Appl Microbiol Biotechnol (2003) 62:446–458DOI 10.1007/s00253-003-1381-9 MINI-REVIEWV. Knight · J.-J. Sanglier · D. DiTullio · S. Braccili ·P. Bonner · J. Waters · D. Hughes · L. ZhangDiversifying microbial natural products for drug discoveryReceived: 10 March 2003 / Revised: 26 May 2003 / Accepted: 30 May 2003 / Published online: 28 June 2003 Springer-Verlag 2003Abstract Historically, nature has provided the source for Cannell 1998). Other examples are the commercializationthe majority of the drugs in use today. More than 20,000 of the antihyperlipidemics lovastatin and the recentmicrobial secondary metabolites have been described, but discovery of guggulsterone (Urizar et al. 2002). Microbialonly a small percentage of these have been carried natural products have also been developed as anti-diabeticforward as natural product drugs. Natural products are in drugs, hormone (ion-channel or receptor) antagonists,tough competition with large chemical libraries and with anti-cancer drugs, and agricultural and pharmaceuticalcombinatorial chemistries. Hence, each step of a natural agents (Grabley and Thiericke 1999). Microorganisms notproduct program has to be more efficient than ever, only produce secondary metabolites that affect cellstarting from the collection of environmental samples and growth but also accumulate bioactive compounds thatthe selection of strains, to metabolic expression, genetic interact with valuable targets of cell metabolism andexploitation, sample preparation and chemical dereplica- signaling that are not directly correlated with cell deathtion. This review will focus on approaches for diversify- (Che et al. 2002).ing microbial natural product strains and extract libraries, Drug discovery strategies for pharmaceutical andwhile decreasing genetic and chemical redundancy. agrochemical applications are in a revolutionary period (Auerbach et al. 2002). The completion of the Human Genome Project and the closure of dozens of microbial pathogen genomes have provided thousands of disease-Introduction related targets for screening. Automated instrument systems, robots, high throughput screening (HTS) plat-As recently as September 2002, natural product drug sales forms, and high throughput chemistry have providedaccounted for 17% of the top 100 best-selling drugs in the powerful tools for screening large compound libraries in aworld, with an approximate value of US $28.9 billion cost-effective manner. Combinatorial chemical, synthetic(including Lipitor and Atorvastatin, whose invention are chemical and natural product libraries provide abundantclearly inspired by natural product HMG-CoA reductase resources for target-based screening. The success andinhibitors such as simvastatin). failure of drug discovery is coupled to the novelty and The most well known examples of natural product are meaningfulness of the applied biological test systems asantibiotics (Demain 1999). The ‘Golden Age of Antibi- well as to the amount and structural diversity of the testotics’ from the 1940s to the 1970s was sparked by the compounds available (Fernandes 2001).serendipitous discovery of penicillin by Alexander Flem- The process of drug discovery for therapeutic anding in 1928 and its development by Chain and Florey in preventive medicines is facilitated by increasing knowl-the 1940s. Another remarkable milestone in the medicinal edge of the biological mechanisms of each challenge,use of microbial metabolites and their derivatives was the allowing factors such as treatment efficiency, potentialintroduction of the immunosuppressants cyclosporin A, side effects, and the growing threat of drug resistance toFK506 and rapamycin (Fig. 1) (VanMiddlesworth and be addressed. A large number of disease-relevant-protein targets are being identified and validated from different “- nomics” and system/computational biology approaches.V. Knight and J.-J. Sanglier contributed equally to this work Novel targets and novel HTS assays and measurementV. Knight · J.-J. Sanglier · D. DiTullio · S. Braccili · P. Bonner · systems are emerging for more sensitive, reliable andJ. Waters · D. Hughes · L. Zhang ()) low-background searches for new natural products suit-Cetek Corporation, able for drug production (Cochran 2000), and have shed260 Cedar Hill St., Marlborough, MA 01752, USA light on exploring new compounds from existing naturale-mail: firstname.lastname@example.org
447Fig. 1 Some representatives of structurally diverse microbial natural products mentioned in the textproduct libraries. For example, Cetek has developed a – Natural products are the main source of pharma-unique approach to screen for high affinity binders, even cophores. Cyclosporins, such as cyclosporin A andto unknown function protein targets. The “CE Assay” FK-506, are active not only as immunosuppressantsdetects potent ligands even in the presence of high but also as antiviral and antiparasitic agents (Bram etconcentrations of weaker, competitive ligands and other al. 1993).nuisance compounds. Potential drug leads for therapeutic – Natural product resources are largely unexplored andtargets can be quickly isolated and characterized using an novel discovery strategies will lead to novel bioactiveintegrated process (Pierceall et al. 2003). compounds. Natural product extracts are complemen- The advantages and challenges of natural-product- tary to synthetic and combinatorial libraries. Aboutbased drug discovery as compared to its synthetic 40% of the natural product diversity is not representedchemistry counterpart can be summarized as follows. in synthetic compounds libraries (Henkel et al. 1999). – Research on natural products has led to the discovery of novel mechanisms of action, for example, theAdvantages discovery of the role of guggulsterone (Urizar et al. 2002).– Natural products offer unmatched chemical diversity – Natural products are powerful biochemical tools, with structural complexity and biological potency serving as “pathfinders” for molecular biology and (Verdine 1996). chemistry and in the investigation of cellular functions– Natural products have been selected by nature for (Hung et al. 1996). specific biological interactions. They have evolved to – Natural products can guide the design of synthetic bind to proteins and have drug-like properties (Nisbet compounds (Breinbauer et al. 2002). and Moore 1997).
448 This review concentrates on the challenges of effi- ciently diversifying a library of microbial compounds and does not deal with other problems such as preparation of samples, scale-up, chemical dereplication of isolated compounds and chemical identification. Systematic ap- proaches to maximize the biodiversity of microorganisms within a natural product library are discussed from the following three perspectives (Fig. 2): (1) isolation and selection of samples from diverse ecosystems, (2) manipulating microbial physiology to activate microbial natural product machinery, and (3) genetically modifying strains for production of unnatural microbial natural products. By manipulating all three of these approaches, the diversity of an extract collection can be maximized and, in so doing, the chance of finding a novel compoundFig. 2 Multi-dimensional diversification of the microbial natural can be increased. However, the quality of a microbialproduct library. Genetic diversity of strains (x-axis) is achieved by natural product library rests on the dynamic equilibriumstringently selecting microbial isolates from diverse ecosystems between diversification and reducing redundancy ofand approaching unculturable microbes. The strains are classifiedby morphological, genetic and biochemical characterization and microbial natural products. Therefore, strategies forprofiling. Only representative strains from each group will be obtaining high quality microbial natural product librariespicked for further physiological manipulations. These strains will are discussed.be fermented in a variety of media with or without elicitorstimulation under various incubation temperatures and incubationtimes (y-axis). Microbial strains will be genetically modified forunnatural microbial natural products (z-axis). When an interesting Isolation and selectioncompound is found, other strains within the same genetic or of samples from diverse ecosystemschemical group will be examined for increased production orpresence of analogs of the compound of interest Microbes sense, adapt and respond to their environment and help compete for survival by generating secondaryMain challenges metabolites. These compounds are produced in response to stress and provide microbes with increased survival– The lack of systematic exploitation of ecosystems for mechanisms. In diversifying microbial natural product the discovery of novel microbial compounds had extract libraries, the greatest influence will undoubtedly resulted in random sampling and has missed the true be the genetic diversity of strains. By maximizing the potential of many regions (Czaran et al. 2002). types of samples collected and diversifying the isolation– Little effort has focused on the isolation and cultiva- strategies a highly diverse microbial collection can be tion of less culturable microorganisms. The discrep- generated. ancy between the number of microbes detected by molecular methods and the number of strains in culture, demonstrate that there remains a relatively Diversifying ecosystems untapped source of novel strains in all ecosystems (Harvey 2000). Collecting environmental samples for isolation of inter-– The selection of strains has traditionally been based on esting microorganisms has often been conducted without morphology, rather than on the more powerful ap- defined strategies (Shrestha et al. 2001). Such programs proaches of chemical diversity and genotype (Firn and need to take into consideration the biogeography of Jones 2000). ecosystems, the number of samples collected, and the– Lack of dereplication has resulted in a redundancy of isolation procedures used. It is important to increase the strains and compounds within many natural product number and diversity of sampling sites (Foissner 1999), extract libraries(Handelsman et al. 1998). and it is especially important to look at under-represented– The characterization and isolation of the active com- sites. Diverse regions such as the deep subsurface, the pounds from natural product extracts are extremely deep sea and sites that have extreme temperature, salinity labor intensive and time consuming (Monaghan et al. or pH often generate novel microorganisms and therefore 1995). provide the potential for novel compounds (Bull 2000).– The production of adequate quantities of the active Temperate ecosystems should not be excluded for they compound needed for drug profiling may require too have the potential to provide many novel species, extensive media optimization and scale-up (Strobel especially when novel isolation strategies are used. 2002). Cyclosporins, rapamycin, penicillin, and rifamycin, among others, were isolated from microorganisms col- lected in temperate regions.
449 It is still debated whether most microorganisms are the oceans provide for discovering new compounds, but itcosmopolitan or endemic to specific geographic areas. also validates the pharmacological value of exploring theThere is a lack of detailed information in the field of oceans for novel compounds. There are some concernsgeographical distribution of microorganisms (Bull 2000). surrounding the isolation of marine microbes. ResearchersIn some cases, the presence of an endemic species can be claim that this resource has not been thoroughly exploreddetected, for example, several groups of bacteria appeared because these organisms are hard to maintain in ato be endemic to an ice microbial community (Staley and laboratory environment. One successful case was theGosink 1999). However, the definition of a microorgan- recent discovery of a new genus of actinomycetes,ism species is difficult, especially for prokaryotes, which Salinospora, found only in the marine environmentexchange part of their genomes with sufficient ease to (Mincer et al. 2002). One isolate produces salinospo-preclude any biologically meaningful definition of a ramide A, a potent anti-cancer agent. This suggests thatspecies (Doolittle 1999). In order to increase the chance the oceans can no longer be ignored (Feling et al. 2003).of constructing a library of microorganisms with high Other regions that warrant further study are locations withdiversity, the first step is to consider different geographic extremes of pH, temperature and salinity. Although theseareas, including biodiversity hot spots. The key criteria environments select for the evolution of unique metabolicfor determining a hotspot are endemism and degree of pathways and enzymes, they also provide less diversity.threat of destruction. Twenty-five current global hotspots The lack of genetic diversity however may negate theare defined at the following web site: http://www.con- need for competition for survival and hence the produc-servation.org/xp/CIWEB/strategies/hotspots/hotspots_map. tion of secondary metabolites (Bull 2000).xml. The question arises if it is necessary for one To comprehensively sample a particular site, multipleindustrial program to obtain samples from all of these discrete locations within the site must be sampled. Ageographical areas, and the answer is no (Tulp and Bohlin defined sampling strategy must be adopted. Many types2002). There are more compounds in nature than possible of sample should be selected within a single ecosystem,requested drugs for all molecular targets. One should such as soils, sediments, organic material, dung, deadconcentrate on sampling in some regions with different animals, plants, and lichens. Soil still remains an impor-climates, fauna, and flora. tant source because it carries a higher population of It is important to properly analyze the various ecosys- microbes than any other habitat (Whitman et al. 1998).tems of a given region. For example, in Massachusetts DNA community analysis has proven that the number ofthere are 13 eco-regions, from the Berkshire Highlands to types of organisms found within a microbial communitythe coastal regions of Cape Cod (http://www.state.ma.us/ is much higher than previously thought. One analysis ofmgis/eco-reg.html), each of which has various sub- the reassociation kinetics of the total bacterial DNA in aecosystems. Microbiologists should work closely with 30 g soil sample found that it contained more thanbotanists and ecologists to obtain as many different 500,000 species (Doolittle 1999).samples and microorganisms as possible from one Plants and lichens offer niches for interaction betweenecosystem to maximize the likelihood of finding novel microorganisms and eukaryotic cells. Many naturalstrains and, in turn, novel chemicals. In almost all products initially isolated from plants and animals, areecosystems, no matter how harsh, a group of organisms actually produced by microbial symbionts found withinwill grow and thrive. In these unique ecosystems, we can the tissue of the host (Jensen and Fenical 1994). In someexpect to find unique metabolic pathways that have instances where the microorganism or symbiont could notevolved to allow the microorganisms to adapt and be cultured axenically, the genes responsible for thesurvive; collection of microbes from diversified ecosys- production of the active compounds were attributed to thetems will provide rich resources for drug discovery. microorganism (Davidson et al. 2001). With the discovery that microbial symbionts were Endophytes are microorganisms including bacteriadriving the metabolism of tubeworms in deep-sea hy- (Zinniel et al. 2002), actinomycetes (Castillo et al.drothermal vents, it was realized that an oasis of rich 2002), and fungi (Ananda and Sridhar 2002) that spenddiversity could be found even in areas that were thought part or all of their life cycle colonizing—either inter- orto be devoid of life. More than 70% of the Earth’s surface intra- cellularly—the healthy tissue of a plant (Tan andis covered with ocean, of which approximately 60% is Zou 2001). Almost all vascular plants and mossesmore than 2,000 m deep (Bull et al. 2000). The deep sea, examined so far have endophytic bacteria or fungi withinonce thought to be devoid of life, is actually a rainforest their tissues (Zinniel et al. 2002). The number of strainswith a diversity of more than 10 million species, more found within the plant tissue can vary from a few tothan 60% of which are unknown (Jensen and Fenical several hundred per plant. This relationship between the1994). In addition to the open ocean, there are also such microorganism and the plant can range from mildlydiverse areas as mangroves, coral reefs, hydrothermal phytopathogenic to symbiotic. Endophytes produce avents and deep-sea sediments, which provide dynamic range of compounds (Strobel 2002), some of which helpareas to search for microbes. Natural products have been the plant survive and thrive in its ecosystem and othersisolated from marine invertebrates such as sponges, that help it fight off infection (Wei et al. 1992). Plantstunicates, molluscs and bryozoans (Proksch et al. 2002). therefore provide an obvious source for isolation ofThis not only demonstrates the numerous opportunities microorganisms that could potentially produce novel
450natural products. Of special interest are the large number Microbial community analysis has revealed that theof alkaloids and taxol produced by endophytic fungi microorganisms in culture not only represent a small part(Strobel 2002). of the population, but also may not be the most prevalent Lichens, a symbiosis between fungi and cyanobacteria in the natural environment (Harvey 2000). It is expectedor algae, are another source of microorganisms living in a that one of the largest pushes in the next decade will beunique and competitive environment. In each lichen exploring means to culture less culturable organisms. It issample, the fungus forms a thallus or lichenized stoma thought that reasons for the enormous discrepancy(Ahmadjian 1965; Rikkinen et al. 2002). Furthermore, in between the total viable cell counts to culturable cellsaddition to the symbiotic fungal strains, other fungi, may include the following: (1) cell damage by oxidativeactinomycetes or bacteria can live as endophytes inside, stress, (2) formation of viable but non-culturable cells, (3)or as epiphytes on surface of the lichens. The fungi within inhibition by high substrate concentrations, (4) inductionthe lichen often produce unique secondary metabolites. of lysogenic phages upon starvation, and (5) lack of cell-Over 800 lichen secondary metabolites have been to-cell communication in laboratory media (Bruns et al.collected so far. 2002). Two main approaches have been used to enhance the resuscitation of less culturable strains. The first is the addition of cell signaling molecules and the second is theIsolation strategies use of oligotrophic isolation media. Microorganisms use pheromones to communicate withThe microbial diversity in the environment is far greater each other both within and across species (Kaeberlein etthan is reflected in most strain collections due to the al. 2002). Microorganisms may require signaling fromnumber of organisms that cannot be cultured using other organisms in order to grow even if provided with thestandard culture conditions (Courtois et al. 2003). There- appropriate nutrients. The addition of growth factors tofore, the vast majority of microorganisms in many culture medium has been used successfully to increase thesamples remain unexplored. Molecular techniques allow resuscitation of greater numbers of microorganisms, thusdetection of organisms that were missed using culture- yielding higher microbial diversity. The addition ofdependant methods. Culture-independent methods, such pyruvate or catalase to reduce oxidative stress duringas DNA clone libraries, have allowed identification of isolation can increase the numbers and diversity of strainsvast numbers of new organisms that are different from isolated (Brewer et al. 1977). The addition of extracellularanything previously cultured. It is estimated that as few as cyclic AMP (Kalish et al. 2001) and N-acyl-homoserine0.1–1% of the organisms living in the biosphere have lactones have both been shown to increase the resusci-been cultured and characterized in a laboratory setting. In tation of starved cultures under laboratory conditionsone study, approximately 107 bacteria were counted in 1 g (Bruns et al. 2002). In enterobacteria, cAMP is involvedsoil (Kellenberger 2001), but as few as 0.1% were in the regulation of the majority of the genes expressedculturable using standard culture techniques. The other under starvation including those coding for high affinity99.9% of the population may represent novel genetic sugar transport systems (Ferenci 1996).diversity (Handelsman et al. 1998). These microorgan- A second approach to increasing resuscitation of lessisms represent a diverse and still undiscovered reservoir culturable strains is the use of oligotrophic isolationof novel strains that may produce novel natural com- media. It has been well documented that conventionalpounds. In 1987, when Carl Woese proposed the five media have extremely high concentrations of complexkingdom phylogenetic tree, the bacteria were divided into organic compounds compared with those present in the12 groups. The initial evaluation was done primarily with natural environment. Most isolation media allow forbacteria in culture. By 2000, the number of groups had growth of only a selected group of strains and inhibit theexpanded to 36, of which 13 do not have a representative majority of the natural population (Connon and Giovanniin culture (Hugenholtz et al. 1998). Approximately 6,000 2002). Oligotrophic medium not only allows the growthbacterial species have been described, but the number of of less culturable microorganisms but also prevents thebacteria that exist in nature is predicted to be as high as overgrowth of fast-growing “microbial weeds” (Zengler600,000 (Davies 1999). The situation is even more et al. 2002). Using unamended site water as growthextreme for fungi. The currently accepted number of medium, unique populations of microorganisms havedescribed species of fungi is 72,000 but the estimated been cultured (Connon and Giovannoni 2002). A varia-figure of fungi that exist in nature could be as high as 1.5 tion of this method is encapsulation of single cells withinmillion (Bull et al. 2000). This suggests that there are gel micro-drops that contain low nutrient medium (Zen-diverse novel microorganisms in the natural environment gler et al. 2002) or within specialized growth chambersthat could be used as sources for drug discovery. The incubated in site water (Kaeberlein et al. 2002).argument against putting effort into culturing less cultur- One argument against applying less culturable strainsable organisms is that it is very time consuming and the to a drug discovery program has been that they could nottechniques used for one organism may not be applicable be cultured at a high enough cell density. The argumentto others. With this in mind, many industrial programs are for including them is that although they may initiallynow seeking to harness the potential of these less require the addition of growth factors or oligotrophicculturable strains. growth conditions there is evidence that, once cultured,
451the organisms can be grown out in nutrient rich medium. However, other biosynthetic pathways, such as productionUsing 960 cells cultured in micro drops, 67% of the of pyrroindomycins by Streptomyces rugosporus (Ab-cultures were able to grow to densities 107 cells per ml banat et al. 1999), are not affected. Optimal production of(Zengler et al. 2002). This allows the cells to be cultured gibberelic acid by Giberella fujikuoi in a defined mediumin a manner that could be easily applied to drug discovery requires a concentration of 22.5 mM ammonium sulfate.platforms. Complex natural sources of nitrogen such as soybeans, An alternate approach to access unculturable microor- and casamino acids are also good. The influence of aminoganisms is to clone DNA directly from uncultured acids on secondary metabolite production is very variablemicroorganisms (see below for more detail). and can depend on the precursor or the natural inducer. Inorganic phosphate suppresses the synthesis of many secondary metabolites, so the optimal phosphate concen-Manipulating microbial physiology to activate tration needed for production of secondary metabolites ismicrobial natural product machinery generally lower than that required for growth. However, the optimal concentration can vary drastically betweenIn order to exploit the true potential of microorganisms, strains. The concentration can be as low as 0.08 mM forthe physiological growth conditions used for generating the synthesis of bacitracin by Bacillus licheniformis, or asextracts need to be diversified. The physiology of high as 8 mM for the production of novobiocin bysecondary metabolism has often been neglected. Very Streptomyces griseus (Gotoh et al. 1982). In somefew of the regulatory features of secondary metabolism instances, high phosphate concentrations can even inducehave been elucidated (Demain 1998a). The global phys- the biosynthesis of some metabolites (Shimada et al.iological regulation situation is very complex due to the 1986). Secondary metabolism often requires trace ele-variety of microbes, the variety of biosynthetic pathways, ments such as iron, zinc, and manganese. Once again, theand the variety of controls. Environmental conditions optimal concentrations vary from process to process, butremain, however, a key element in the discovery and often range from less than 0.1 to 1103 M (Weinbergproduction of secondary metabolites. Strategies that 1978).exploit the full metabolic potential of each microorganism Optimal temperatures for the production of secondaryhave to be developed in order to maximize chemical metabolites are in general lower than for growth but candiversity. Biochemical pathways, induction and regula- vary considerably. For example, an optimal temperaturetion of secondary metabolism by internal molecules (such of 21C is required for cyclosporin by Tolypocladiumas autoregulators) have been reviewed previously (De- inflatum, 25C for the synthesis of streptomycine by S.main 1998b; Horinoushi 2002). griseus or 28C for nebramycine by Streptomyces tene- brarius. Most of the known secondary metabolites are produced under standard aeration conditions, but someVarious optima require lower, and some higher, dissolved oxygen concentrations (Barberel and Walker 2000). ExtremeThe optimal conditions for biosynthesis of secondary aeration is required for optimal production of secondarymetabolites are not necessarily identical to those for metabolites by Streptosporangium (Pfefferle et al. 2001).growth. In general, the optimal zones are narrower for Incubation time is another key point and is dependentsecondary metabolism production. Physiological regula- on the growth characteristics of the microorganism andtion varies with different microorganisms and different the culture conditions. For example, actinomycetes canmetabolic pathways. The qualitative and quantitative vary from 3 days for the maximum production ofaspects of secondary metabolite production must be taken arylomycins by a strain of Streptomyces (Schimana etinto consideration. al. 2002) to 12 days for maximum production of There are usually differences between the optimal pramicidin S by a strain of Actinomadura (Saitoh et al.carbon sources for growth and those that are good for 1993). The addition of adsorbents such as XAD-16 resinsecondary metabolism (Betina 1994). For example, to liquid cultures can also enhance the concentration ofglucose is an excellent carbon source for growth in most secondary metabolites produced (Gerth et al. 1996). Mostcases but depresses the production of a series of programs for the discovery of novel metabolites fromsecondary metabolites such as actinomycin, cephalospor- microorganisms use liquid shaken cultures for cultivationine, ergot alkaloids, and tylosin. However, glucose does of microorganisms. This provides an easy and well-not interfere with the production of aflatoxin, aminogly- controlled system. Solid phase fermentation allows thecosides or chloramphenicol (Luchese and Harrigan 1993), biosynthesis of other metabolites, mainly related to theand the production of anticapsine by Streptomyces sporulation process (Calvo et al. 2002). Both types can begriseoplanus is maximal at a concentration of glucose scaled-up effectively (Robinson 2001).as high as 100 g/l (Boeck et al. 1971). Secondary metabolic pathways are often negativelyaffected by nitrogen sources favorable to rapid growth,for example, ammonium salts inhibit the production ofcephamycin, fusidine, and rifamycin (Aharonowitz 1980).
452Selection of culture conditions chemical methods. Myxobacteria and fungi are also considered talented microbes (Reichenbach 2001).The optimal conditions for secondary metabolite produc- The genomes of actinomycetes (8 Mb) (Hopwoodtion vary from microbe to microbe. Medium composition 1999), fungi (13–42 Mb) (Kupfer et al. 1997) andand culture conditions have a great impact on the myxobacteria (12 Mb) (Pradella et al. 2002) are muchproduction of secondary metabolites. In a discovery larger than needed for all basic functions. Therefore, it isprogram, one is working with a large series of unique widely supposed that part of the genome may encodeand ubiquitous microorganisms. Multiple conditions are genes for alternative metabolic pathways. For example,necessary in order to allow the expression of secondary Streptomyces coelicolor A (3) 2 is designated as a potentmetabolites. Both static and shaken liquid cultures should producer of secondary metabolites. It producesbe incorporated. Different incubation temperatures must methylenomycin, prodigiosin, actinorhodin, and otherbe chosen. Addition of elicitor compounds such as heavy calcium-dependent antibiotics. In addition, several for-metals, oils (Sandor 2001), microbial or fungal cell wall merly unknown gene clusters (polyketide syntheses type Icomponents and dimethylsulfoxide (Chen et al. 2000) can and II, non-ribosomal peptide syntheses) have been foundincrease the biosynthesis of secondary metabolites. The in its genome (Bentley and Chater 2002). In Streptomycesmedium can be devised choosing carbon and nitrogen avermitilis ATCC 31267, the producer of avermectine, 24sources at different concentrations, as well as other additional gene clusters have been sequenced (Omura etnutrients such as phosphate and trace elements or elicitors al. 2001). Genomic data has suggested that the myxobac-at various levels and using a Greco-Latin square format. terium Stigmatella aurantiaca DW4/3-1 has a muchThe goal is to achieve a good ratio between the number of broader capacity to produce natural products than thosestrains (genetic diversity) and the number of culture isolated to date from this organism (Silakowski et al.conditions for each one (metabolic expression). 2001). The genes responsible for production of many Another powerful tool is the pre-selection of strains compounds can be found in the genomes of non-based upon growth in a series of media in micro-cultures producing strains. Several questions arise: Are theseor in small vials (Minas et al. 2000). A standard format genes non-functional? Are the detection methods notthat is amenable to automation allows over 20 media to be powerful enough? Are these genes not expressed undereasily tested with each microorganism, at different standard growth conditions? Do the genes require externaltemperatures, and in liquid as well as on solid medium. signaling to turn them on? More efficient detectionThis allows the selection of the best conditions for each methods, biochemical assays such as capillary electro-strain, which can then be used for scale-up to obtain the phoresis (Pierceal et al. 2003), or chemical methodslarger volumes required for initial structure determina- (Cochran 2000) will allow the discovery of large numberstion. This system is also adequate for a quick optimization of novel compounds (Bode et al. 2002). As brieflyprogram. described above, small changes in culture conditions can For each group of strains a series of conditions can be have a major influence in the spectrum of secondarychosen incorporating both shaken liquid cultures and metabolites synthesized. For example, the fungal strainstationary solid cultures. Typically, five medium types are Sphaeropsidales strain F-24’707 is a producer of theused for each batch of cultures, and from three to five antifungal compound cladospirone bisepoxyde. When thisincubation conditions are chosen to include various strain was grown in combinations of different media andtemperatures and different incubation periods. In order cultivation methods, eight new spironaphtalenes wereto enhance the chance of success in such a random isolated. There were previously only six known membersprocess, the conditions used for one group must be of this class of compounds. The addition of inhibitors,rotated. The results of chemical profiling and scores in such as tricyclazole, inhibits some pathways and thereforescreening should be constantly analyzed in order to allowed the production of other compounds, includingimprove the system. two new bisnaphtenes and a rare macrolide, mutolide (Bode et al. 2002). A systematic fermentation program should be con-Physiological exploitation of talented strains ducted with “talented” strains and with representative strains of poorly known genera in order to maximize theSome microorganisms are able to produce a variety of number of compounds produced by each strain. Such ancompounds from different chemical families and are approach should include:termed “metabolically talented” (Trujillo et al. 1997).Most recently described microbial compounds are pro- – cultures grown in shaken liquid vessels and on solidduced by actinomycetes, mostly Streptomyces strains, and mediaby saprophytic filamentous fungi. One metabolically – cultures grown with media of different compositiontalented microorganism, Streptomyces sp. strain Go.40/ – incubation at two or more temperatures10, synthesizes at least 30 different secondary metabo- – incubation at two or more shaker speedslites, many of which are new compounds (Schiewe and – incubation for at least two different time periodsZeeck 1999). Some strains can synthesize more than 50 – media with at least two pH levelscompounds, which can be detected only by classical
453– absorbents, enzyme inhibitors, elicitors, precursors, thesis. These GST are used as probes to screen for the precursor analogs and high concentration of salts presence of these genes within a clonal library. Any clone should be added to the most productive fermentation that contains a GST can then be screened for novel natural medium product gene clusters. More than 450 natural product clusters have been identified in this manner (Zazopoulos After selection of one or two potent media, the et al. 2003).influence of the other factors can be analyzed using anexperimental design such as fractionated factorial orPlackett-Burmann design (Wieling et al. 1993; Abdel- Gene shufflingFattah et al. 2002). The traditional way to diversify unnatural microbial natural products is by random mutagenesis or by culturingCo-cultivation microbes with non-natural precursors. However, the discovery in prokaryotes that the genes for naturalMicrobial communities also hold the potential for products are usually clustered, made it possible to cloneproduction of novel compounds. In nature, microorgan- an entire pathway into a vector (Handelsman et al. 1998).isms do not exist alone; they are part of tiny ecosystems. Many natural product genes are modular and produceThere is expected to be diverse signaling and cross- multifunctional enzymes. They have a high degree offeeding going on between organisms that will elicit plasticity. By interchanging and moving genes aroundproduction of novel compounds. Although the longstand- within these clusters, hybrid enzymes can be produceding argument against this type of research has been that that are capable of synthesizing an unlimited set of newgetting a stable mixed culture is almost impossible, it may molecules (Kennedy and Hutchinson 1999).provide a means for exploiting the true potential of the The modular nature of many secondary metaboliteconsortium as a whole. An example using co-cultivation genes provides an ideal system to genetically engineerof two microorganisms producing related products has unnatural microbial natural products by incorporatingbeen suggested as a suitable route towards diversification genes from different pathways. An example of such anof microbial structures (Degenkolb 2002). approach is the polyketide synthases (PKS), which are large multi-domain enzymes that produce polyketides including antibacterials, immunosuppressants and choles-Genetically modifying strains for unnatural microbial terol lowering agents (Xue et al. 1999). PKS are encodednatural products by a cluster of continuous genes and have a linear modular organization of similar catalytic domains thatCloning the metagenome build and modify a polyketide backbone (McDaniel et al. 1999). Microbial genes can be engineered to produceCulturable organisms provide only a finite pool of enzymes with novel catabolic activities (Kennedy andsecondary metabolites (Kennedy and Hutchinson 1999). Hutchinson 1999). The cloning of biosynthetic pathwayOne approach to maximize the diversity of natural genes from Streptomyces has allowed the production ofproduct extract libraries has been to access the DNA novel compounds by mixing the antibiotic systems ofdirectly from uncultured microorganisms. DNA can be different antibiotic-producing strains (Hopwood 1999).isolated directly from an environmental sample digested Novel compounds were produced by gene transferinto large fragments with restriction enzymes and cloned between strains producing the isochromanquinone antibi-into an artificial vector (Handelsman et al. 1998). The otics actinorhodin, granaticin, and medermycin (Hop-vector is then transformed into a surrogate host (Stokes et wood et al. 1985). This pioneering work has beenal. 2001). Environmental DNA libraries can be prepared developed by many others for the production of novelwith large fragments of DNA from a wide range of enzymes and unnatural natural products (Seow et al.uncultivated bacteria within an environmental sample 1997; Stokes et al. 2001; Christiansen et al. 2003). These(Courtois et al. 2003). This is described as screening the modular PKS clusters have been manipulated throughmetagenome, the genomes of the total microbiota in an introduction of different loading domains that specify aenvironmental sample (Rondon et al. 2000). Expression branched chain or cyclic substrate and directed inactiva-of the heterologous genes from unculturable microbes tion or insertion of individual catalytic domains tomay lead to the production of novel compounds. How- produce new enzymes (McDaniel et al. 1999). Combina-ever, it must be noted that these biosynthetic and torial biosynthesis has also been used effectively toregulatory genes could be dormant in the host and generate novel compound and enzymes in Type I andoptimal induction conditions may be required for the Type II PKS systems. This type of approach can beproduction of novel natural products. applied to many other modular enzyme systems (Rix et al. A high-throughput genome scanning method has 2002; Walsh 2002).recently been developed that allows discovery of meta- At a molecular level, DNA shuffling mimics, yetbolic loci-independent of expression. Genome sequence accelerates, evolutionary processes, and allows the im-tags (GST) are genes involved in natural product biosyn- provement of individual genes and sub-genomic DNA
454fragments (Zhang et al. 2002). DNA shuffling can be used reached a consensus for defining the fundamental criteriafor directed evolution and pathway engineering by of biological diversity to the species (Cohan 2001).recombination, and for combining useful mutations from Prokaryotes exchange chunks of their genomes tooindividual genes (Crameri et al. 1998). An example of frequently to make any meaningful species definitiongene shuffling with four individual cephalosporine genes (Doolittle 1999). An accurate definition of a fungalgave a yield improvement of between 270 and 540-fold. species is also problematic. Certainly for all types ofThe best clone had eight segments from three of the four microbes, the basic unit is the “ecotype” (Cohan 2001)genes and 33 point mutations (Crameri et al. 1998). This also called “geovar” (Staley and Gosink 1999). This is thetype of approach has now been used at the genome level reason why dereplication of strains has to be consideredand can efficiently generate combinatorial libraries of only within populations isolated from a geographical-new strains (Zhang et al. 2002). This type of approach has ecological region. A strain of Streptomyces hygroscopicusthe potential to facilitate cell and metabolic engineering, isolated from California is not necessarily identical to oneand to provide a non-recombinant alternative to the rapid isolated from China. Indeed, more than 200 secondaryproduction of improved organisms. metabolites have been isolated from various Streptomyces In order to target the full potential of genetic hygroscopicus strains.engineering for industrial strain development, methods When building a library of microorganisms for use inmust be developed to understand the genetic control of HTS, thousands of strains are isolated and have to bemetabolite production and to identify potential routes for selected and characterized. The first step remains carefulgenetic engineering. An example of such an approach is morphological observation. This allows the cultures to beintegration of genomic, transcriptional array and metab- separated into taxonomic groups. Further speciation canolite profiling to guide yield improvement of lovastatin be done using molecular or biochemical methods.(Askenazi et al. 2003). Strains of Aspergillus terrus were Biochemical culture-dependent techniques such as fattyconstructed to produce enhanced or reduced yields of acid analysis, pyrolysis mass spectrometry (Goodfellow etlovastatin. Comparison of genomic microarrays, tran- al. 1997) or FT-IR analysis (Bastert et al. 1999) werescription profiles and HPLC analysis of the fermentation developed initially for clinical isolates and cannot bebroth allowed insight into the genetic and physiological easily transferred to environmental samples that requirecontrol of lovastatin production (Askenazi et al. 2003). prolonged growth periods. Changes in the medium, Genetic engineering and pathway modification will incubation temperature and growth period can alter theundoubtedly be important in strain optimization in the profile of the organism and hence results can befuture. These methods provide a targeted approach for effectively compared to one another only within a singleconstruction of novel pathways and, in turn, the potential experiment. However, if the culture conditions can beof novel natural products. standardized, the use of Pyrole-mass spectroscopy anal- ysis can reflect similar clustering of taxonomic groups as molecular methods (Brandao et al. 2002b).Monitoring diversity while reducing redundancy The morphotype groups can be separated further usingof microbial strains and extracts molecular methods such as restriction fragment length polymorphism (RFLP), which can differentiate strains toIn order to increase the quality of the above-diversified the subspecies level (Schloter et al. 2000; Brandao et al.microbial natural products, it is important to spend more 2002a; Vermis et al. 2002). Ribosomal genes, includingtime upfront on the pre-screening of the strains and the the intragenic spacer regions, have been used routinely toextracts to remove redundancy and select for novelty. differentiate both fungal and actinomycete strains. Thou- sands of sequences are available in GenBank and the Ribosomal Database Project that can be used to phylo-Characterization and selection of microbial strains genetically identify interesting organisms. The pitfalls of relying on PCR-based rRNA analysis as a measure ofAny library of microorganisms is likely to have a high microbial diversity in environmental samples have beennumber of duplicate strains. While identical strains from emphasized (von Wintzingerode et al. 2002). Sequencingthe same site may be excluded on isolation, many strains of other molecular markers, while costly, does howeverof the same species may be collected over time from a allow identification to the species level. When used inwide range of collection sites. While these strains may be combination with RFLP, strains can be separated to theuseful later in strain optimization, they are redundant and subspecies level (Schloter et al. 2000).costly in the initial screening phase and decrease theprobability of finding novel compound. Thus, it isimportant to dereplicate the culture collection. However, Characterization and selection of microbial extractsone must consider that strains of the same subspecies mayproduce different compounds. Strains of the same species may generate different Many methods can be used to dereplicate cultures, but chemistry in the same medium. The first physicalmolecular techniques are especially well suited to this characteristics of unknown natural products are deter-type of analysis. However, bacterial taxonomy has not yet mined during the chemical extraction and concentration
455steps. Solvent-based and acid-base partitioning experi- ture determination methods could implement data fromments can help define hydrophobicity and types of various spectra to raise the confidence level.functional groups of the natural product structures. A discussion of natural product chemicals screening, The most effective selection from the metabolic aspect purification and dereplication to prevent repeated discov-is chemical profile analysis by high performance liquid ery of a given natural product or its producing organismchromatography (HPLC) and thin layer chromatography will be presented elsewhere (MS in preparation).(TLC) (Nielsen et al. 1999). Identical HPLC retentiontime or TLC Rf values may not tell you if two compoundsare exactly the same, but different values definitely Conclusions and perspectivesindicate they are different. Micro-scale extraction proce-dures have been developed (Smedsgaard 1997) and can be One prerequisite to natural-product discovery that re-automated. The first selection criterion is the metabolic mains paramount is the range and novelty of molecularcreativity of strains, as number of peaks analyzed by diversity. Currently, natural product chemistry is goingHPLC with detectors of evaporative light scattering through a phase of reduced interest in the drug discoverydetection (ELSD), vis-photodiode array (PDA), chemilu- field. However, new developments may turn around thisminescent nitrogen (CLND) and time-of-flight mass negative perception:spectrometer (TOFMS) (Yurek et al. 2002). Nielsen etal. (1999) proposed a method for identification and – The systematic exploitation of selected ecosystemsconfirmation of chemical compounds classification, based combined with the development of new techniques andon single- or multiple-wave length chromatographic media for isolation of novel microorganisms will allowprofiles. Chromatographic matrices from analysis of the collection of representative strains from large partspreviously identified samples are used for generating of the micro-population. This maximized biodiversityreference chromatograms and new samples are compared will deliver chemical diversity for a given ecosystem.with all chromatograms by calculating resemblance – The direct expression of environmental DNA inindexes. In addition, the method allows identification of heterologous surrogate hosts is progressing. There ischaracteristic sample components by local similarity a need for rapid and sensitive detection and charac-calculations. terization of new metabolites as well as their corre- The basic methods used to compare microbial extracts sponding gene clusters.are HPLC-DAD (diode-array-detection) and HPLC-MS. – Manipulation of physiology should be based onResearchers at Lilly developed a rapid (about one sample experimental design and measurement of secondaryper minute) surrogate measure of microbial secondary metabolism. Co-cultures will give novel insight intometabolite production computed from the electrospray secondary metabolism and will require the develop-mass spectra of samples injected directly into a spec- ment of new vessels for stable mixed-culture fermen-trometer (Higgs et al. 2001); Zahn et al. 2001). tation. The development of a multi-channel mass spectrom- – Gene-shuffling coupled with the genetic engineeringetry interface has allowed analysis at high-throughput power of, for example, PKS, will allow the generationlevel (Eldridge et al. 2002). In most cases, LC-MS (liquid of hybrid or unnatural microbial natural products.chromatography-mass spectrometry) is the most sensitive – Total synthesis of natural products with interestingmethod for obtaining dereplication information about a biological activities is paving the way for preparationcompound. A recent development is an 8-way fully of new and improved analogs. Combinatorial chemis-automated parallel LC-MS-ELSD system for the analysis try permits the selection of the best drug from a largeof natural products (Cremin and Zeng 2002). LC-NMR number of candidates. Beyond synthesis and evalua-(liquid chromatography-nuclear magnetic resonance) tion of organic molecules, a number of new bioorganicshould become operational in the near future, allowing methods are emerging on the horizon.the on-line identification of organic molecules (Bobzin et – In natural product chemistry, the rapid and accurateal. 2000a, 2000b). LC-NMR, despite its lower sensitivity differentiation of chemical compound profiles is basedcompared to LC-MS, provides a powerful tool for rapid on on-line measurement by LC-ELSD, DAD, MS, andidentification of known compounds and identification of NMR.structure classes of novel compounds. LC-NMR isespecially useful in instances where the data from LC- Today, more than 30,000 diseases are clinicallyMS are incomplete or do not allow confident identifica- described, but less than one-third of these can be treatedtion of the active component of a sample. symptomatically, and only a few can be cured (Schultz For strains and chemical tracking, an in-house and Tsaklakidis 1997). New chemical entities as thera-database has to be built up to integrate commercial peutic agents and for agricultural applications are urgent-available databases such as Antibase (Chemical Concepts, ly needed. Natural products can continue to play a majorWeinheim, Germany) or the Dictionary of Natural role in drug discovery. New strategies to natural-product-Compounds (2002). There is no single technique that based drug discovery will increase chemical diversity andgives 100% confidence to differentiate any two natural reduce redundancy. Maximizing the discovery of newproduct chemical profiles, but computer-enhanced struc-
456compounds and minimizing the re-evaluation of already Brewer DG, et al (1977) Beneficial effects of catalase or pyruvateknown natural products will be crucial. in a most-probable-number technique for the detection of Staphylococcus aureus. Appl Environ Microbiol 34:797–800 Bruns A, et al (2002) Cyclic AMP and acyl homoserine lactonesAcknowledgements The authors would like to thank Drs. Andy increase the cultivation efficiency of heterotrophic bacteriaStaley, Xiaoyu Shen, Eugenie Boutin, Hyedong Yoo, Alexei from the Central Baltic Sea. Appl Environ Microbiol 68:3978–Belenky, Bill Pierceall, Baoliang Cui and Shuhui Chen for critical 3987reading and comments on the manuscript. Additional gratitude is Bull AT (2000) Clean technology: industry and environment, aextended to the drug discovery teams at Cetek, Inc. viable partnership? Biologist (London) 47:61–64 Bull AT, et al (2000) Search and discovery strategies for biotechnology: the paradigm shift. 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