strain collections, and even collections held in the public domain. While their unifying characteristics link collections, at another level they are as different from one another as a backyard garden is different from industrial agriculture. Culture collections holding fungal germplasm serve a diverse and growing constituency. Beyond providing fungal strains, culture collections often serve as clearinghouses for taxonomic information, for genetic information, and increasingly for genomic resources.

Many world-caliber institutions began as private culture collections and grew through many years by collaborative effort into the diverse and robust Biological Resource Centers on which modern research depends. ^{2 –4} Thus, many collections are faced with the prospect of outgrowing their origins. This growth is both natural and predicated on international pressures, including the mandate of the Convention on Biological Diversity that every member state establish ex situ microbial germplasm repositories. ⁵ Similarly, the Organization for Economic Cooperation and Development has established the opinion that culture collections, through the structure of a biological resource center, establish the foundation of unprecedented economic growth, which they call the coming bioeconomy. ⁶ In this view, microbial processes constitute the white biotechnology revolution, distinguishing microbe-based industry from plant and agricultural economy and reflecting the image that the practitioners of microbiological sciences wear white lab coats.

Microbial repositories, having existed for many years, find themselves in the position of curating and distributing an increasing diversity of materials to clients not only in molecular and taxonomic research, industry, and medicine, but also in agriculture, culinary, and food processing technologies, and biofuel development schemes. The informatics requirements both for managing collections and for integrating a growing universe of taxonomic, genetic, physiological, and genomic data require culture collections to develop shared resources using international standards. Increasing professionalism of culture collections leverages work by previous researchers and allows saltatory rather than incremental progress.

Collection Holdings

Many diverse entities hold culture collections, from academic research laboratories to corporate and governmental entities. ^2,7,8 Collections are typically focused on one area, such as industrial or agricultural applications, taxonomic diversity, diversity within one phylogenetic or geographic area, or medical or genetic research. For example, the ex situ microbial germplasm repositories required by the Convention on Biological Diversity are expected to emphasize the microbial diversity of the host country. Fungal collections encompass all of these different emphases, although the specific type of material in any one collection is typically associated with their source of funding. Health ministries typically fund medical collections, and agricultural agencies fund agriculturally relevant collections. Many fungal collections have overlapping missions and, while some exist in the context of mycological herbaria, others are a small part of a larger, taxonomically broad collection such as the American Type Culture Collection (ATCC) or the German Collection of Microorganisms and Cell Cultures (DSMZ). ⁹ Most collections have fewer than 10,000 isolates and many hold thousands of different species including type strains for taxonomic use, reference strains for validation and quality control, and strains with unique or desirable characteristics. In the US, many collections exist without formal support (Table 1), which limits the ability to coordinate activities or formalize relationships among collections. On a global scale, many countries, especially in Europe, Asia, and South America, have growing collection programs that operate on a variety of scales.

The World Data Centre for Micro-organisms (WDCM) maintains databases of collection holdings on behalf of the World Federation for Culture Collections (WFCC). ¹⁰ Host to some of the largest collections in the world, it is reported that collections in the US hold over 200,000 isolates (Table 1). This breadth includes collections dedicated to wild or wine yeast, insect pathogens, mycorrhizal fungi, genetically characterized fungi, Fusarium, Oomycetes, and forest fungi. Other US collections not listed in the WDCM include endophytic fungi, mushroom-producing fungi, as well as clinical reference collections, and the Microbiology and Infectious Diseases Biological Research Repository established by the US National Institutes of Allergy and Infectious Diseases with broad holdings including medically relevant fungi. ^{11 –13}

A special type of collection exists specifically for the management of microbial material associated with patented processes. These collections, formally called International Depository Authorities (IDA), are required to meet international standards spelled out in the 1980 Budapest Treaty. ¹⁴ While the materials in the collection may be searchable via publicly accessible catalogs, use of the materials has special requirements. In the US, there are two IDA collections. The ATCC was both the first IDA and the largest in the world. ¹⁵ The US Department of Agriculture's (USDA's) collection at Peoria, IL (Table 1) has a patent collection in addition to its public collection.

Many collections have diverse holdings beyond their accessioned isolates. For example, in the 1990's the Fungal Genetics Stock Center (FGSC) held thousands of genomic DNA clones, both as ordered genome libraries and also as anonymous libraries. Similarly, special groups of strains are often transferred to established collections, although they are not formally accessioned. Collections of this type are frequently transferred to a collection on the retirement or demise of the scientist responsible for the assembly of the collection. Examples of these sorts of materials include the Tatum and Perkins Neurospora collections at the FGSC. ^16,17 These strains are made available on a “deposit-storage-distribution” format. ¹⁸ Many of these collections are not well documented and were collected prior to establishment of current intellectual property laws. Moreover, many resources with transitory value are made available through collections. During the Sanger-genome era, cosmid, fosmid, and bacterial artificial chromosome libraries were used to enable long-distance assembly of short reads. These libraries, along with libraries of cDNA, were valuable for map-based cloning among other applications. In the most well-established research systems these libraries are still used.

Use of Collections

Because the ability to identify novel drugs from microbes is considered central to the value of a collection, and with an estimated success rate for a new compound reaching the market of one chance in 2.5 million, the scope of the bioprospecting problem becomes clear. ¹⁹ A simple search of every fungal isolate held in culture collection—the WDCM database currently estimates that there are just over half a million fungal isolates held by collections around the world—would have only a fraction of a chance at finding a bioactive molecule. Fortunately, this is not an accurate estimate of the value of fungal cultures in formal collections as many other perspectives are important. The possibility that a strain would possess enzymes for bioprocessing, or enzymes with a desirable characteristic, is higher than the chance that it produces a novel chemical, and would also be amenable to manipulation by genetic or molecular techniques. ²⁰ Estimates of the cost of maintaining strains in collections range from $0.15–$0.30 per year, and this cost is several orders of magnitude lower than the cost of identifying a new sample from nature (especially for strains collected legally under the Convention for Biological Diversity). ⁸ Since the “majority of pharmacologically relevant molecules have been derived from the filamentous fungi,” it is tempting to assume that fungal culture collections would be greatly valued and protected from fluctuations in support. ⁸ While the WFCC estimates that there are only approximately 0.5 million fungal isolates in culture collections, this number is both an underestimate of the total number of fungal strains—many isolates are held in multiple collections—and also only a minute fraction of the total number of fungi in the environment.

Most collections have a specific focus, whether it be genetics such as the FGSC, industrial applications like the USDA NRRL collection (Agricultural Research Service culture collection), entomopathogens, human pathogens, or even mycorrhizal fungi. ²¹ Most collections, and by extension the total holdings of fungal collections, do not represent sufficient phylogenetic depth for significant and sustained discovery activities. ⁸ At the same time, the number of well-characterized isolates being made available for deposit into collections has increased. Many collections are forced to decline or limit acceptance of new isolates of contemporary strains, especially as collectors are not always aware of or compliant with regulations regarding taking materials from sovereign states.

Distributions from collections are a strong indicator of the value of the materials in these collections, and technological developments are enabling broader distribution of materials from collections. At the FGSC, distribution grew from approximately 1,000 strains per year in the mid- to late-1990's to well over 100,000 strains per year at present. This is due to the ability to distribute materials in arrayed sets in which each plate carries as many as 96 unique strains. Because of the requirements of high-throughput processing, this format is best for distributing groups of similar strains. It has therefore been used to distribute arrayed sets of Neurospora, Candida, Cryptococcus, and Pichia. ^{22 –27} Additionally, this format has been used to distribute functional sets of mutants, such as the Pichia strains carrying genes for cell-wall-degrading enzymes, the Aspergillus nidulans kinase gene deletion set, or functionally grouped mutants of Candida or Neurospora. ^25,27,28

International Programs for Collections

Many entities exist at national, regional, and international scales to promote the interests and activities of culture collections. ² The WFCC has a long history speaking for culture collections, and this authority comes from being a multidisciplinary commission of the International Union of Biological Sciences and a federation within the International Union of Microbiological Societies (IUMS). Originally established in 1970, the WFCC statutes were formally published in 1972 and have been updated by membership several times since. ^29,30 As a federation of the IUMS, the WFCC is afforded a voice at international discussions and has been proactive in discussions of “access and benefit sharing” relevant to the Convention on Biological Diversity and specifically on the negotiations relevant to Nagoya Protocol. This is a very important contribution, and users of microbial material will benefit by rational and practical guidelines on the use of fungal germplasm and the sharing of benefits from that use. Meanwhile, discussions regarding the standing of microbial collections have been undertaken from the perspective of the legal community. Legal scholars, economists, collection managers, collection users, and interested observers have joined forces to engage across disciplines with an interest in fungal and other microbial germplasm. ³¹ They described options for governing what they call a “Microbial Commons,” referring to it as being in danger by virtue of its being of value to a wide community but without an explicit owner.

The WFCC interacts with regional and national networks including the European Culture Collection Organization, the Asian Biological Resource Center Network, and the newly established US Culture Collection Network. These entities promote the interests of collections through the publication of standard documents such as material transfer agreements and data standards, and they work to lobby for stable or increased support for collections. They also promote collaborative interaction among collections and enable efforts such as straininfo.net that provides access to strain information, and the microbial commons effort that establishes standards for material sharing. ^31,32

The WDCM is an activity of the WFCC and as such has a long history of integrating its activities with the needs of the research community. The first version of the World Directory of Collections of Cultures of Microorganisms was published in 1972 and has been through several print editions prior to the current electronic, open access version. ^10,33,34 Recent activities at the WDCM include the development of informatics to associate every strain with the taxonomic and physiological characteristics described in published literature referencing each strain. For industrial microbiology, this is an important trend for a number of reasons. The WDCM Reference Strain Catalog is used by numerous entities to fulfill their unique requirement for unequivocal strain identifiers. Independent certification requires authoritative identification, for example the ISO technical committee on Food Microbiology-Culture Media codifies the WDCM strain identifiers in its guidelines. One significant advantage of referring to the WDCM strain number is in that it is independent of any one culture collection or national government. It can also make equivalent strains with shared origin but that are held in different collections. Additional informatics tools being developed at the WDCM include an analyzer of bioresource citations, which aggregates citations to unique strains held in public culture collections. Using publicly available and proprietary datasets this tool has cataloged over 94,000 strains held in 573 culture collections and described in more than 107,000 publications from 2,800 journals. Because of the depth of this resource, it offers multiple opportunities to small- and medium-sized culture collections. It has the capability of providing a virtual home to collections that do not individually have the resources to build an Internet presence and e-commerce features. ¹⁰

The straininfo.net project—a similar effort but without the imprimatur of authority represented by the WDCM—seeks to use online tools to present additional information about strains by identifying material held in multiple collections. ³² This effort also exists in a framework of a proposed Microbiological Common Language, which is intended to facilitate data exchanges and comparisons among collections and to promote the use of defined language and semantic rules to allow integration of data pertaining to habitat and location in microbial datasets. ^35,36 These data are foundational to exploitation of microbes for industrial and pharmaceutical technology, and in a mature biotechnological economy, will assure both that scientists can find the materials they need for myriad applications and that the appropriate benefits from the identification and provision of these materials can return to the inventor, discoverer, or country of origin.

Impact of Genome Data on Collections

While many collections hold type specimens for taxonomic purposes, other collections hold genetically or molecularly characterized materials. As high-throughput DNA sequencing impacts fungal biology, it becomes reasonable to consider that some collections will associate whole genome sequences with strains in the collection. A pilot study using whole genome sequencing to identify otherwise anonymous classical mutations is being extended to provide an association between strains and the polymorphic alleles they harbor. ³⁷ This capability, still in its infancy, has the potential to enable a genome-based taxonomy, replacing the trait-based taxonomy currently universally employed. ³⁸

Moreover, the ability to search strains in a culture collection based on the genes available from strains in that collection will radically change the ability to screen microbial material for desirable industrial, pharmaceutical, or other biologically relevant traits. While this capability exists already for many collections, it has not been exploited; this is a function of informatics tool development lagging behind resource characterization. The intersection of two unique programs may provide the answer to this lag. The Genomic Encyclopedia of Bacteria and Archaea is being undertaken as a foundational project between the US Department of Energy Joint Genome Institute (JGI) and the DSMZ while the bacterial pan-genome theory is the culmination of work by many different laboratories. ^{39 –44} Together these may provide a framework, with obvious extension to more complex eukaryotic systems, for both a core, phylogenetically meaningful genome, and a dispensable genome with high biodiversity and industrial or biotechnological value. ^45–46

Similarly, the newly established 1000 Fungal Genomes program sponsored by the JGI promises to launch a similar era for fungal biotechnology. The number of strains held by the FGCS that have been subject to whole genome analysis has grown rapidly over the last 10 years to include nearly 50 reference strains and more than 20 classic mutant strains (Table 2). The reference strains are typically wild-type strains sequenced as the one representative of the species. The classic mutant strains are sequenced to represent genetically characterized mutations that have been studied using Mendelian genetic, biochemical, and physiological approaches. ³⁷ In some future, a researcher who wishes to find strains with a particular combination of traits could search for these using a gene ontology to select families of genes with overlapping function and to mine the culture collection's strain information database to find strains with the suite of genes with unique biotechnological traits.

Future prospects

The Convention on Biological Diversity explicitly mandates that all parties to the convention establish ex situ microbial germplasm repositories to satisfy the requirements to preserve and characterize their microbial diversity. ⁵ This is foundational to any possible exploitation of this biological diversity. Indeed, the recently negotiated Nagoya Protocol on Access and Benefit Sharing pays special attention to the benefits from use of microbial material. ⁴⁷ It is anticipated that most tangible benefits apart from tourism will come not from mega fauna, but rather from microbial and plant diversity. For many stakeholders, the issue of access to microbial culture collections becomes one of identifying the materials, determining who has rights to the materials, and obtaining the materials through established channels. The growing professionalism of ex situ microbial germplasm repositories provides a foundation for the legitimate implementation of fungal biotechnology in the increasingly active, valuable, and promising area of fungal biotechnology.