Chemistry at the extremes

Life exists in gold mines, hot springs, oil wells and in cold Arctic waters. Extremophiles, the microbes that can survive such harsh conditions, often contain molecules that have applications in all areas of biotechnology. ‘Extreme temperatures and life conditions give rise to unique properties,’ says Helga Pedersen, Norway’s minister of fisheries and coastal affairs, speaking at the recent BIOPROSP conference in Tromsø, Norway, home of the world’s most northerly university. Pedersen was referring to the high salinity, high pressures and cold temperatures of the Arctic, where many extremophiles exist. However, they are found in other ecological niches around the world too – mainly characterised by high or low temperature, high salt concentrations, extremes of pH or high pressure – conditions, in other words, outside of those traditionally thought to support life.

Norway has built its country’s wealth on marine industry and is now turning its attention to marine or ‘blue’ biotechnology. Bioprospecting – searching the country’s fjords and cold seas for unusual and interesting microbes and invertebrates – could help to build the ‘industry of the future,’ according to Pedersen. The Norwegian government is hoping to kick-start the commercialisation of marine bioprospecting by putting NOK30m (£3m) this year, as initial funding, into its new national bioprospecting initiative including MabCent, which brings together bio-banking, genomics, high throughput screening and structural biology. MabCent is one of Norway’s researchbased innovation centres, set up by the Research Council of Norway and set up in late 2007. Based at the University of Tromsø, MabCent’s mission is to analyse and characterise bioactives from Arctic and sub-Arctic marine organisms that show potential for commercialisation.

In the short term, it is the enzymes that extremophiles produce which are likely to find most use in the biotech industry, particularly those adapted to temperatures above and below the range of conventional enzymes. The enzyme market is worth US$3-5bn/year and is growing at 10%/year, with applications in the pharmaceutical, starch production, food and beverage, research, cleaning, diagnostics and animal feed sectors. Arne Smalås director of the Norwegian Structural Biology Centre, NorStruct, has carried out studies comparing cold and regular versions of well-known enzymes like trypsin, which shed light on why cold-adapted enzymes are so efficient.

Tromsø-based Biotec Pharmacon has built a successful business from cold-adapted enzymes. Its shrimp alkaline phosphatase, shrimp nuclease and cod uracil-DNA N-glycosylase are used in molecular biology labs around the world. These enzymes are rapidly and irreversibly inactivated – which allows elimination of the usual heat inactivation step in the polymerase chain reaction (PCR) and gives a cleaner product. PCR, a method of amplifying DNA, is probably the most widely performed process in molecular biology. Meanwhile, ZyGEM, based in New Zealand and the US, has developed a range of research, forensic, agricultural and livestock applications for its thermophilic EAI proteinase enzyme, which produces very clean DNA samples. The company has a collection of hundreds of other ‘extremozymes’ awaiting commercial development.

Jenny Littlechild of the Exeter Biocatalysis Centre at the University of Exeter, UK, says there are many advantages to heat-adapted extremozymes. For instance, they are better able to tolerate organic solvents and are more resistant to proteolysis, which makes them more stable and robust for applications. Furthermore, some non-natural substrates are more soluble at higher temperatures. Her team isolates extremozymes from marine or terrestrial archaea, then clones and over-expresses them in a soluble form from E.coli for large-scale production. Heat-adapted enzymes are easy to purify because heat removes the host proteins.

Exeter group extremozymes used in the pharmaceutical industry include an alcohol dehydrogenase isolated from Aeropyrum pernix for production of optically pure alcohols; this has optimal activity at a temperature above 75°C and is also resistant to solvents such as acetonitrile. There is also an L-aminoacylase from Thermococcus litoralis for resolution of amino acids and amino acid analogues into optically pure forms. This is being used by Dow Pharma/Chirotech for the production of optically pure L-amino acids and analogues. Sulfolobus solfataricus has yielded a gamma-lactamase for the synthesis of optically pure gamma lactam, which is the building block for antiviral carbocyclic nucleotide drugs in multi-tonne amounts and has been used in the synthesis of GSK’s new anti-HIV drug abacavir (Ziagen).

‘Many companies are interested because industry wants chiral intermediates for optically pure drugs,’ Littlechild explains.

One of the most exciting near-term applications of extremophiles will be in the oil industry. Hans Kristian Kotlar, senior biotechnology specialist at StatoilHydro believes that oil-loving microbes could help both to make more of existing oil reserves and drive the development of a new generation of biofuels. His team has compiled a library of around 5000 microbial isolates from oil deposits around the world; these extremophiles live 2–5km below the land or sea bed surface at a temperature range between 50 and 120°C and a pressure of 200- 300 bars – equivalent to the pressure exerted by an elephant on the head of a needle.

‘Statoil Hydro is looking at the whole value chain for oil, from exploration to the end product,’ Kotlar says. The company is constructing DNA probes that will detect the presence of oil without having to drill – which is especially important in environmentally sensitive regions. The probes can detect specific microbial ‘fingerprints’ associated with hydrocarbon seepages from oil deposits, which differ from patterns found in the surrounding soil or sea bed.

Meanwhile, oil-associated microbes could also be applied to improving the properties of oil. Around two thirds of the world’s oil reserves are heavy to extra-heavy grades and their viscosity makes them difficult to handle and transport. In practice, only around 7-8% of heavy oil deposits are actually recovered. StatoilHydro is searching its library for microbes that can degrade the heavy components of oil, like n-alkanes and aromatic rings, and has found certain strains capable of transforming heavy grades of oil to give a proportion of lighter grades. Results from sand columns and radial reservoir models, which are more like ‘real life’ than lab experiments, look promising, so it may not be too long before oil-loving microbes are set to work on the heavy grades, where just a few percent improved yield could mean billions of dollars of extra revenue.

Like all oil companies these days, Statoil Hydro is also interested in biofuels, particularly second generation processes that do not use cereals to produce biodiesel or bioethanol. ‘We will not go into competition with the food chain,’ promises Kotlar. ‘We want to co-mingle various raw materials, such as animal fats, fish oil and plant oil.‘ Thermophilic enzymes can get to work on converting fats above their melting points of 50°-60°C, making these a viable feedstock. Although applications within the oil industry are the main focus, Statoil Hydro is keeping an open mind on also discovering novel anti-cancer and anti-malarial drugs in its oil-loving microbes.

But getting new medicines from extremophiles is probably a longer-term prospect, although the Norwegian researchers have made a promising start. Magdalena fjord in Svalbard, close to the North Pole, is being investigated for anti-cancer, diabetic, antiviral and immune-modulator compounds. Also, Valentin Stonik of the Pacific Institute of Bioorganic Chemistry at the Russian Academy of Sciences is looking at North-Western Pacific cold water organisms – mainly from the Sea of Okhotsk and also off the coast of Maine, in the North Atlantic. His group has recently isolated six new triterpene glycosides from the sea cucumber Cucumaria okhotensis – with potent immunostimulatory properties – as well as polar steroids from starfish that stimulate the differentiation and development of the body’s nerve cells. It is likely that there will be many surprises, and useful applications, as the tools of molecular biology are applied to those organisms that live on the margins of life – particularly those in the Arctic and other hostile environments.

Susan Aldridge is a freelance medical writer based in London, UK.