Structure, Mechanism and Application of Hydratase/Dehydratases: Flavours, Fragrances and Polymer Precursors

Lead Research Organisation: University of York
Department Name: Chemistry

Abstract

Biocatalysis is the application of enzymes and microorganisms to the production of chemicals for the pharmaceutical, agrochemical and bulk chemical industries. As part of the wider field of 'Industrial Biotechnology' (IB) that is making an increasing contribution to the production of essential chemicals. Biocatalysis is an attractive alternative to traditional methods of chemical synthesis in some applications as it provides processes that are environmentally benign and highly selective, in a way that many conventional catalysts are not. One example of this is the ability of biocatalysts to generate single optical isomers, or 'enantiomers' of otherwise identical chemical products, where the properties of different isomers can have vastly different effects in a biological context, such as in a drug. Researchers in the chemical industry are always looking for new biocatalysts to replace established chemical processes, and these new enzymes are often discovered in microbes. Hydratase enzymes are biocatalysts that are capable of turning one form of abundant petrochemically-derived hydrocarbons, known as alkenes, into synthetically valuable alcohols, in single isomer form, which can act as precursors for the pharmaceutical and flavour/fragrance industries. These new enzymes offer great promise, but being only recently discovered, little is known about what they look like or how they work, and further knowledge of these aspects is essential if the enzymes are to be engineered for improved activity and process suitability. In this project, we will study a new class of hydratase enzymes that not only catalyse the production of alcohols from alkenes, but are also able to take naturally occurring alcohols, and, in the reverse reaction, turn them into non-natural alkenes such as isoprene for the production of polymers including rubber. We will determine the structures of the enzymes using X-ray crystallography, and use the information to study how the enzymes work, and to inform protein engineering studies that will help us change and improve the enzymes for different applications. Finally we will, with the assistance of commercial partners, apply the improved enzymes to the transformation of useful molecules, with a view to providing new selective and sustainable methods of chemistry for industrial processes.

Technical Summary

The asymmetric hydration of alkenes to form optically active alcohols would be a valuable biocatalytic process, but enzymes that catalyse this transformation have until now largely been restricted to those that work on electron deficient alkenes, such as maleate, or the CoA thioesters of carboxylic acids. Recent research has unearthed new hydratase enzymes that catalyse the asymmetric hydration of electron-rich alkenes; such biocatalysts hold great promise for the generation of chiral alcohols from abundant alkene precursors. One enzyme, linalool dehydratase-isomerase, catalyses the asymmetric hydration of the triene myrcene, to yield a single enantiomer of the product (S)-linalool, a valuable flavour-fragrance additive, but also the reverse dehydration reaction, and the isomerisation of linalool to the product geraniol. In addition to the production of chiral flavour terpenols, LinD has been shown to catalyse the dehydration of naturally accessible alcohols, such as isoprenol and crotyl alcohol, to the bulk polymer precursors isoprene and butadiene, opening up new prospects for synthetic biology routes to sustainable polymers. LinD is representative of a new sub-family of enzymes with no close sequence homologs in the databases, and there is little structural or mechanistic information that would inform engineering of these enzymes for improved properties. In order to enable LinD-type enzymes for applications in industrial biotechnology, we have recently determined the structure of the enzyme, and are now in a position to conduct detailed mechanistic studies and rationally informed engineering of enzymes within this new group. Together with our commercial partner, we will employ X-ray crystallography, mutagenesis and enzyme assay, electrochemical methods and biotransformation studies to characterise the LinD-type enzymes, engineer them for improved performance, and explore their suitability for applications in industrial biotechnology.

Planned Impact

The project addresses the investigation and development of new biological catalysts for the production of terpenes, terpene alcohols and other alkenes, and has potential benefits therefore to biotechnologists working in academia and in in the flavour, fragrance and polymer industries. There are also potential benefits to the pharmaceutical sector, given the demonstrated bioactivity of terpenes and their derivatives. The development of biological routes to chemical products also has benefits for the wider community in industrial chemistry as such methods can supplant conventional chemical methodology, being superior in aspects such as efficiency, selectivity, but also assisting in the move towards a more sustainable platform for the chemical industry, rooted in bio-based feedstocks. The chemicals sector in the UK is estimated to contribute £15 bn per year to UK GDP (source: Chemical Industries Association, 2015) so improved processes for the chemical synthesis can make a clear contribution to economic competitiveness. Scientists will benefit from the proposed research through the development and dissemination of new methods of biological conversion of terpenes and other alkenols, as well as associated analytical tools and data. These benefits are delivered through publication in scientific journals, but also through presentation at conferences and academic-industrial networks such as the BBSRC-funded NIBBs, such as BIOCATNET and the the Centre of Excellence for Biocatalysis, Biotransformations and Biomanufacture (CoEBio3), of which the University of York is a member. The PI collaborates extensively with representatives of major UK and other international chemical companies (GSK, Dr Reddy's, Merck, Codexis et al.) both through CoEBio3 and also company-specific collaborations. Our industrial contacts are often laboratory scientists in biotransformation laboratories who will be able to quickly transfer information on new techniques and developments to their own grups. Further direct benefits will of course be experienced by the commercial partner, Genomatica, who will obtain protocols and data on enzymes of proprietary interest that feeds directly into the development of industrial biocatalytic processes.
Beyond the academic and industrial researchers that benefit most directly from new technology advances within the project, relevant UK government stakeholders also derive benefit in general from successful developments in Industrial Biotechnology (IB), as these help to publicise the benefits of investing in IB and also inform the development of future policy. IB makes a significant contribution to the UK economy, with an estimate of 225 companies and 8,800 employees involved in generating £2.9 billion of sales in 2013-14 alone (source BBSRC). Organisations such as the BiS-funded Bioscience Knowledge Transfer Network are well-placed to employ these case studies to champion IB in the presence of companies who had not previously considered using this technology. The general benefits of IB such as those described in the project can be communicated through Bioscience KTN activities such as visits, newsletters, webinars and workshops.
Finally, the wider benefits of the work are experienced by the general public. As consumers and users of chemical products, they will benefit from their more efficient production. In the longer term, the incorporation of IB into chemicals manufacture directly benefits the public in terms of the improved environment associated with a more efficient, sustainable and environmentally benign chemicals industry. The impact of these developments on the public can be communicated at local and international level using press releases, public engagement events and articles in popular science journals.

Publications

10 25 50
 
Description LinD is an enzyme that converts cheap and readily available myrcene into valuable products such as the flavour component Linalool. As part of the project, we have determined the structure of the enzyme. Mutation of the enzyme using the structure can give an improved enzyme for industrial processes. We have constructed a mutant library of the enzyme and been able to shed light on the role of amino acid residues involved in catalysis. We have also begun to study other LinD enzymes from Nature and started to explore their potential for the chemical reactions of interest.
Exploitation Route Engineering of LinD for altered or improved activity
Sectors Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Genomatica 
Organisation Genomatica Inc
Country United States 
Sector Private 
PI Contribution Research Collaboration Structural Biology
Collaborator Contribution Research Collaboration Analytical Techniques
Impact Publications in progress
Start Year 2017
 
Description Modelleing collaboration 
Organisation University of Girona
Country Spain 
Sector Academic/University 
PI Contribution Provision of data for use in computer modelling
Collaborator Contribution QM/MM modelling of reaction catalysed by LinD (de)hydratase
Impact None yet
Start Year 2019
 
Description Technical University of Stuttgart 
Organisation University of Stuttgart
Country Germany 
Sector Academic/University 
PI Contribution Research collaboration - Structural Biology
Collaborator Contribution Research Collaboration - Enzyme Evolution
Impact Publications in Progress
Start Year 2017
 
Description York Festival of Ideas: Festival Fringe Family Fun Afternoon 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The session was called 'The Value of Vitamins'. Games were designed and prepared for children to participate, which focused on, for example, the modelling of chemical structures and the importance of vitamins to the diet.
Year(s) Of Engagement Activity 2018
URL http://yorkfestivalofideas.com/2018/community/festival-fringe/