Natural products Extreme potential LIBRARY PHOTO LARSEN/SCIENCE CALVIN The world’s least hospitable environments are treasure troves for synthetic organic chemists and drug developers, reports Bea Perks

In 1995, a flock of 300 snow geese unravel the structures of previously migrating across the US state of In short unknown molecules produced by Montana landed on a lake near the  Temperature, pressure, these life forms. former mining town of Butte and toxicity and salinity died. Berkeley Pit lake marked the extremes can sustain Life from the jaws of death site of an abandoned open-pit copper unusual organisms The full potential of those molecules mine that had shut down 13 years  Extremophile enzymes remains unknown, but there is plenty earlier. Tests on the birds revealed can be more suitable of evidence to suggest they will be exposure to high concentrations of for use in biotechnology significant. For example berkelic copper, cadmium and arsenic. than those from regular acid, isolated from a species of fungus The appeal of the lake – 115 billion species, and their unusual living in the Berkeley Pit lake, has litres of acidic (pH2) metal-laden metabolites are a source been shown to have selective activity water – is not immediately obvious. of potential new drugs against ovarian cancer. But living in that toxic pool are  Growing extremophiles The race is on – there is clearly a organisms that could help treat a in the lab is difficult and need to clean up a lake which will kill spectrum of illnesses, from ovarian expensive, so synthesis any passing wildlife. But, perversely, cancer to Alzheimer’s disease. is often required to fully the lake may be harbouring life- Berkeley Pit lake is clearly an evaluate new compounds saving agents for mankind. extreme environment. The idea And it’s a race on several levels. that organisms could thrive in such While the Berkeley Pit lake is a environments wasn’t fully recognised dangerous manmade lake in serious until the 1980s and 1990s. The term need of remediation, there are ‘extremophile’ was first proposed naturally extreme environments in the 1970s by Robert MacElroy, a that require protection in their biochemist working at Nasa’s Ames natural state (see ‘Protecting Research Center in Moffat Field, natural extremes’, p51). One way or US. MacElroy predicted that the another, extremophiles are under inhospitable environment on Mars threat. There is a growing need to could be one day be colonised by devise ways of synthesising the Earth-based life. unusual, but potentially very useful, Biologists have been secondary metabolites produced by busy discovering ever more these organisms. extraordinary organisms inhabiting Among the best studied molecules a range of extreme environments on produced by extremophiles are Earth (see ‘Extreme extremophiles’, enzymes, sometimes referred to p50), prompting chemists to as extremozymes. The enzymes 48 | Chemistry World | June 2011 www.chemistryworld.org The acidic and metal- produced by organisms that inhabit laden waters of Berkeley extreme environments are, not Pit lake in Montana, US, surprisingly, quite unlike the enzymes may seem inhospitable, you will find in a standard textbook but they are home to (see Chemistry World, September bacteria and fungi that 2005, p51). They catalyse reactions produce unique molecules far outside the long-established like berkelic acid temperature, pH and pressure norms, suiting them to a range of biotechnological applications, particularly in the oil industry. More recently, the natural products produced by extremophiles have been attracting attention. These are usually secondary metabolites – molecules that are not directly required to keep the organism alive, but perform some kind of helpful function, such as defence mechanisms and colourings.

Hot and cold Several secondary metabolites isolated from thermophilic sources Psychrophilin D (organisms that thrive in the was isolated from a heat) have been shown to have cold-loving fungus in antibacterial activity. For example, Greenland one species of the thermophilic

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off southern Japan; to a group of ‘It’s a really toxic potential antifoulants capable of environment, fending off barnacle attachment, produced by the bacteria there are unique Streptomyces fungicidicus discovered microbes 5000m deep in the Pacific.

and they’re Sturdy old salts producing some The halophiles, organisms that thrive in high salt conditions, counter the really unusual osmotic stress of their surroundings metabolites’ either by accumulating or maintaining concentrations of osmolytes such as KCl close to that of their surroundings. Relatively little is known about the secondary metabolites produced by these organisms, and rather than (+)-Formylanserinone B offering cures for intractable human was isolated from fungi diseases, some of them seem more living 1335m below sea likely to make us ill. A halophilic level, but shows promise Gram-negative bacteria, Vibrio for treating leukaemia parahaemolyticus, was first isolated in 1950 as the cause of seafood poisoning. fungus Streptomyces thermoviolaceus barophiles; organisms that thrive Even less is known about secondary was shown to be a source not only under high pressure). These metabolites produced by organisms of a previously isolated antibiotic organisms normally have to be that thrive at high pH, the alkaliphiles. molecule called granaticin, but also able to withstand extreme cold as The enzymes from these organisms a source of a previously unknown well as high pressure, except for have attracted more attention though, biosynthetic precursor of that the few that live near hot vents. particularly for their use in biological antibiotic called dihydrogranaticin. Secondary metabolite production detergents. Among the secondary metabolites by piezophiles is particularly isolated from psychrophilic sources well studied. A mixture of two From lake to lab (organisms that thrive in the cold), Penicillium corylophilum strains The secondary metabolites produced three cyclic peptides – mixirins A, collected 1335m below sea level by organisms that thrive at low B and C – collected from a Bacillus produced five previously unknown pH, the acidophiles, are a different strain living in the Arctic, were shown compounds. Among these was matter, taking us back to the Berkeley to inhibit the growth of human colon (+)-formylanserinone B, which was Pit lake. Over 40 different bacteria, cancer cells. A species of cold-loving shown to selectively inhibit growth fungi, algae and protozoans, have fungus, Penicillium algidum – which in mouse leukaemia cells. been discovered in this one polluted, was found under a redcurrant bush in Apart from compounds with manmade lake. Despite the ecological Greenland – produces the previously antitumour activity, secondary disaster that the lake represents, unknown compound psychrophilin D, metabolites produced by over 20 previously unknown natural which shows some activity against a piezophiles have been linked to products have been isolated from its mouse leukaemia cell line. a range of applications: from a resident microbe population. previously unknown violet pigment Living in this toxic soup are life Deep, deep down isolated from a species of bacteria forms that researchers predict could Down at the bottom of the (Shewanella violacea), living help treat migraine, high blood ocean are the piezophiles (or 5110m down in an ocean trench pressure, ovarian cancer and lung cancer. One team is beginning to investigate the possibility that there Extreme extremophiles might also be compounds to help Extremophiles turn up in Arctic sea bed – arriving by treat neurological disorders like surprising places. We now some kind of passive transport Alzheimer’s disease. expect to find extraordinary mechanism. He is now figuring ‘It’s a really toxic environment, organisms on the sea floor, out why they are there, and there are some pretty unique in hydrothermal vents, or in what this might tell us about, microorganisms and they’re geysers and hot pools, but for example, the presence of oil producing some really unusual researchers were surprised reserves deep below the sea bed. secondary metabolites,’ says recently to find heat-loving And when they’re not Margaret Brimble, professor of thermophiles in the Arctic. Casey turning up in the ‘wrong’ place, organic and medicinal chemistry at Hubert, a geomicrobiologist at extremophiles can turn up the University of Auckland in New

JOEL E KOSTKA E JOEL the University of Newcastle, in just odd places. A couple Zealand. ‘We’re looking at making UK, found thermophiles lying Casey Hubert has found heat- of years ago a Japanese some of those metabolites because dormant in arctic marine loving thermophiles in the Arctic research team discovered a they’ve got some really unusual sediments, where it is 50°C new species of extremophile, structures that we haven’t seen colder than they would need and they sprang back to life. Microbacterium hatanonis, before in the chemical world.’ to grow. Hubert put them in a Importantly, he showed a stable living in hairspray. The bacteria Brimble’s group was the first thermophile-friendly incubator, supply of thermophiles to the grows best at 30°C, pH7. to synthesise berkeleyamide A, a secondary metabolite isolated from 50 | Chemistry World | June 2011 www.chemistryworld.org a Penicillium rubrum Stoll species living in the lake. Considering that the natural source of berkelyamide wouldn’t be available once the lake was cleaned up, a total synthesis of the molecule became a pressing need after it was found to have anti-inflammatory and potential antitumour activity. It inhibits the proteases caspase-1 and matrix metalloproteinase 3 (MMP3), which are implicated in the mechanisms by which some cancers spread. Her group is now working on improving the synthesis of the more complex berkelic acid, a highly competitive area worldwide. ‘Everyone’s trying to make it ... it’s a bit like the new taxol,’ says Brimble. Berkelic acid, and berkeleyamide A, were both first isolated by Donald and Andrea Stierle at the University of Montana, US. Berkelic acid has a very unusual tetracyclic structure. It too inhibits both caspase-1 and MMP-3, and it shows selective activity against an ovarian cancer cell line. As well as the anti-cancer KIDD HAMISH activity of secondary metabolites discovered in the lake, Stierle’s or near hydrothermal vents and other There is already a collection team is interested in targeting their extreme environments, but taking the of metagenomes from extreme antimicrobial potential. ‘Of particular organisms back to the lab is a different environments, which have been used interest are compounds with matter. Recreating these extreme Metagenomic analysis of to study extremozymes. Brimble antifungal activity, as opportunistic environments in vitro, in order to bulk DNA from extreme hopes that these extremophile DNA mycoses are a growing problem in study the molecules produced by locations can turn up new libraries could be transferred into immunocompromised individuals, these extraordinary organisms, is both enzymes that could be host organisms, which would then be including Aids patients and patients difficult and prohibitively expensive. employed industrially tested to see whether they produce on immunosuppressive therapy,’ says One way to get round this, suggest interesting secondary metabolites. Donald Stierle. Stierle and Brimble, is to go down a Currently, Brimble is working ‘metagenomic’ route. Metagenomics with a biotech firm looking at the Metabolite mining is the sequencing of bulk genetic DNA from extremophiles and using Despite their obvious potential, material from whole environmental it as a template ‘to produce the relatively little is known about the samples rather than from individual peptides normally produced by those secondary metabolites produced by organisms. Genomics techniques microorganisms’. A library can be extremophiles. Most of what is known are used to study communities of constructed using those peptides, about extremophiles relates to their microbial organisms where they which can then be screened against biology and ecology. It is one thing to live, rather than having to cultivate different drug targets. ‘It’s a new study these life forms at great depths, individual species back in the lab. approach,’ says Brimble, ‘looking back at the DNA level of unusual organisms and then creating the Protecting natural extremes proteome from that.’ Natural extreme environments its adaptation methods; by Nasa, the US Department Brimble’s team is making – from deep inside desert rocks understanding biodiversity and of Energy, and the US National analogues of the compounds to hydrothermal vents on the interactions within extreme Science Foundation, she says, discovered in the Berkeley Pit lake ocean floor – are threatened by environments; and finding but the roadmap will be useful and recently teamed up with another human activities and changing limits of habitability which to those agencies as well. research group at the University of climate. In April this year, could inform the search for ‘There’s a heightened payoff Auckland working on neurological the European Commission’s extraterrestrial life. scientifically of having different assays. ‘We’re going to screen these international coordination Mary Voytek, senior scientist groups work together,’ says compounds in neurological assays, action for research on life in for Nasa’s astrobiology Voytek. A prime example is which hasn’t been done before,’ she extreme environments (Carex) programme, says this is the hydrothermal vents, which says. ‘People tend to focus on cancer project launched a roadmap first effort by the European were originally discovered by and antiviral activity. We’re going identifying the most important Commission to mine its geologists and then capitalised to take these extremophile-derived questions for extreme community of extreme on by microbiologists, molecules and put them into assays environment researchers. environment researchers to astrobiologists, and synthetic that involve the brain.’ Research priorities include: identify the best topics to fund. organic chemists among life’s response to climate The report’s findings are not others. ‘No one group has Further reading and environmental change; dissimilar to those identified claimed them for itself.’ Z E Wilson and M A Brimble, Nat. Prod. Rep., 2009, 26, 44 www.chemistryworld.org Chemistry World | June 2011 | 51