Exploiting extremophile proteins could mean new era for industrial processes
|Organisms that can survive in extreme conditions, such as volcanic vents, have opened up many research fields|
Organisms of the Archaea Kingdom have attracted a great deal of interest in the biotechnology industry over the last few years. Many of these ancient prokaryotes have adapted to thrive in extremes of temperature, pressure, pH, salinity, radiation or oxygen tension, such as volcanic hot springs, alkaline lakes, deep seas black smokers and highly acidic solutions that seep from ores and mine refuse piles.
To survive and thrive in such harsh environments, extremophiles have developed ingenious strategies for stabilising their nucleic acids and other macromolecules in vivo, including novel chemical transformation and energy transduction processes, and extremely heat-stable proteins. Understanding the proteins involved in the molecular stability of these organisms has opened up new fields of research into their potential microbiological and technical applications in industrial processes.
Many extremophiles have already proved their commercial suitability, and applications that use them include manufacturing liposomes for drug delivery and cosmetics, waste treatments and environmental cleaning, food production and molecular biology. Despite their potential, the use of extremophiles has so far not been as straightforward as hoped. They can suffer from low cell yields when grown outside their natural habitat, so understanding their physiological characteristics and optimal cultivation conditions is essential to obtain the highest possible yields and realising their potential.
Using Archaea metabolism to help the environment
The production of biofuels, like ethanol, is a good example of how extremophile proteins can be used to optimise existing industrial processes. Making biofuels has traditionally been based on industrial batch fermentation, a costly and time-consuming procedure. Cooling of the fermentation vessels for catalysis reactions is expensive and frequent periods of downtime are needed to remove products and add more plant material. By using the enzymes of extremophiles that need temperatures above 80oC to work instead of traditional enzymes, catalysis can take place at high temperatures. No expensive cooling steps are required and the increased heat allows the ethanol to be collected much more easily. In addition, the vessels can be fed constantly with new material, which eliminates downtime.
Another good example of how organisms can be used to benefit the environment is bioremediation. Toxic materials like industrial effluents, crude oil and other xenobiotics are not easily treated by conventional biological methods. However, some extremophiles can convert these materials into harmless products like CO2, making them an effective and inexpensive method of environmental cleaning following leaks and spills. In addition, understanding the metabolic pathways of the organisms means ways can be found to promote the multiplication of those already present in the environment and speed up the whole cleaning process naturally.
Taking proteomics to extremes – extremophiles that is!
This type of extremophile research has only been possible in the last few years because of the emergence of protein quantitation technology. Proteomics laboratories have traditionally had to finely balance the accuracy of protein quantitation with the amount of proteins identified. Despite being able to successfully characterise and identify peptides, quantitating them has not been nearly so easy. This frustrating situation was partially resolved with the development of chemical labelling reagents in 1999, which allowed a rough estimate to be made of the relative protein abundance between two proteomes. It was not until the introduction of labelling reagents with tags in 2003 that specific signals could be generated on mass spectra and major headway in protein quantitation could finally begin.
Here in the Department of Chemical and Process Engineering, University of Sheffield, we have been studying a variety of extremophiles using high throughput protein quantitation technology for the last three years. The department’s mission is to develop metabolic engineering and proteomics techniques to solve biomedical, biochemical and environmental engineering problems. The Biological and Environmental Systems Group is especially interested in studying how extremophiles, like the hyperthermophile Sulfolobus solfataricus, function and whether their proteins hold the key to advancements in drug discovery, biofuel production and bioremediation.
We approach our research from a systems biology perspective, which aims to understand the subtle interplay between the components of an organism and the biochemical reactions of those components. It is important to study an organism as an integrated network because the intrinsic properties that determine structure and function are not obvious when analysed in isolation. At all stages, quantitative information is needed so modelling and simulation software can produce probable in silico behaviours. These are then compared with those observed through experiments to eventually predict which experimental changes and manipulations have the most worthwhile effects. In theory this approach should result in an accurate representation of the in vivo behaviour of the system or organism under study.
Expanding to cope with success
With such a dependence on quantifying everything we do, it is not hard to imagine how the development of high throughput protein quantitation technology answered some big questions in our research. In 2003, the department created a brand new £800,000 Systems Biology Laboratory so we could expand our work on extremophiles and install a wide variety of state-of-the-art quantitation instruments, including an Applied Biosystems QSTAR XL System.
This system is used for ‘shotgun’ proteomics based on multi-dimensional protein identification technology, or MudPIT for short. Our aim is to generate as much quantitative data as possible and we have found the most effective way to do this is to differentially label our peptides with iTRAQ reagents, again from Applied Biosystems. The new iTRAQ reagents allow us to run four separately labelled samples in a single MS experiment and do four quantifications simultaneously. Because we are interested in the overall dynamics of the systems we study, we have been practicing nested multiplexing with the iTRAQ reagents to give up to 14 data points at a time. In addition, the isotopes can really help us with peptide identification, which is sometimes more interesting than the quantification! The isotopes allow us to identify proteins with greater confidence and prove useful for de novo sequencing.
Once we have got all the quantitative information we need, we overlay mathematical models on top of the data to identify what components have been quantitatively up- and down-regulated in the various metabolic pathways, tissues, cells etc. This integration of high throughput experimentation with bioinformatics is essential for making sense of the large quantities of data generated from the proteomics experiments. By combining the available genomic, proteomic, transcriptomic, metabolomic, fluxomic and kinetic data, from both our experiments and public databases, with data generated by computational modelling and simulation, we can begin to build a picture of a metabolic system in its true context. In the case of our Archaea studies, we can use the knowledge to develop new productive strains or improve old strains of organisms for industrial, medical and pharmaceutical purposes.
New directions for research
Because of what we learnt about proteomics during our first years working on Sulfolobus, we have been able to take on diverse proteomics projects in areas we never expected to be working on, including:
• the identification and quantitative analysis of differentially expressed proteins in human kidney disease relative to healthy tissue counterparts
• the identification and quantitative analysis of differentially expressed proteins in oviductal tissues of pigs with NCP to help draw parallels with human infertility
• the identification and quantitative analysis of stem cell cultures for therapeutic use.
In addition, we have been quantifying novel secondary metabolic compounds in Cyanobacteria that could be developed into chemotherapeutic drugs and biochemical research tools. Marine-dwelling Cyanobacteria naturally produce hydrogen gas and numerous secondary metabolic compounds, such as non-ribosomal peptides (NRP) and polyketide (PK) structures, which demonstrate antibacterial, antiviral and antiprotozoal activity. These could prove to be new novel targets for development in the pharmaceutical and life science industries.
We are particularly keen to expand our work in the biomedical area and are being increasingly drawn towards studying post-translational modifications like phosphorylation, so we may invest in more technology and focus in that area. However, with the QSTAR XL System running around the clock, even on Christmas Day, and the group tripling in size in as many years, we might need to think about building another lab for ourselves first!
By Professor Phillip Wright
Head of the Biological and Environmental Systems Group,
Department of Chemical and Process Engineering,
University of Sheffield