A generic biocatalytic reaction platform for the stereospecific reduction and oxidation of alkenes

Lead Research Organisation: University of Manchester

Abstract

We propose an ambitious, interdisciplinary research programme to develop a viable manufacturing process, independent of redox coenzymes, to exploit a wide range of novel and industrially relevant redox biotransformations. Our approach is based on the development of (i) microfabricated electrode systems and arrayed microfluidic reactors, (ii) specifically tailored and robust redox catalysts and (iii) the integration and process optimisation of 'engineered catalysts' within the microreactor environment, incorporating on-line quantification of reaction products and facile removal of the desired product by microfluidic liquid-liquid extraction. The technology platform is designed to accommodate a wide range of redox biocatalysts by employing generic methods of electron delivery, and to by-pass the requirement for coenzyme recycling/regeneration. The outcome will be a viable manufacturing process for the production and rapid recovery of high valuable enantiomeric products for the pharmaceutical and synthetic chemistry communities. For the first time, our programme will drive the diverse catalytic potential of redox enzymes towards industrial exploitation, and will thus make a substantial contribution to the field of biocatalysis. The proposed programme is highly innovative and provides generic solutions to the key problems associated with the exploitation of redox enzymes in industrial biocatalytic processes. The programme capitalises on the unique combination of expertise available within the UK Centre of Excellence in Biocatalysis, Bioprocessing and Biomanufacturing (CoEBio3) at the University of Manchester and the excellent research infrastructure available to this group of research workers in the Manchester Interdisciplinary Biocentre. This direct involvement of the 13 member companies of CoEBio3 will provide an invaluable route for effective dissemination and implementation of the project findings.

Technical Summary

Redox biotransformations should provide many new, versatile routes to manufacture both chiral and non-chiral speciality chemicals. In practice, it is extremely difficult to operate these reactions economically at scale, since the reactions depend on the provision of stoichiometric quantities of NAD(P)H. Since the cofactors are extremely expensive, economic viability depends on recycling the cofactors. In vitro cofactor recycling using coupled enzymes is generally impractical at large scale (although there are a few exceptions), and alternative approaches, such as artificial cofactors or chemical, electrochemical or photochemical reduction of cofactors, are not industrially viable. As a result, nearly all redox biotransformations are operated using whole cells, exploiting their in-built capacity for cofactor recycling. Even this approach suffers from problems, since cells are notoriously prone to poisoning by substrate/product toxicity, and side reactions may consume substrate or product. Redox biotransformations would be a much more attractive prospect if there were general methods to use isolated enzymes instead of whole cells. In this application, we propose to develop robust, cofactor-independent systems to deliver reducing power to redox enzymes in vitro using microfabricated electrochemical reactors which offer the prospect of a realistic approach to bioelectrocatalytic manufacturing. Our approach is to construct a generic platform technology based on a small number of well characterised catalytic frameworks that enables tailoring of reaction and substrate specificity to target reagents. The platform technology will provide a 'step change' in biocatalysis and allow us to develop a viable manufacturing process, independent of redox coenzymes, to exploit a wide range of novel and industrially relevant redox biotransformations.

Publications

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Heyes DJ (2009) Enzymes - Nature's catalytic machines in Biological Sciences Review

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Toogood H (2013) Enzyme engineering toolbox - a 'catalyst' for change in Catalysis Science & Technology

 
Description We established routes to bioelectrocatalysis using our published microfluidic devices, identified novel biocatalysts with an extensive range of specificities, enantioselectivities and group chemistries. We were able to evolve many variants of ene reductases towards new target substrates.
Sectors Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other

 
Description Several publications have emerged from the grant funded work documenting efficient laboratory based evolution of ene reductases (mainly PETNR) using our high throughput robotics platform. The work has established the general utility of ene reductase biocatalysts and their evolved variants as useful industrial biocatalysts for activated carbon-carbon double bond reductions. The technology has been transferred to industrial partners (GSK and Dr Reddy's) through licensing agreements. We have developed novel microreactors for reduction of unsaturated compounds based on microfluidic concepts that will facilitate these biotransformations in an industrial context. The designs enable enzyme/substrate recycling, efficient product removal in biphasic systems and avoid the use of expensive reducing coenzymes such as NADPH.
First Year Of Impact 2007
Sector Chemicals,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other
Impact Types Economic

 
Title Robot 
Description Liquid handling robotics platform for directed evolution of enzyme activity. 
Type Of Material Improvements to research infrastructure 
Provided To Others? No  
Impact Publications 
 
Description DrReddys 
Organisation Dr. Reddy's Laboratories
Country India 
Sector Private 
PI Contribution Provided expertise, research time and equipment.
Collaborator Contribution Provided expertise and funded a CASE studentship
Impact Developed ezymatic methods for production of fine chemicals.
Start Year 2010
 
Description GSK 
Organisation GlaxoSmithKline (GSK)
Country Global 
Sector Private 
PI Contribution Provided expertise, research time and equipment.
Collaborator Contribution Provided expertise and funding.
Impact Synthetic biology methods for production of fine chemicals.
Start Year 2011
 
Title METHODS FOR PREPARING A HYDROCARBON 
Description Method for preparing a mono-unsaturated alkene comprising contacting an aliphatic mono-unsaturated carboxylic acid with an Fdc1 polypeptide comprising an amino acid sequence with at least 21% sequence identity to SEQ ID NO: 1 and a Pad1 polypeptide comprising an amino acid sequence with at least 17% sequence identity to SEQ ID NO: 2. 
IP Reference US2013330795 
Protection Patent application published
Year Protection Granted 2013
Licensed Yes
Impact .
 
Description Science Spectacular 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Members of research group presented exhibit on topics of enzymes and proteins. Event was well received by both students and their teachers and seemed to inspire interest in the subject.

No defined impacts realised to date
Year(s) Of Engagement Activity 2013,2014,2015,2016