Metabolomics – the final frontier

Yeast, to the great delight of Dr Paul Chambers, is now king. A wine yeast that Anthony Borneman, one of Chambers’ team members at the Australian Wine Research Institute in Adelaide, has sequenced has just been chosen as the model organism on which a truly systems biology approach in the Australian life sciences can focus.

The simple yeast, sequenced at the Australian Genome Research Facility (AGRF) recently, will bring together the different strands of BioPlatforms Australia, the overarching group set up through NCRIS funds to co-ordinate research capabilities in four different areas.

They are the Australian Bioinformatics Facility (ABF), based at Murdoch University in WA; Genomics Australia (GA), managed by the AGRF; and Proteomics Australia (PA), managed by the Australian Proteome Analysis Facility at Macquarie University.

The last piece in the jigsaw is Metabolomics Australia (MA), which was established in February last year to encourage and assist research using the new science of metabolomics. MA’s Victorian node, headquartered at the University of Melbourne’s School of Botany and at the Bio21 Institute for Molecular Science and Biotechnology, was officially opened by Victoria’s innovation minister, Gavin Jennings, on November 12.

(Another part of the jigsaw, a web-based network called Proteomics and Metabolomics Victoria, established to provide information and education to local scientists and to link them to industry and new technologies, was also launched on November 12. See www.pmv.org.au for more information.)

The platform convenor for the Victorian node, the School of Botany’s Professor Tony Bacic, said the facility had been six years in the planning and was now fully operational and open for business.

Part of that planning involved a bit of forward thinking on Bacic’s part: several years ago he head-hunted Dr Ute Roessner from the Max Planck Institute for Molecular Plant Physiology in Golm, Germany, and brought her out to Australia.

It was scientists at the Max Planck Institute who were credited with devising the field of plant metabolomics, particularly Dr Richard Trethewey and Professor Lothar Willmitzer, who was Roessner’s supervisor.

“It was always part of our physiological studies, measuring metabolites using enzymatic assays,” Roessner says. “But we were measuring one metabolite at a time using a different assay.

“Then new technology came on the market using mass spectrometry, and one of our senior people, Richard Trethewey, thought up the idea of using gas chromatography-mass spectrometry (GC-MS) to try to measure as much stuff in one go. That’s when he bought the first GC-MS.”

That was back in 1997, and since then metabolomics has developed into a field all of its own. Roessner was recruited to Australia in 2003, and now spends half her time with MA and half with the Australian Centre for Plant Functional Genomics, where she continues her research interest in abiotic stresses in cereals.

While she concentrates on the plant side, the Bio21 Institute’s Professor Malcolm McConville concentrates on microbial pathogens.

His group has used metabolomic approaches to study how Leishmania parasites survive in their animal and human hosts. Leishmania are an extremely important group of parasites, second only to the malarial parasite in terms of morbidity.

“It infects around 12 million people worldwide, in 88 countries, and there are no vaccines against Leishmaniasis, nor for any other protozoan parasites,” McConville says. “And it’s the usual story: drug therapies are appalling at the moment. Most of them were developed 50 years ago.”

The frontline drugs against Leishmania and related parasites are based on heavy metals, particularly arsenic and antimony, which can have severe side effects. There is obviously a great deal of interest in identifying new drug targets, and in his early research McConville concentrated on an unusual set of polysaccharide antigens that coat the Leishmania cell surface, which were thought to be vital for infectivity.

“We got a bit of a shock a couple of years ago when we and others got around to knocking out the genes that are involved in making the surface coat and found that the loss of these molecules had absolutely no effect on the long-term infectivity of the parasite,” McConville says.

“It did affect the initial stages of infection but once the parasites had survived the initial onslaught of the host they caused disease in the same way as wild-type parasites. This is becoming a reoccurring theme for many other metabolic pathways in Leishmania.

“It became apparent that we didn’t know what the parasite was doing in the body as distinct from what it was doing in the culture flask. So there was a need to develop methods for probing parasite metabolism or metabolic processes that were switched on during infection.

“People have used microarrays and proteomic approaches to identify genes and proteins that are switched on in other microbial pathogens during infection. However, Leishmania appears to be constantly on the alert for opportunities and has everything switched on all the time.

“It transcribes its entire genome and most of the proteins are constitutively expressed and yet it goes through these amazing morphological and metabolic transformations.

“So it was clear that we had to look at the metabolites themselves, which reflect the end product of all of these changes. Then the idea of metabolomics came. We needed to find out what was being switched on at the coalface.”

---PB--- Yea for yeast

Over at the Australian Wine Research Institute, Paul Chambers is very keen on a systems biology approach to study wine yeast. Chambers and his team have worked on sequencing a spore from the industrial strain N96 of Saccharomyces cerevisiae, named AWRI1631. This spore’s genome has been compared to that of the well-studied laboratory strain of S. cerevisiae and to another strain isolated from the lungs of an AIDS patient.

“We figured that the yeast that comes from what is effectively an opportunistic pathogen and the yeast that’s used in laboratory work and the yeast that’s used in wine production are about as distant as you can get within a species,” Chambers says.

“And that’s given us a really good insight into the extent of genetic variation across this species. We hope by sequencing other industrial yeasts we’ll get a clearer picture about what’s special about wine yeasts.”

And what is special about wine yeasts? When Chambers talks of these yeasts he is talking about a rather large group of different strains. Most of them are Saccharomyces cerevisiae but not all, and they perform differently, he says.

“We’ve done experiments here where we can take exactly the same grape juice and just inoculate it with different yeasts and we get quite different wines at the other end. So we are trying to get a handle on what it is about wine yeasts and the differences about wine yeasts that impact so much on the quality of wine and the efficiency of fermentation.”

This is where metabolomics is proving so useful. Wine is considered a mixed metabolome in that it has a grape-yeast metabolic interaction.

“Wine is a very complex mix of chemicals that come from the grape, and then they are modified and changed in fairly complex ways by the yeast cell,” Chambers says.

“So far all we’ve been able to do at a chemical level is look at a number of clearly definable compounds. There’s maybe 50 or more chemicals that we can target with standard metabolite analysis.

“In our typical assays we may look at 30 or thereabouts. Metabolomic analysis, we hope, will take us into the realm of hundreds that so far we haven’t even seen.”

One of his aims is to use metabolic engineering to build yeasts that will work in the way wine makers want them to work, and to use metabolomics to understand the complexity of the different compounds that affect flavour, aroma and stability.

“That’s why we’re in it,” he says. “The facility that we are running here as part of MA is not just going to be about yeast, although we have a vested interest at AWRI in bringing our yeast research into it. The facility will be open to research throughout Australia.

“But I think there is a level of complexity with metabolomics that means if we start with simpler organisms we might be able to make more rapid progress in the early stages.

“That’s another plug for yeast, I suppose. If you could climb into a yeast cell and have a walk around you’d see much the same sort of structure and activities as you would in human cell. They are designed quite similarly in many ways, but in yeast there are about 6000 genes so there are fewer to work with, there’s a lot less intergenic sequence, and of course we don’t have the complexity of multi-cellularity.”

---PB--- GC-MS – the workhorse

According to Ute Roessner, GC-MS is still the workhorse of metabolomics, while liquid chromatography-mass spectrometry (LC-MS) is still in its infancy. She believes, however, that LC-MS will eventually take over.

“There are a lot of compounds that are not detectable by GC-MS, and it is limited to small molecules and molecules that you can make volatile,” she says.

“Still, in its robustness and reproducibility, GC-MS is the best around, which is why a lot of people use it. With LC-MS, it is better to measure larger molecules but there is a lot of development with it. You can do a crude run which checks everything without really caring what you measure, but that’s not really what we want.”

Other techniques such as Fourier- transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) are also being further developed. One node of MA, the Separations Science Laboratory at Murdoch University’s School of Pharmacy, has FT-ICR-MS capability as well as standard GC-MS.

This lab is run by Professor Robert Trengove, who predominantly works in environmental as well as plant science.

“FT-ICR-MS is very high mass accuracy mass spectrometry, and it’s good to give you an idea of the accurate mass of the molecule you are measuring,” Roessner says. “But for metabolites, they are either very different or they are so similar that they have the same mass anyway.

“So with the high-resolution Q-TOF that I am working with now, you have enough mass accuracy. But if we have a compound of interest identified as a marker we may be able to identify its chemical structure by utilising Murdoch’s FT-ICR-MS.”

MA also has a node at the University of Western Australia, run by Professor Steven Smith at the ARC Centre of Excellence in Plant Energy Biology and the Centre of Excellence for Plant Metabolomics, and there is a node at the University of Queensland, run by Professor Lars Nielsen.

“The UQ node is more specialised in doing flux analysis, where you measure the flow of carbon between the different metabolite pathways,” Roessner says. “This is useful in metabolic engineering, especially in microbiology, and in plants like sugar cane to produce biodegradable plastics. It is very powerful and sophisticated technology.”

For Malcolm McConville, the reason why metabolomics has been a late developer in comparison to genomics and proteomics is both the analytical challenge and the very complexity of metabolic pathways.

“There is so much diversity in the chemistry of various metabolites, and huge variation in the concentration of different metabolites in the cell, so you need to use a wide variety of different analytical approaches,” he says.

Paul Chambers agrees. While proteomics researchers may argue otherwise, he says metabolomics takes biological complexity to a whole new level.

“Some of this will settle out in the coming years when we get the technologies and different platforms up and running, and we may well find that there is a sufficient level of overlap so we can offer similar services.

“But metabolomics, to get started, needs to have its own identity and its own people to troubleshoot problems. That’s what Metabolomics Australia is all about.”