Novel thermostable enzymes for industrial biotechnology (THERMOGENE)

There is an increasing demand for new thermostable enzymes with enhanced performance and/or novel functionalities that could provide savings in time, money and energy for industrial processes in the areas of high value chemical production and many other "white" biotechnology applications.
Enzyme chemistry can make reactions feasible that are currently unavailable using conventional chemical methods. Use of enzymes for chemical processes is a route to lower energy consumption and reduced waste generation. In addition the selectivity of enzymatic processes reduces raw material costs and the safety issues surrounding the production of wasteful bi-products. Optimised enzyme production will lead to economically viable and cost effective, sustainable production.
THERMOGENE will focus on the discovery of selected transferase enzymes with known and potential commercial applications. These include enzymes able to transfer 2-carbon units, transketolases; able to transfer amine groups, transaminases; able to transfer isoprenyl or prenyl groups, prenyltransferases and able to transfer methyl and hydroxymethyl groups, methyl and hydroxymethyl transferases.
The use of enzymes in green chemistry and biotechnology is increasingly important. This is partially driven by the pharmaceutical sector in the development of new therapeutic agents that are required to be optically pure compounds. The number of new drug intermediates produced by enzymes is expected to rise significantly in the next few years.
One disadvantage of using enzymes is that proteins are often not stable during the reaction conditions required for the industrial process. This is still a major bottleneck in the commercial use of enzyme catalysts.
This proposal will concentrate on discovery of novel enzymes from marine and terrestrial environments. This will use newly sequenced genomes of thermophilic bacteria and archaea and metagenomes isolated from thermophilic environments. The latter will allow us to sample the DNA from organisms not able to be cultured in the laboratory and also the genetic material from viruses. The enzymes isolated from organisms able to grow at high temperatures will have higher stability in water based and solvent based conditions which are required for the industrial processes. It will also seek to discover enzymes with new and novel activities which are often found in these organisms that have evolved to have different fats in their cell membrane and different pathways in their metabolism.
At the end of the project we will identify many new biological catalysts that can be used to replace and work together with traditional chemical processes. This will lead to a greener and more sustainable environment for future generations.

The application of enzymes in 'white biotechnology' for the synthesis of industrially important chiral compounds is becoming increasingly important for the pharmaceutical industry. Many companies who were traditionally not incorporating biocatalysis in their drug production programmes are now very keen to develop the technology. The wealth of genome data now available makes searching for enzymes using both advanced bioinformatic and substrate screening approaches an area for development. Also more of the classified enzyme groups are being investigated for their application in industrial biocatalytic processes which are often used with a traditional chemistry synthetic step.
This project will concentrate on the transferase class of enzymes. These include the transaminase enzymes that are already accepted for commercial production of chiral amines and amino alcohols. These are key building blocks in many important drugs. The transketolase enzymes that have also been demonstrated for use in large scale biocatalysis for the production of unusual sugars (Lilly et al., 1996). In addition other transfer enzymes including the soluble prenyl transferases (some unique to Archaea; Ohnuma et al.,2000) include trans-polyprenyl diphosphate synthases, cis-polyprenyl diphosphate synthases and ABBA aromatic prenyltransferases. The aromatic prenyltransferases found in bacteria allow a promiscous prenylation of different aromatic substrates which is advantageous for their chemoenzymatic synthesis of bioactive compounds (Heide, 2009). The methyl and hydroxymethyl transferases have potential for development as commercial biocatalyts (Vidal et al., 2005, Koehl, 2005).

The THERMOGENE project will discover and develop enzymes that can transfer a variety of chemical groups from one molecule to another. The proposal will develop more industrially stable counterparts of two already studied transfer enzymes, that can transfer two carbon groups, transketolases and amine groups, transaminases. It has already been shown that these enzymes can work together in tandem to product high value chemical compounds. In particular the less studied class IV and class VI will be studied. In addition other transferase enzymes studied in any detail as regard for their application for commercial biocatalysis namely soluble prenyl transferases and methyl and hydroxymethyl transferases. This combination of different transferase enzymes will ensure a wealth of new thermally stable enzymes will be discovered with industrially relevant applications.
The enzyme discovery in WP2 will develop new bioinformatic approaches to search for different transferase enzymes in recently sequenced thermophilic genomes which have not been investigated to date. This will produce an impact into evolution of different transferase enzymes and their mechanism. It will also have an impact on the identification of new stable enzymes that can be used to drive the bio-economy in Europe.
The cloning, expression and enzyme production in WP 3 will develop new expression hosts and vectors that can be used to produce industrial scale quantities of soluble robust biocatalysts. This will have an impact on the over-expression of other thermophilic enzymes of industrial interest. The screening in this WP will have an impact on the development of new screening techniques where the technology can be transferred to screening of other biocatalysts.
The overall information gained will have an impact on the supply of enzyme preparations by partner 4 and other major pharmaceutical companies.
The biochemical and structural studies in WP4 will have an impact academically on our understanding of the structures of the different transferase enzymes and how this can help in our understanding of 'natural' methods of protein stabilization. It will also impact on our understanding of enzyme mechanism and substrate specificity.
Overall the results from the THERMOGENE project will make a major advance in enzyme discovery and will bring a variety of new transferase biocatalysts to the European industrial community in the area of 'white biotechnology'. The project will therefore be of the upmost importance to help European companies maintain their expertise and leading role in this area.
This project will provide cutting-edge tools and resources to the European and international scientific/biotechnological community, and also nucleate a European network of laboratories/industry aiming to exploit these tools and resources for biotechnological innovation.
The project will train early career scientists and graduate students in this multidiscplinary area and promote exchange between countries within Europe. It is anticipated for each partner to host at least one meeting in their own country and to encourage personnel employed and associated with the project to visit each the different laboratories in order to exchange expertise as appropriate at different stages in the research programme.